Antigen binding molecules comprising a TNF family ligand trimer and a Tenascin binding moiety (2024)

U.S. patent number 11,149,083 [Application Number 16/186,443] was granted by the patent office on 2021-10-19 for antigen binding molecules comprising a tnf family ligand trimer and a tenascin binding moiety.This patent grant is currently assigned to Hoffmann-La Roche Inc.. The grantee listed for this patent is Hoffmann--La Roche Inc.. Invention is credited to Maria Amann, Peter Bruenker, Christina Claus, Claudia Ferrara Koller, Sandra Grau-Richards, Christian Klein, Viktor Levitski, Ekkehard Moessner, Pablo Umana.

Antigen binding molecules comprising a TNF family ligand trimer anda Tenascin binding moiety

Abstract

The invention relates to novel TNF family ligandtrimer-containing antigen binding molecules comprising (a) at leastone antigen binding moiety capable of specific binding toTenascin-C (TnC) and (b) a first and a second polypeptide that arelinked to each other by a disulfide bond, wherein the antigenbinding molecules are characterized in that the first polypeptidecomprises two ectodomains of a TNF ligand family member or twofragments thereof that are connected to each other by a peptidelinker and in that the second polypeptide comprises only oneectodomain of said TNF ligand family member or a fragment thereof.The invention further relates to methods of producing thesemolecules and to methods of using the same.

Prior Publication Data

Related U.S. Patent Documents

Foreign Application Priority Data

References Cited [Referenced By]

U.S. Patent Documents

Foreign Patent Documents

Other References

Primary Examiner: Landsman; Robert S
Attorney, Agent or Firm: Wu; Linda

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2017/060870, filed May 8, 2017, which claims priority fromEuropean Patent Application No. 16169244.7, filed May 11, 2016. Thecontents of each of the foregoing applications are incorporatedherein by reference in their entireties.

Claims

The invention claimed is:

1. A TNF (tumor necrosis factor) family ligand trimer-containingantigen binding molecule comprising (a) at least one antigenbinding moiety capable of specific binding to Tenascin-C(TnC) and(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises twoectodomains of a TNF ligand family member or a fragment thereofthat are connected to each other by a peptide linker and in thatthe second polypeptide comprises only one ectodomain of said TNFligand family member or a fragment thereof.

2. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, further comprising (c) an Fc domain composed of a firstand a second subunit capable of stable association.

3. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the antigen binding molecule is furthercharacterized in that (i) the first polypeptide contains a CH1 orCL domain and the second polypeptide contains a CL or CH1 domain,respectively, wherein the second polypeptide is linked to the firstpolypeptide by a disulfide bond between the CH1 and CL domain, or(ii) the first polypeptide contains a CH3 domain and the secondpolypeptide contains a CH3 domain, respectively, or (iii) the firstpolypeptide contains a VH-CL or a VL-CH1 domain and the secondpolypeptide contains a VL-CH1 domain or a VH-CL domain,respectively, wherein the second polypeptide is linked to the firstpolypeptide by a disulfide bond between the CH1 and CL domain.

4. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the TNF ligand family member costimulates humanT-cell activation.

5. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the TNF ligand family member is selected from4-1BBL and OX40L.

6. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the TNF ligand family member is 4-1BBL.

7. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the moiety capable of specific binding to TnCis selected from the group consisting of an antibody fragment, aFab molecule, a crossover Fab molecule, a single chain Fabmolecule, a Fv molecule, a scFv molecule, a single domain antibody,an aVH and a scaffold antigen binding protein.

8. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the molecule comprises one or two moietiescapable of specific binding to TnC.

9. The TNF family ligand trimer-containing antigen binding moleculeof claim 1, wherein the antigen binding moiety capable of specificbinding to TnC is a Fab molecule capable of specific binding toTnC.

10. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding moiety capable ofspecific binding to TnC comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO: 67, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO: 68, and (iii)CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69, and aVL domain comprising (iv) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 55, (v) CDR-L2 comprising the amino acid sequence ofSEQ ID NO: 56, and (vi) CDR-L3 comprising the amino acid sequenceof SEQ ID NO: 57.

11. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding moiety capable ofspecific binding to TnC comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO: 70, (ii) CDR-H2comprising the amino acid sequence of SEQ ID NO: 71, and (iii)CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72, and aVL domain comprising (iv) CDR-L1 comprising the amino acid sequenceof SEQ ID NO: 58, (v) CDR-L2 comprising the amino acid sequence ofSEQ ID NO: 59, and (vi) CDR-L3 comprising the amino acid sequenceof SEQ ID NO: 60.

12. The TNF family ligand trimer-containing antigen bindingmolecule of claim 2, wherein the Fc domain is an IgG1 Fc domaincomprising the amino acid substitutions at positions 234 and 235(EU numbering) and/or 329 (EU numbering).

13. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding molecule comprisesa first heavy chain and a first light chain, both comprising a Fabmolecule capable of specific binding to TnC, a first peptidecomprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkerfused at its C-terminus by a second peptide linker to a secondheavy or light chain, and a second peptide comprising oneectodomain of said TNF ligand family member fused at its C-terminusby a third peptide linker to a second light or heavy chain,respectively.

14. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the first peptide comprising twoectodomains of a TNF ligand family member or fragments thereofconnected to each other by a first peptide linker is fused at itsC-terminus by a second peptide linker to a CH1 domain that is partof a heavy chain, and the second peptide comprising one ectodomainof said TNF ligand family member or a fragment thereof is fused atit* C-terminus by a third peptide linker to a CL domain that ispart of a light chain.

15. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the first peptide comprising twoectodomains of a TNF ligand family member or fragments thereofconnected to each other by a first peptide linker is fused at itsC-terminus by a second peptide linker to a CL domain that is partof a heavy chain, and the second peptide comprising one ectodomainof said TNF ligand family member or a fragment thereof is fused atit* C-terminus by a third peptide linker to a CH1 domain that ispart of a light chain.

16. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the first peptide comprising twoectodomains of a TNF ligand family member or fragments thereofconnected to each other by a first peptide linker is fused at itsC-terminus by a second peptide linker to a VH domain that is partof a heavy chain, and the second peptide comprising one ectodomainof said TNF ligand family member or a fragment thereof is fused atit* C-terminus by a third peptide linker to a VL domain that ispart of a light chain.

17. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding moiety binds tohuman TnC, and cross-reacts to mouse and cynomolgus TnC.

18. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding moiety binds to atleast one of human, mouse and cynomolgus TnC with a K.sub.D rangeof about 1 .mu.M to about 0.001 nM.

19. The TNF family ligand trimer-containing antigen bindingmolecule of claim 17, wherein the antigen binding moiety binds tohuman TnC with a K.sub.D range of about 1 .mu.M to about 0.001 nM,and cross-reacts to mouse and cynomolgus TnC with a bindingaffinity within the K.sub.D range of a factor of 20 relative to theK.sub.D for binding human TnC.

20. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, for use as a medicament.

21. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, for use in the treatment of cancer.

22. The TNF family ligand trimer-containing antigen bindingmolecule of claim 1, wherein the antigen binding moiety binds tohuman TnC with a K.sub.D range of about 1 .mu.M to about 0.001 nM,and cross-reacts to mouse and cynomolgus TnC with a bindingaffinity within the K.sub.D range of a factor of 20 relative to theK.sub.D for binding human TnC.

23. A pharmaceutical composition comprising the TNF family ligandtrimer-containing antigen binding molecule of and at least onepharmaceutically acceptable excipient wherein the TNF family ligandtrimer-containing antigen binding molecule comprises: (a) at leastone antigen binding moiety capable of specific binding toTenascin-C(TnC) and (b) a first and a second polypeptide that arelinked to each other by a disulfide bond, wherein the antigenbinding molecule is characterized in that the first polypeptidecomprises two ectodomains of a TNF ligand family member or afragment thereof that are connected to each other by a peptidelinker and in that the second polypeptide comprises only oneectodomain of said TNF ligand family member or a fragmentthereof.

24. The TNF family ligand trimer-containing antigen bindingmolecule of the pharmaceutical composition of claim 23, for use asa medicament.

25. The pharmaceutical composition of claim 23 for use in thetreatment of cancer.

Description

SEQUENCE LISTING

This application contains a Sequence Listing submitted via EFS-Weband hereby incorporated by reference in its entirety. Said ASCIIcopy, created on Dec. 20, 2018, is named P33586-US_SL.txt and is538,620 bytes in size.

FIELD OF THE INVENTION

The invention relates to novel TNF family ligand trimer-containingantigen binding molecules comprising (a) at least one antigenbinding moiety capable of specific binding to Tenascin-C (TnC) and(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecules arecharacterized in that the first polypeptide comprises twoectodomains of a TNF ligand family member or two fragments thereofthat are connected to each other by a peptide linker and in thatthe second polypeptide comprises only one ectodomain of said TNFligand family member or a fragment thereof. The invention furtherrelates to methods of producing these molecules and to methods ofusing the same.

BACKGROUND

Ligands interacting with molecules of the TNF (tumor necrosisfactor) receptor superfamily have pivotal roles in the organizationand function of the immune system. While regulating normalfunctions such as immune responses, hematopoiesis andmorphogenesis, the TNF family ligands (also called cytokines) playa role in tumorgenesis, transplant rejection, septic shock, viralreplication, bone resorption, rheumatoid arthritis and diabetes(Aggarwal, 2003). The TNF ligand family comprises 18 genes encoding19 type II (i.e. intracellular N terminus and extracellularC-terminus) transmembrane proteins, characterized by the presenceof a conserved C-terminal domain coined the `TNF hom*ology domain`(THD). This domain is responsible for receptor binding and is thuscritical for the biological activity of the TNF ligand familymembers. The sequence identity between family members is about20-30% (Bodmer, 2002). Members of the TNF ligand family exert theirbiological function as self-assembling, noncovalent trimers (Banneret al, Cell 1993, 73, 431-445). Thus, the TNF family ligands form atrimer that is able to bind to and to activate the correspondingreceptors of TNFR superfamily.

4-1BB (CD137), a member of the TNF receptor superfamily, has beenfirst identified as a molecule whose expression is induced byT-cell activation (Kwon and Weissman, 1989). Subsequent studiesdemonstrated expression of 4-1BB in T- and B-lymphocytes (Snell etal., 2011; Zhang et al., 2010), NK-cells (Lin et al., 2008),NKT-cells (Kim et al., 2008), monocytes (Kienzle and von Kempis,2000; Schwarz et al., 1995), neutrophils (Heinisch et al., 2000),mast (Nishimoto et al., 2005) and dendritic cells as well as cellsof non-hematopoietic origin such as endothelial and smooth musclecells (Broil et al., 2001; Olofsson et al., 2008). Expression of4-1BB in different cell types is mostly inducible and driven byvarious stimulatory signals, such as T-cell receptor (TCR) orB-cell receptor triggering, as well as signaling induced throughco-stimulatory molecules or receptors of pro-inflammatory cytokines(Diehl et al., 2002; von Kempis et al., 1997; Zhang et al.,2010).

Expression of 4-1BB ligand (4-1BBL or CD137L) is more restrictedand is observed on professional antigen presenting cells (APC) suchas B-cells, dendritic cells (DCs) and macrophages. Inducibleexpression of 4-1BBL is characteristic for T-cells, including both.alpha..beta. and .gamma..delta.T-cell subsets, and endothelialcells (reviewed in Shao and Schwarz, 2011).

CD137 signaling is known to stimulate IFN.gamma. secretion andproliferation of NK cells (Buechele et al., 2012; Lin et al., 2008;Melero et al., 1998) as well as to promote DC activation asindicated by their increased survival and capacity to secretcytokines and upregulate co-stimulatory molecules (Choi et al.,2009; Futagawa et al., 2002; Wilcox et al., 2002). However, CD137is best characterized as a co-stimulatory molecule which modulatesTCR-induced activation in both the CD4+ and CD8+ subsets ofT-cells. In combination with TCR triggering, agonistic4-1BB-specific antibodies enhance proliferation of T-cells,stimulate lymphokine secretion and decrease sensitivity ofT-lymphocytes to activation-induced cells death (reviewed in Snellet al., 2011).

In line with these co-stimulatory effects of 4-1BB antibodies onT-cells in vitro, their administration to tumor bearing mice leadsto potent anti-tumor effects in many experimental tumor models(Melero et al., 1997; Narazaki et al., 2010). However, 4-1BBusually exhibits its potency as an anti-tumor agent only whenadministered in combination with other immunomodulatory compounds(Curran et al., 2011; Guo et al., 2013; Morales-Kastresana et al.,2013; Teng et al., 2009; Wei et al., 2013), chemotherapeuticreagents (Ju et al., 2008; Kim et al., 2009), tumor-specificvaccination (Cuadros et al., 2005; Lee et al., 2011) orradiotherapy (Shi and Siemann, 2006). In vivo depletion experimentsdemonstrated that CD8+ T-cells play the most critical role inanti-tumoral effect of 4-1BB-specific antibodies. However,depending on the tumor model or combination therapy, which includesanti-4-1BB, contributions of other types of cells such as DCs,NK-cells or CD4+ T-cells have been reported (Melero et al., 1997;Murillo et al., 2009; Narazaki et al., 2010; Stagg et al.,2011).

In addition to their direct effects on different lymphocytesubsets, 4-1BB agonists can also induce infiltration and retentionof activated T-cells in the tumor through 4-1BB-mediatedupregulation of intercellular adhesion molecule 1 (ICAM1) andvascular cell adhesion molecule 1 (VCAM1) on tumor vascularendothelium (Palazon et al., 2011).

4-1BB triggering may also reverse the state of T-cell anergyinduced by exposure to soluble antigen that may contribute todisruption of immunological tolerance in the tumor microenvironmentor during chronic infections (Wilcox et al., 2004).

It appears that the immunomodulatory properties of 4-1BB agonisticantibodies in vivo require the presence of the wild type Fc-portionon the antibody molecule thereby implicating Fc-receptor binding asan important event required for the pharmacological activity ofsuch reagents as has been described for agonistic antibodiesspecific to other apoptosis-inducing or immunomodulatory members ofthe TNFR-superfamily (Li and Ravetch, 2011; Teng et al., 2009).However, systemic administration of 4-1BB-specific agonisticantibodies with the functionally active Fc domain also inducesexpansion of CD8+ T-cells associated with liver toxicity (Dubrot etal., 2010) that is diminished or significantly ameliorated in theabsence of functional Fc-receptors in mice. In human clinicaltrials (ClinicalTrials.gov, NCT00309023), Fc-competent 4-1BBagonistic antibodies (BMS-663513) administered once every threeweeks for 12 weeks induced stabilization of the disease in patientswith melanoma, ovarian or renal cell carcinoma. However, the sameantibody given in another trial (NCT00612664) caused grade 4hepatitis leading to termination of the trial (Simeone andAscierto, 2012).

Collectively, the available pre-clinical and clinical data clearlydemonstrate that there is a high clinical need for effective 4-1BBagonists. However, new generation drug candidates should not onlyeffectively engage 4-1BB on the surface of hematopoietic andendothelial cells but also be capable of achieving that throughmechanisms other than binding to Fc-receptors in order to avoiduncontrollable side effects. The latter may be accomplished throughpreferential binding to tumor-specific or tumor-associatedmoieties.

Tenascins are a highly conserved family of large multimericextracellular matrix (ECM) glycoproteins, which is found invertebrates. Four Tenascin paralogues have been identified inmammals, termed Tenascin-C, Tenascin-R, Tenascin-X and Tenascin-W.Tenascin family proteins have a common primary structure,comprising N-terminal heptad repeats, epidermal growth factor(EGF)-like repeats, fibronectin type III domain repeats and aC-terminal fibrinogen-like globular domain. Via an N-terminaloligomerization domain, individual subunits assemble into trimersor, as is the case for TnC, even hexamers. Indeed, theoligomerization domain of TnC was reported to improve trimerzationof CD27L, CD40L, 41BBL, and glucocorticoid-induced TNF receptorligand when fused to the respective TNF receptor ligand (see Wyzgolet al., J Immunol. 183(3), 1851-61 (2009) and Berg et al., CellDeatch and Differentiation 14(12, 2021-2034 (2007)). These studiesused fragments of the TnC polypeptide chain to stabilice the activetrimeric form of the TNF ligands in TnC-TNF-ligand fusionpolypetides.

Mammalian Tenascin-C (TnC) monomers typically have 14.5 EGF-likerepeats and 8 fibronectin type III domain repeats that are sharedby all Tenascin-C isoforms. However, up to 9 additional fibronectintype III domain repeats (domains A1 to D) can be independentlyincluded or excluded by alternative splicing, giving rise to alarge number of Tenascin-C isoforms (see e.g. Hsia andSchwarzbauer, J Biol Chem 280, 26641-26644 (2005)).

Tenascin-C is transiently expressed in the developing embryo, butvirtually absent from adult tissues. It reappears, however, intissues undergoing remodeling processes, including certainpathological conditions such as wound healing, inflammation andcancer (reviewed in Chiquet-Ehrismann & Chiquet, J Pathol 200,488-499 (2003)).

Importantly, Tenascin-C is highly expressed in the majority ofmalignant solid tumors, including tumors of the brain, breast,colon, lung, skin and other organs (reviewed in Orend andChiquet-Ehrismann, Cancer Letters 244, 143-163 (2006)), where itmay be expressed by transformed epithelial cells as well as stromalcells in the tumor microenvironment (Yoshida et al., J Pathol 182,421-428 (1997), Hanamura et al., Int J Cancer 73, 10-15 (1997)). Inparticular, the "large isoform" of Tenascin-C, containing thealternatively spliced domains A1 to D, is expressed in invasivecarcinomas while being nearly undetectable in healthy adult tissues(Borsi et al., Int J Cancer 52, 688-692 (1992), Carnemolla et al.,Eur J Biochem 205, 561-567 (1992)).

SUMMARY OF THE INVENTION

There remains a need for active and safe 4-1BB agonists. Thebispecific molecules of the invention with at least one TnC antigenbinding moiety targeting the molecules of the invention to tumorcells of a broad range of malignancies, and a trimeric and thusbiologically active TNF ligand, provide a unique targeting tool toaddress tumor cells. The antigen binding molecules of the inventionhave sufficient stability to be pharmaceutically useful and can beused to treat different cancer with increased efficiency andsafety.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to Tenascin-C(TnC) and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, and (c) an Fc domain composed of a first and a secondsubunit capable of stable association.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that

(i) the first polypeptide contains a CH1 or CL domain and thesecond polypeptide contains a CL or CH1 domain, respectively,wherein the second polypeptide is linked to the first polypeptideby a disulfide bond between the CH1 and CL domain, and wherein thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or fragments thereof that are connected to each other and tothe CH1 or CL domain by a peptide linker and wherein the secondpolypeptide comprises one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCL or CH1 domain of said polypeptide, or (ii) the first polypeptidecontains a CH3 domain and the second polypeptide contains a CH3domain, respectively, and wherein the first polypeptide comprisestwo ectodomains of a TNF ligand family member or fragments thereofthat are connected to each other and to the C-terminus of the CH3domain by a peptide linker and wherein the second polypeptidecomprises only one ectodomain of said TNF ligand family member or afragment thereof connected via a peptide linker to C-terminus ofthe CH3 domain of said polypeptide, or (iii) the first polypeptidecontains a VH-CL or a VL-CH1 domain and the second polypeptidecontains a VL-CH1 domain or a VH-CL domain, respectively, whereinthe second polypeptide is linked to the first polypeptide by adisulfide bond between the CH1 and CL domain, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to to VHor VL by a peptide linker and wherein the second polypeptidecomprises one ectodomain of said TNF ligand family member or afragment thereof connected via a peptide linker to VL or VH of saidpolypeptide.

In a particular aspect, the TNF ligand family member is one thatcostimulates human T-cell activation. Thus, the TNF family ligandtrimer-containing antigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, wherein the TNF ligand family member costimulates humanT-cell activation. More particularly, the TNF ligand family memberis selected from 4-1BBL and OX40L.

In one aspect, the TNF ligand family member is 4-1BBL.

In a further aspect, the ectodomain of a TNF ligand family membercomprises the amino acid sequence selected from the groupconsisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO: 193 and SEQID NO: 194, particularly the amino acid sequence of SEQ ID NO: 172or SEQ ID NO: 183.

In another aspect, the ectodomain of a TNF ligand family member orfragment thereof comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:174, SEQ ID NO: 175 and SEQ ID NO: 183, particularly the amino acidsequence of SEQ ID NO: 172 or SEQ ID NO: 183. More particularly,the ectodomain of a TNF ligand family member comprises the aminoacid sequence of SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO:185 and SEQ ID NO: 186 and in that the second polypeptide comprisesthe amino acid sequence selected from the group consisting of SEQID NO: 172, SEQ ID NO: 174 and SEQ ID NO: 175 and SEQ ID NO:183.

In one aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:176 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 177.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:176 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 187.

In yet a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:184 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 188 or SEQ ID NO: 189.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 or CL domain and a secondpolypeptide containing a CL or CH1 domain, respectively, whereinthe second polypeptide is linked to the first polypeptide by adisulfide bond between the CH1 and CL domain,

and wherein the antigen binding molecule is characterized in thatthe first polypeptide comprises two ectodomains of a TNF ligandfamily member or two fragments thereof that are connected to eachother and to the CH1 or CL domain by a peptide linker and in thatthe second polypeptide comprises only one ectodomain of said TNFligand family member or a fragment thereof connected by a peptidelinker to the CL or CH1 domain of said polypeptide.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 domain and a secondpolypeptide containing a CL domain, wherein the second polypeptideis linked to the first polypeptide by a disulfide bond between theCH1 and CL domain, and wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises twoectodomains of a TNF ligand family member or fragments thereof thatare connected to each other and to the CH1 domain by a peptidelinker and in that the second polypeptide comprises one ectodomainof said TNF ligand family member or a fragment thereof connectedvia a peptide linker to the CL domain of said polypeptide.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CL domain and a secondpolypeptide containing a CH1 domain, wherein the second polypeptideis linked to the first polypeptide by a disulfide bond between theCH1 and CL domain, and wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises twoectodomains of a TNF ligand family member or fragments thereof thatare connected to each other and to the CL domain by a peptidelinker and in that the second polypeptide comprises one ectodomainof said TNF ligand family member or a fragment thereof connectedvia a peptide linker to the CH1 domain of said polypeptide.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined hereinbefore, wherein the moiety capable of specific binding to TnC is anantibody fragment.

In particular, the moiety capable of specific binding to TnC is anantigen binding moiety.

In particular, the moiety capable of specific binding to TnC isselected from the group consisting of an antibody fragment, a Fabmolecule, a crossover Fab molecule, a single chain Fab molecule, aFv molecule, a scFv molecule, a single domain antibody, and aVH anda scaffold antigen binding protein.

In particular, the TNF family ligand trimer-containing antigenbinding molecule comprises one or two moieties capable of specificbinding to TnC.

In a particular aspect, the invention is concerned with a TNFfamily ligand trimer-containing antigen binding molecule as definedabove, wherein the moiety capable of specific binding to TnC is aFab molecule.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined hereinbefore, wherein the moiety capable of specific binding to TnCantigen is a scaffold antigen binding protein.

The invention provides a TNF family ligand trimer-containingantigen binding molecule that comprises at least one moiety capableof specific binding to TnC. In a particular aspect, the TNF familyligand trimer-containing antigen binding molecule comprises onemoiety capable of specific binding to TnC. In another aspect, theinvention provides a TNF family ligand trimer-containing antigenbinding molecule comprising two moieties capable of specificbinding TnC.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the moietycapable of specific binding to TnC comprises a VH domain comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 orSEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence ofSEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VLdomain comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:60.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the moietycapable of specific binding to TnC comprises a VH domain comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67,(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 and(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69,and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO: 55, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO: 56 and (vi) CDR-L3 comprising the amino acidsequence of SEQ ID NO: 57.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the moietycapable of specific binding to TnC comprises a VH domain comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 70,(ii) CDR-H2 comprising the amino acid sequence SEQ ID NO: 71, and(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 72,and a VL domain comprising (iv) CDR-L1 comprising the amino acidsequence of SEQ ID NO: 58, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO: 59, and (vi) CDR-L3 comprising the aminoacid sequence of SEQ ID NO: 60.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as defined herein before, wherein themoiety capable of specific binding to TnC comprises a variableheavy chain comprising an amino acid sequence of SEQ ID NO: 46 anda variable light chain comprising an amino acid sequence of SEQ IDNO: 45 or wherein the moiety capable of specific binding to TnCcomprises a variable heavy chain comprising an amino acid sequenceof SEQ ID NO: 48 and a variable light chain comprising an aminoacid sequence of SEQ ID NO: 47.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein a peptide comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker is fused at its C-terminus to the CH1 orCL domain of a heavy chain by a second peptide linker and whereinone ectodomain of said TNF ligand family member or a fragmentthereof is fused at the its C-terminus the CL or CH1 domain on alight chain by a third peptide linker.

In a particular aspect, the invention relates to a TNF familyligand trimer-containing antigen binding molecule as defined above,wherein the peptide linker is (G4S).sub.2, i.e. a peptide linker ofSEQ ID NO: 150. In one aspect, the first peptide linker is(G4S).sub.2, the second peptide linker is GSPGSSSSGS (SEQ ID NO:153) and the third peptide linker is (G4S).sub.2. In anotheraspect, the first, the second and the third peptide linker is(G4S).sub.2.

The invention is further concerned with a TNF family ligandtrimer-containing antigen binding molecule as defined hereinbefore, comprising an Fc domain composed of a first and a secondsubunit capable of stable association.

In particular, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprising (c) an Fc domaincomposed of a first and a second subunit capable of stableassociation further comprises (a) a Fab molecule capable ofspecific binding to a target cell antigen, wherein the Fab heavychain is fused at the C-terminus to the N-terminus of a CH2 domainin the Fc domain.

In a further aspect, the Fc domain is an IgG, particularly an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fc domain isan IgG1 Fc domain. In a particular aspect, the Fc domain comprisesa modification promoting the association of the first and secondsubunit of the Fc domain.

In another aspect, the invention is concerned with a TNF familyligand trimer-containing antigen binding molecule as defined hereinbefore, comprising

(c) an Fc domain composed of a first and a second subunit capableof stable association, wherein the Fc domain comprises one or moreamino acid substitution that reduces binding to an Fc receptor, inparticular towards Fc.gamma. receptor.

In particular, the Fc domain comprises amino acid substitutions atpositions 234 and 235 (EU numbering) and/or 329 (EU numbering) ofthe IgG heavy chains. More particularly, provided is a trimeric TNFfamily ligand-containing antigen binding molecule according to theinvention which comprises an IgG1 Fc domain with the amino acidsubstitutions L234A, L235A and P329G (EU numbering).

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises a first heavy chain and a first lightchain, both comprising a Fab molecule capable of specific bindingto TnC, a first peptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof connected to each other by afirst peptide linker fused at its C-terminus by a second peptidelinker to a second heavy or light chain, and a second peptidecomprising one ectodomain of said TNF ligand family member fused atit* C-terminus by a third peptide linker to a second light or heavychain, respectively.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a CH1domain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a CL domain that is part of a light chain.

In yet another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a CLdomain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a CH1 domain that is part of a light chain.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a VHdomain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a VL domain that is part of a light chain.

Provided is further a TNF family ligand trimer-containing antigenbinding molecule, wherein in the CL domain adjacent to the TNFligand family member the amino acid at position 123 (EU numbering)has been replaced by arginine (R) and the amino acid at position124 (EU numbering) has been substituted by lysine (K), and whereinin the CH1 domain adjacent to the TNF ligand family member theamino acids at position 147 (EU numbering) and at position 213 (EUnumbering) have been substituted by glutamic acid (E).

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described hereinbefore, wherein the antigen binding molecule comprises

(a) a first heavy chain and a first light chain, both comprising aFab molecule capable of specific binding to TnC,

(b) a second heavy chain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ IDNO: 185 and SEQ ID NO: 186, and a second light chain comprising anamino acid sequence selected from the group consisting of SEQ IDNO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule, wherein the antigen binding moleculecomprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 48 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 47, (ii) a second heavy chain comprising theamino acid sequence of SEQ ID NO: 178, and (iii) a second lightchain comprising the amino acid sequence of SEQ ID NO: 179.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 48 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 47, (ii) a second heavy chain comprising theamino acid sequence selected from the group consisting of SEQ IDNO: 102, SEQ ID NO: 108, SEQ ID NO: 116 and SEQ ID NO: 120, and(iii) a second light chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 103, SEQ ID NO:109, SEQ ID NO: 117 and SEQ ID NO: 121.

In yet another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide contains a CH3 domain and the second polypeptidecontains a CH3 domain, respectively, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to theC-terminus of the CH3 domain by a peptide linker and wherein thesecond polypeptide comprises one ectodomain of said TNF ligandfamily member or a fragment thereof connected via a peptide linkerto C-terminus of the CH3 domain of said polypeptide.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain and wherein the firstpolypeptide comprising two ectodomains of a TNF ligand familymember or fragments thereof comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 176 and SEQ ID NO:184 and the second polypeptide comprising one ectodomain of saidTNF ligand family member or a fragment thereof comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 172and SEQ ID NO: 183. In one aspect, the first polypeptide comprisingtwo ectodomains of a TNF ligand family member or fragments thereofcomprises an amino acid sequence SEQ ID NO: 176 and the secondpolypeptide comprising one ectodomain of said TNF ligand familymember or a fragment thereof comprises the amino acid sequence ofSEQ ID NO: 172. In a particular aspect, the first polypeptidecomprising two ectodomains of a TNF ligand family member orfragments thereof comprises an amino acid sequence of SEQ ID NO:184 and the second polypeptide comprising one ectodomain of saidTNF ligand family member or a fragment thereof comprises the aminoacid sequence of SEQ ID NO: 183.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) one Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain. The invention thus relates to aTNF family ligand trimer-containing antigen binding molecule,wherein TNF family ligand trimer-containing antigen bindingmolecule is monovalent for the binding to the target cellantigen.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined beforefurther comprising (d) a Fab domain that is not capable of specificbinding to TnC. Thus, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association,

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain, and (d) a Fab domain that isnot capable of specific binding to a target cell antigen.

In particular, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQID NO: 127, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 125, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO:130, a second heavy chain comprising the amino acid sequenceof SEQ ID NO:131, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 124, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 133, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 136, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 137, and one light chain comprising the amino acidsequence of SEQ ID NO: 77.

In particular, such a TNF family ligand trimer-containing antigenbinding molecule comprises two moieties capable of specific bindingto TnC. In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising two moietiescapable of specific binding to TnC. In particular, provided is aTNF family ligand trimer-containing antigen binding molecule asdescribed herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQID NO: 112, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 113, and two light chains comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 124, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 125, and two light chains comprising the amino acidsequence of SEQ ID NO: 77.

In one aspect, the TNF ligand family member is OX40L. In a furtheraspect, the ectodomain of a TNF ligand family member comprises theamino acid sequence selected from the group consisting of SEQ IDNO: 181 or SEQ ID NO: 182, particularly the amino acid sequence SEQID NO: 181.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described hereinbefore, comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:190 or SEQ ID: 191 and in that the second polypeptide comprises theamino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 182.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as describe herein,wherein the target cell antigen is Tenascin-C (TnC) and the moietycapable of specific binding to TnC comprises a VH domain comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 orSEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence ofSEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VLdomain comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:60.

Particularly, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein, wherein the antigenbinding molecule comprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO:45 or a first heavy chain comprising the VH domain comprisingthe amino acid sequence of SEQ ID NO: 48 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 47, (ii) a second heavy chain comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 195, and(iii) a second light chain comprising the amino acid sequence ofSEQ ID NO: 196.

According to another aspect of the invention, there is provided anisolated polynucleotide encoding an antigen binding molecule asdefined herein before. The invention further provides a vector,particularly an expression vector, comprising the isolatedpolynucleotide of the invention and a host cell comprising theisolated polynucleotide or the vector of the invention. In someembodiments the host cell is a eukaryotic cell, particularly amammalian cell.

In another aspect, provided is a method for producing the antigenbinding molecule of the invention, comprising the steps of (i)culturing the host cell of the invention under conditions suitablefor expression of the antigen binding molecule, and (ii) recoveringthe antigen binding molecule. The invention also encompasses anantigen binding molecule produced by the method of theinvention.

The invention further provides a pharmaceutical compositioncomprising the antigen binding molecule of the invention and atleast one pharmaceutically acceptable excipient.

Also encompassed by the invention is the antigen binding moleculeof the invention, or the pharmaceutical composition of theinvention, for use as a medicament. In one aspect is provided theantigen binding molecule of the invention, or the pharmaceuticalcomposition of the invention, for use in the treatment of a diseasein an individual in need thereof. In a specific embodiment,provided is the antigen binding molecule of the invention, or thepharmaceutical composition of the invention, for use in thetreatment of cancer.

Also provided is the use of the antigen binding molecule of theinvention for the manufacture of a medicament for the treatment ofa disease in an individual in need thereof, in particular for themanufacture of a medicament for the treatment of cancer, as well asa method of treating a disease in an individual, comprisingadministering to said individual a therapeutically effective amountof a composition comprising the antigen binding molecule of theinvention in a pharmaceutically acceptable form. In a specificembodiment, the disease is cancer. In any of the above embodimentsthe individual is preferably a mammal, particularly a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C shows immunohistological staining in LS174Txenografts tumors at 100.times. magnification as stained withanti-TnC clone 18D4 (FIG. 1B) and anti-TnC clone 11C7 (FIG. 1C).The pattern of staining corresponds to specific TnC stroma fibers.The TnC staining, for both clones 18D4 and 11C7, is overallexpressed with moderate intensity. Negative isotype control signalvalidates the specificity of the technique (FIG. 1A).

FIG. 2 shows the results of the immunohistological staining inhuman tumor array with a rabbit isotype control. Negative isotypecontrol signal in all tissues tested validates the specificity ofthe technique.

FIG. 3 shows the results of the immunohistological staining inhuman tumor array with anti-TnC clone 18D4. The pattern of stainingcorresponds to specific TnC stroma fibers. The TnC staining isexpressed at higher levels in most tumor tissues compared tocontrol normal pair tissue.

FIG. 4 shows the results of the immunohistological staining inhuman tumor array with anti-TnC clone 11C7. The pattern of stainingcorresponds to specific TnC stroma fibers. The TnC staining isexpressed at higher levels in most tumor tissues compared tocontrol normal pair tissue.

FIGS. 5A and 5B show components for the assembly of monovalent TnCtargeted split trimeric human 4-1BB ligand (71-254) (construct6.1). FIG. 5A shows a dimeric ligand fused to human IgG1-CL domain.FIG. 5B shows a monomeric ligand fused to human IgG1-CH1 domain."GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 6 shows the 4-1BBL-trimer-containing antigen binding moleculeconstructs 6.1 of the invention (monovalent TnC (18D4) targetedsplit trimeric 4-1BB ligand (71-254) Fc (kih) PGLALA fusioncontaining CH-CL cross with charged residues). The preparation andproduction of this construct is described in Example 1.1. The VHand VL domains are those of anti-TnC antibody 18D4, the thick blackpoint stands for the knob-into-hole modification. * symbolizesamino acid modifications in the CH1 and CL domain (so-calledcharged residues).

FIGS. 7A and 7B show the components for the assembly of monovalentTnC targeted split trimeric human 4-1BB ligand (71-254) (construct6.2). FIG. 7A shows dimeric ligand fused to human IgG1-CL domain.FIG. 7B shows monomeric ligand fused to human IgG1-CH1 domain."GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 8 shows monovalent TnC (18D4) targeted split trimeric 4-1BBligand (71-254) Fc (kih) fusion containing CH-CL cross withoutcharged residues (construct 6.2). The preparation and production ofthis construct is described in Example 1.2. The VH and VL domainsare those of anti-TnC antibody 18D4, the thick black point standsfor the knob-into-hole modification.

FIGS. 9A and 9B show components for the assembly of bivalent TnCtargeted split trimeric human 4-1BB ligand (71-254) (construct6.3). FIG. 9A shows dimeric ligand fused to human IgG1 Fc holechain. FIG. 9B shows monomeric ligand fused to human IgG1 Fc knobchain. "GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 10 shows bivalent TnC (18D4) targeted split trimeric 4-1BBligand Fc (kih) PGLALA fusion (construct 6.3). The preparation andproduction of this construct is described in Example 1.3. The VHand VL domains are those of anti-TnC antibody 18D4, the thick blackpoint stands for the knob-into-hole modification.

FIGS. 11A and 11B show components for the assembly of monovalentTnC targeted split trimeric human 4-1BB ligand (71-248) (construct6.4). FIG. 11A shows dimeric ligand fused to human IgG1-CL domain.FIG. 11B shows monomeric ligand fused to human IgG1-CH1 domain."GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 12 monovalent TnC (18D4) targeted split trimeric 4-1BB ligand(71-248) Fc (kih) fusion containing CH-CL cross with chargedresidues (construct 6.4). The preparation and production of thisconstruct is described in Example 1.4. The VH and VL domains arethose of anti-TnC antibody 18D4, the thick black point stands forthe knob-into-hole modification.

FIGS. 13A and 13B show components for the assembly of monovalentTnC targeted split trimeric human 4-1BB ligand (71-248) (construct6.5). FIG. 13A shows dimeric ligand fused to human IgG1-CL domain.FIG. 13B shows monomeric ligand fused to human IgG1-CH1 domain."GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 14 shows monovalent TnC (18D4) targeted split trimeric 4-1BBligand (71-248) Fc (kih) fusion containing CH-CL cross withoutcharged residues (construct 6.5). The preparation and production ofthis construct is described in Example 1.5. The VH and VL domainsare those of anti-TnC antibody 18D4, the thick black point standsfor the knob-into-hole modification.

FIGS. 15A and 15B show components for the assembly of bivalent TnCtargeted split trimeric human 4-1BB ligand (71-248) (construct6.6). FIG. 15A shows dimeric ligand fused to human IgG1 Fc holechain. FIG. 15B shows monomeric ligand fused to human IgG1 Fc knobchain. "GGGGSGGGGS" is disclosed as SEQ ID NO: 150.

FIG. 16 shows bivalent TnC (18D4) targeted split trimeric 4-1BBligand (71-248) Fc (kih) fusion (construct 6.6). The preparationand production of this construct is described in Example 1.6. TheVH and VL domains are those of anti-TnC antibody 18D4, the thickblack point stands for the knob-into-hole modification.

FIGS. 17A and 17B show components for the assembly of monovalenttargeted split trimeric human 4-1BB ligand fused at the C-terminusof a human Fc. FIG. 17A shows dimeric ligand fused to human IgG1 Fchole or knob chain. FIG. 17B shows monomeric ligand fused to humanIgG1 Fc knob or hole chain. "GGGGSGGGGS" is disclosed as SEQ ID NO:150.

FIGS. 18A and 18B show monovalent targeted split trimericC-terminal 4-1BB ligand Fc (kih) knob fusion. FIG. 18A showsconstruct 6.11, dimeric 4-1BBL fused to hole Fc chain and monomeric4-1BBL fused to knob Fc chain. FIG. 18B shows construct 6.12,dimeric 4-1BBL fused to knob Fc chain and monomeric 4-1BBL fused tohole Fc chain. The preparation and production of these constructsis described in Example 1.7. The VH and VL domains are those ofanti-TnC antibody 18D4, the thick black point stands for theknob-into-hole modification.

FIGS. 19A and 19B show monovalent targeted (hole chain) splittrimeric C-terminal 4-1BB ligand Fc (kih) fusion. FIG. 19A showsconstruct 6.13, dimeric 4-1BBL fused to hole Fc chain and monomeric4-1BBL fused to knob Fc chain. FIG. 19B shows construct 6.14,dimeric 4-1BBL fused to knob Fc chain and monomeric 4-1BBL fused tohole Fc chain. The preparation and production of these constructsis described in Example 1.8. The VH and VL domains are those ofanti-TnC antibody 18D4, the thick black point stands for theknob-into-hole modification.

FIGS. 20A, 20B, and 20C relate to simultaneous binding of targetedsplit trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusionmolecules. FIG. 20A shows setup of the SPR experiments. FIG. 20Bshows simultaneous binding of human 4-1BB and human TnC forconstruct 6.5. FIG. 20C shows simultaneous binding of human 4-1BBand human TnC for construct 6.6.

FIGS. 21A, 21B, 21C, and 21D show binding of TnC-targeted 4-1BBsplit trimeric ligand Fc fusion antigen binding molecules tofreshly isolated PBMCs. Shown is the geo mean of fluorescenceintensity of PE-conjugated secondary detection antibody (y-axis)versus the concentration of tested constructs (x-axis). Binding wasmonitored on fresh human CD45+CD3+ CD8neg CD4+ T cells (FIG. 21A),fresh human CD45+ CD3+ CD4neg CD8+ T cells (FIG. 21), fresh humanCD45+ CD3neg CD19+ B cells (FIG. 21C) and CD45+ CD3+ CD4neg CD8neg.gamma..delta.T cells (FIG. 21D).

FIGS. 22A, 22B, 22C, and 22D show binding of TnC-targeted 4-1BBsplit trimeric ligand Fc fusion antigen binding molecules toactivated PBMCs. Shown is the geo mean of fluorescence intensity ofPE-conjugated secondary detection antibody (y-axis) versus theconcentration of tested constructs (x-axis). Binding was monitoredon activated human CD45+ CD3+ CD8neg CD4+ T cells (FIG. 22A),activated human CD45+ CD3+ CD4neg CD8+ T cells (FIG. 22B), CD45+CD3neg CD19+ B cells (FIG. 22C) and activated human CD45+ CD3+CD4neg CD8neg .gamma..delta.T cells (FIG. 22D).

FIG. 23 shows binding of TnC-targeted or untargeted split trimerichuman 4-1BB ligand Fc (kih) fusion molecules to human-TnCexpressing U87-MG tumor cell line. Binding was detected withR-Phycoerythrin-fluorochrome conjugated anti-human IgGFc.gamma.-specific goat IgG F(ab')2 fragment and the geo meanmeasured by flow cytometry is shown on the y-axis. On the x-axisthe used concentration of TnC-targeted 4-1BB split trimeric ligandFc fusion antigen binding molecules and their controls areshown.

FIG. 24 shows an illustration of an activation assay set up withhuman 4-1BB expressing HeLa reporter cell line. A crosslinking of4-1BB, expressed on the reporter cells, induces NF.kappa.Bactivation and NF.kappa.B-mediated Luciferase expression. Afterlysis of the cells, luciferase can catalyze the oxidation ofLuciferin to Oxyluciferin. This chemical reaction correlatespositively with the strength of NF.kappa.B-mediated luciferaseexpression and can be measured by the strength of light emission(units of released light).

FIGS. 25A and 25B show the NF.kappa.B-activation-induced luciferaseactivity. Units of released light (URL) are measured for 0.5 s/welland plotted against the used concentration of split trimeric 4-1BBligand containing fusion molecules and controls. Human4-1BB-expressing HeLa-reporter cells were incubated for 6 h in theabsence (FIG. 25A) or presence of crosslinking via human-TnCexpressing U87MG cells (FIG. 25B). The cell ratio of4-1BB-expressing HeLa reporter cell to TnC-expressing tumor cellswas 1:5.

FIG. 26 shows an illustration of the Schematic set-up of the humanPBMC activation in the presence of TnC-expressing U87MG, agonisticanti-human CD3 antibody and bispecific anti-TnC split trimeric4-1BBL fusion molecule construct 6.5.

FIGS. 27A and 27B shows the human PBMC activation assay in thepresence of TnC+U87MG, agonistic CD3 antibody and TnC-targetedsplit trimeric 4-1BBL fusion molecules. FIG. 27A shows expressionlevels of CD25 as frequency of total CD8+ T cells are plottedagainst the used concentration of construct 6.5 and 6.6 or controls(control F, control L and control E). FIG. 27B shows levels ofCD137 (4-1BB) as frequency of total CD8+ T cells are plottedagainst the used concentration of construct 6.5 and 6.6 or controls(control F, control L and control E).

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, technical and scientific terms usedherein have the same meaning as generally used in the art to whichthis invention belongs. For purposes of interpreting thisspecification, the following definitions will apply and wheneverappropriate, terms used in the singular will also include theplural and vice versa.

As used herein, the term "antigen binding molecule" refers in itsbroadest sense to a molecule that specifically binds an antigenicdeterminant Examples of antigen binding molecules are antibodies,antibody fragments and scaffold antigen binding proteins.

As used herein, the term "moiety capable of specific binding"refers to a polypeptide molecule that specifically binds to anantigenic determinant. In one aspect, the antigen binding moiety isable to activate signaling through its target cell antigen. In aparticular aspect, the antigen binding moiety is able to direct theentity to which it is attached (e.g., the TNF family ligand trimer)to a target site, for example to a specific type of tumor cell ortumor stroma bearing the antigenic determinant Moieties capable ofspecific binding to a target cell antigen include antibodies andfragments thereof as further defined herein. In addition, moietiescapable of specific binding to a target cell antigen includescaffold antigen binding proteins as further defined herein, e.g.,binding domains which are based on designed repeat proteins ordesigned repeat domains (see e.g., WO 2002/020565).

In relation to an antibody or fragment thereof, the term "moietycapable of specific binding" refers to the part of the moleculethat comprises the area which specifically binds to and iscomplementary to part or all of an antigen. A moiety capable ofspecific antigen binding may be provided, for example, by one ormore antibody variable domains (also called antibody variableregions). Particularly, a moiety capable of specific antigenbinding comprises an antibody light chain variable region (VL) andan antibody heavy chain variable region (VH).

The term "antibody" herein is used in the broadest sense andencompasses various antibody structures, including but not limitedto monoclonal antibodies, polyclonal antibodies, monospecific andmultispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desiredantigen-binding activity.

The term "monoclonal antibody" as used herein refers to an antibodyobtained from a population of substantially hom*ogeneous antibodies,i.e., the individual antibodies comprising the population areidentical and/or bind the same epitope, except for possible variantantibodies, e.g., containing naturally occurring mutations orarising during production of a monoclonal antibody preparation,such variants generally being present in minor amounts. In contrastto polyclonal antibody preparations, which typically includedifferent antibodies directed against different determinants(epitopes), each monoclonal antibody of a monoclonal antibodypreparation is directed against a single determinant on anantigen.

The term "monospecific" antibody as used herein denotes an antibodythat has one or more binding sites each of which bind to the sameepitope of the same antigen. The term "bispecific" means that theantigen binding molecule is able to specifically bind to at leasttwo distinct antigenic determinants Typically, a bispecific antigenbinding molecule comprises two antigen binding sites, each of whichis specific for a different antigenic determinant. In certainembodiments the bispecific antigen binding molecule is capable ofsimultaneously binding two antigenic determinants, particularly twoantigenic determinants expressed on two distinct cells.

The term "valent" as used within the current application denotesthe presence of a specified number of binding sites in an antigenbinding molecule. As such, the terms "bivalent", "tetravalent", and"hexavalent" denote the presence of two binding sites, four bindingsites, and six binding sites, respectively, in an antigen bindingmolecule.

The terms "full length antibody", "intact antibody", and "wholeantibody" are used herein interchangeably to refer to an antibodyhaving a structure substantially similar to a native antibodystructure. "Native antibodies" refer to naturally occurringimmunoglobulin molecules with varying structures. For example,native IgG-class antibodies are heterotetrameric glycoproteins ofabout 150,000 daltons, composed of two light chains and two heavychains that are disulfide-bonded. From N- to C-terminus, each heavychain has a variable region (VH), also called a variable heavydomain or a heavy chain variable domain, followed by three constantdomains (CH1, CH2, and CH3), also called a heavy chain constantregion. Similarly, from N- to C-terminus, each light chain has avariable region (VL), also called a variable light domain or alight chain variable domain, followed by a light chain constantdomain (CL), also called a light chain constant region. The heavychain of an antibody may be assigned to one of five types, called.alpha. (IgA), .delta. (IgD), .epsilon. (IgE), .gamma. (IgG), or.mu. (IgM), some of which may be further divided into subtypes,e.g., .gamma.1 (IgG1), .gamma.2 (IgG2), .gamma.3 (IgG3), .gamma.4(IgG4), .alpha.1 (IgA1) and .alpha.2 (IgA2). The light chain of anantibody may be assigned to one of two types, called kappa(.kappa.) and lambda (.lamda.), based on the amino acid sequence ofits constant domain.

An "antibody fragment" refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that bindsthe antigen to which the intact antibody binds. Examples ofantibody fragments include but are not limited to Fv, Fab, Fab',Fab'-SH, F(ab').sub.2; diabodies, triabodies, tetrabodies,cross-Fab fragments; linear antibodies; single-chain antibodymolecules (e.g., scFv); and single domain antibodies. For a reviewof certain antibody fragments, see Hudson et al., Nat Med 9,129-134 (2003). For a review of scFv fragments, see e.g.,Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab')2 fragments comprisingsalvage receptor binding epitope residues and having increased invivo half-life, see U.S. Pat. No. 5,869,046. Diabodies are antibodyfragments with two antigen-binding sites that may be bivalent orbispecific, see, for example, EP 404,097; WO 1993/01161; Hudson etal., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc NatlAcad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat Med 9, 129-134 (2003).Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion ofthe light chain variable domain of an antibody. In certainembodiments, a single-domain antibody is a human single-domainantibody (Domantis, Inc., Waltham, Mass.; see e.g., U.S. Pat. No.6,248,516 B1). Antibody fragments can be made by varioustechniques, including but not limited to proteolytic digestion ofan intact antibody as well as production by recombinant host cells(e.g., E. coli or phage), as described herein.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called "Fab" fragments containing eachthe heavy- and light-chain variable domains and also the constantdomain of the light chain and the first constant domain (CH1) ofthe heavy chain. As used herein, thus, the term "Fab fragment"refers to an antibody fragment comprising a light chain fragmentcomprising a VL domain and a constant domain of a light chain (CL),and a VH domain and a first constant domain (CH1) of a heavy chain.Fab' fragments differ from Fab fragments by the addition of a fewresidues at the carboxy terminus of the heavy chain CH1 domainincluding one or more cysteins from the antibody hinge region.Fab'-SH are Fab' fragments in which the cysteine residue(s) of theconstant domains bear a free thiol group. Pepsin treatment yieldsan F(ab').sub.2 fragment that has two antigen-combining sites (twoFab fragments) and a part of the Fc region.

The term "cross-Fab fragment" or "xFab fragment" or "crossover Fabfragment" refers to a Fab fragment, wherein either the variableregions or the constant regions of the heavy and light chain areexchanged. Two different chain compositions of a crossover Fabmolecule are possible and comprised in the bispecific antibodies ofthe invention: On the one hand, the variable regions of the Fabheavy and light chain are exchanged, i.e., the crossover Fabmolecule comprises a peptide chain composed of the light chainvariable region (VL) and the heavy chain constant region (CH1), anda peptide chain composed of the heavy chain variable region (VH)and the light chain constant region (CL). This crossover Fabmolecule is also referred to as CrossFab.sub.(VLVH). On the otherhand, when the constant regions of the Fab heavy and light chainare exchanged, the crossover Fab molecule comprises a peptide chaincomposed of the heavy chain variable region (VH) and the lightchain constant region (CL), and a peptide chain composed of thelight chain variable region (VL) and the heavy chain constantregion (CH1). This crossover Fab molecule is also referred to asCrossFab.sub.(CLCH).

A "single chain Fab fragment" or "scFab" is a polypeptideconsisting of an antibody heavy chain variable domain (VH), anantibody constant domain 1 (CH1), an antibody light chain variabledomain (VL), an antibody light chain constant domain (CL) and alinker, wherein said antibody domains and said linker have one ofthe following orders in N-terminal to C-terminal direction: a)VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptideof at least 30 amino acids, preferably between 32 and 50 aminoacids. Said single chain Fab fragments are stabilized via thenatural disulfide bond between the CL domain and the CH1 domain. Inaddition, these single chain Fab molecules might be furtherstabilized by generation of interchain disulfide bonds viainsertion of cysteine residues (e.g., position 44 in the variableheavy chain and position 100 in the variable light chain accordingto Kabat numbering).

A "crossover single chain Fab fragment" or "x-scFab" is a is apolypeptide consisting of an antibody heavy chain variable domain(VH), an antibody constant domain 1 (CH1), an antibody light chainvariable domain (VL), an antibody light chain constant domain (CL)and a linker, wherein said antibody domains and said linker haveone of the following orders in N-terminal to C-terminal direction:a) VH-CL-linker-VL-CH1 and b) VL-CH1-linker-VH-CL; wherein VH andVL form together an antigen-binding site which binds specificallyto an antigen and wherein said linker is a polypeptide of at least30 amino acids. In addition, these x-scFab molecules might befurther stabilized by generation of interchain disulfide bonds viainsertion of cysteine residues (e.g., position 44 in the variableheavy chain and position 100 in the variable light chain accordingto Kabat numbering).

A "single-chain variable fragment (scFv)" is a fusion protein ofthe variable regions of the heavy (V.sub.H) and light chains(V.sub.L) of an antibody, connected with a short linker peptide often to about 25 amino acids. The linker is usually rich in glycinefor flexibility, as well as serine or threonine for solubility, andcan either connect the N-terminus of the V.sub.H with theC-terminus of the V.sub.L, or vice versa. This protein retains thespecificity of the original antibody, despite removal of theconstant regions and the introduction of the linker. scFvantibodies are, e.g. described in Houston, J. S., Methods inEnzymol. 203 (1991) 46-96). In addition, antibody fragmentscomprise single chain polypeptides having the characteristics of aVH domain, namely being able to assemble together with a VL domain,or of a VL domain, namely being able to assemble together with a VHdomain to a functional antigen binding site and thereby providingthe antigen binding property of full length antibodies.

"Scaffold antigen binding proteins" are known in the art, forexample, fibronectin and designed ankyrin repeat proteins (DARPins)have been used as alternative scaffolds for antigen-bindingdomains, see, e.g., Gebauer and Skerra, Engineered proteinscaffolds as next-generation antibody therapeutics. Curr Opin ChemBiol 13:245-255 (2009) and Stumpp et al., Darpins: A new generationof protein therapeutics. Drug Discovery Today 13: 695-701 (2008).In one aspect of the invention, a scaffold antigen binding proteinis selected from the group consisting of CTLA-4 (Evibody),Lipocalins (Anticalin), a Protein A-derived molecule such asZ-domain of Protein A (Affibody), an A-domain (Avimer/Maxibody), aserum transferrin (trans-body); a designed ankyrin repeat protein(DARPin), a variable domain of antibody light chain or heavy chain(single-domain antibody, sdAb), a variable domain of antibody heavychain (nanobody, aVH), V.sub.NAR fragments, a fibronectin(AdNectin), a C-type lectin domain (Tetranectin); a variable domainof a new antigen receptor beta-lactamase (V.sub.NAR fragments), ahuman gamma-crystallin or ubiquitin (Affilin molecules); a kunitztype domain of human protease inhibitors, microbodies such as theproteins from the knottin family, peptide aptamers and fibronectin(adnectin).

CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is aCD28-family receptor expressed on mainly CD4+ T-cells. Itsextracellular domain has a variable domain-like Ig fold. Loopscorresponding to CDRs of antibodies can be substituted withheterologous sequence to confer different binding properties.CTLA-4 molecules engineered to have different binding specificitiesare also known as Evibodies (e.g. U.S. Pat. No. 7,166,697B1).Evibodies are around the same size as the isolated variable regionof an antibody (e.g., a domain antibody). For further details seeJournal of Immunological Methods 248 (1-2), 31-45 (2001).

Lipocalins are a family of extracellular proteins which transportsmall hydrophobic molecules such as steroids, bilins, retinoids andlipids. They have a rigid beta-sheet secondary structure with anumber of loops at the open end of the conical structure which canbe engineered to bind to different target antigens. Anticalins arebetween 160-180 amino acids in size, and are derived fromlipocalins. For further details see Biochim Biophys Acta 1482:337-350 (2000), U.S. Pat. No. 7,250,297B1 and US20070224633.

An affibody is a scaffold derived from Protein A of Staphylococcusaureus which can be engineered to bind to antigen. The domainconsists of a three-helical bundle of approximately 58 amino acids.Libraries have been generated by randomization of surface residues.For further details see Protein Eng. Des. Sel. 17, 455-462 (2004)and EP 1641818A1.

Avimers are multidomain proteins derived from the A-domain scaffoldfamily. The native domains of approximately 35 amino acids adopt adefined disulfide bonded structure. Diversity is generated byshuffling of the natural variation exhibited by the family ofA-domains. For further details see Nature Biotechnology 23(12),1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6),909-917 (June 2007).

A transferrin is a monomeric serum transport glycoprotein.Transferrins can be engineered to bind different target antigens byinsertion of peptide sequences in a permissive surface loop.Examples of engineered transferrin scaffolds include theTrans-body. For further details see J. Biol. Chem 274, 24066-24073(1999).

Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrinwhich is a family of proteins that mediate attachment of integralmembrane proteins to the cytoskeleton. A single ankyrin repeat is a33 residue motif consisting of two alpha-helices and a beta-turn.They can be engineered to bind different target antigens byrandomizing residues in the first alpha-helix and a beta-turn ofeach repeat. Their binding interface can be increased by increasingthe number of modules (a method of affinity maturation). Forfurther details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4),1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) andUS20040132028A1.

A single-domain antibody is an antibody fragment consisting of asingle monomeric variable antibody domain. The first single domainantibodies were derived from the variable domain of the antibodyheavy chain from camelids (nanobodies or V.sub.HH fragments).Furthermore, the term single-domain antibody includes an autonomoushuman heavy chain variable domain (aVH) or V.sub.NAR fragmentsderived from sharks.

Fibronectin is a scaffold which can be engineered to bind toantigen. Adnectins consists of a backbone of the natural amino acidsequence of the 10th domain of the 15 repeating units of humanfibronectin type III (FN3). Three loops at one end of the.beta.-sandwich can be engineered to enable an Adnectin tospecifically recognize a therapeutic target of interest. Forfurther details see Protein Eng. Des. Sel. 18, 435-444 (2005),US20080139791, WO2005056764 and U.S. Pat. No. 6,818,418B1.

Peptide aptamers are combinatorial recognition molecules thatconsist of a constant scaffold protein, typically thioredoxin(TrxA) which contains a constrained variable peptide loop insertedat the active site. For further details see Expert Opin. Biol.Ther. 5, 783-797 (2005).

Microbodies are derived from naturally occurring microproteins of25-50 amino acids in length which contain 3-4 cysteinebridges--examples of microproteins include KalataBl and conotoxinand knottins. The microproteins have a loop which can be engineeredto include up to 25 amino acids without affecting the overall foldof the microprotein. For further details of engineered knottindomains, see WO2008098796.

An "antigen binding molecule that binds to the same epitope" as areference molecule refers to an antigen binding molecule thatblocks binding of the reference molecule to its antigen in acompetition assay by 50% or more, and conversely, the referencemolecule blocks binding of the antigen binding molecule to itsantigen in a competition assay by 50% or more.

The term "antigen binding domain" refers to the part of an antigenbinding molecule that comprises the area which specifically bindsto and is complementary to part or all of an antigen. Where anantigen is large, an antigen binding molecule may only bind to aparticular part of the antigen, which part is termed an epitope. Anantigen binding domain may be provided by, for example, one or morevariable domains (also called variable regions). Preferably, anantigen binding domain comprises an antibody light chain variableregion (VL) and an antibody heavy chain variable region (VH).

As used herein, the term "antigenic determinant" is synonymous with"antigen" and "epitope", and refers to a site (e.g., a contiguousstretch of amino acids or a conformational configuration made up ofdifferent regions of non-contiguous amino acids) on a polypeptidemacromolecule to which an antigen binding moiety binds, forming anantigen binding moiety-antigen complex. Useful antigenicdeterminants can be found, for example, on the surfaces of tumorcells, on the surfaces of virus-infected cells, on the surfaces ofother diseased cells, on the surface of immune cells, free in bloodserum, and/or in the extracellular matrix (ECM). The proteinsuseful as antigens herein can be any native form the proteins fromany vertebrate source, including mammals such as primates (e.g.humans) and rodents (e.g. mice and rats), unless otherwiseindicated. In a particular embodiment the antigen is a humanprotein. Where reference is made to a specific protein herein, theterm encompasses the "full-length", unprocessed protein as well asany form of the protein that results from processing in the cell.The term also encompasses naturally occurring variants of theprotein, e.g., splice variants or allelic variants.

The terms "capable of specific binding to TnC" refer to an antigenbinding molecule or antigen binding moiety that is capable ofbinding Tenascin-C (TnC) with sufficient affinity such that theantigen binding molecule is useful as a diagnostic and/ortherapeutic agent in targeting TnC. The antigen binding moleculesinclude but are not limited to, antibodies, Fab molecules,crossover Fab molecules, single chain Fab molecules, Fv molecules,scFv molecules, single domain antibodies, and VH and scaffoldantigen binding protein. In one embodiment, the extent of bindingof an anti-TnC antigen binding molecule to an unrelated, non-TnCprotein is less than about 10% of the binding of the antigenbinding molecule to TnC as measured, e.g., by a radioimmunoassay(RIA). In certain embodiments, an antigen binding molecule thatbinds to TnC has a dissociation constant (K.sub.D) of .ltoreq.1.mu.M, .ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.5 nM, .ltoreq.2 nM,.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM(e.g., 10.sup.-8 M or less, e.g., from 10.sup.-8 M to 10.sup.-13 M,e.g., from 10.sup.-9M to 10.sup.-13 M, e.g., from 10 nM to 0.1 nM,e.g., from 5 nM to 0.1 nM, e.g., from 2 nM to 0.1 nM). In certainembodiments, an anti-TnC antigen binding molecule binds to anepitope of TnC that is conserved among TnC from different species.In certain embodiments, an antigen binding molecule that binds toan epitope of TnC is specific for at least one of the domainsselected from the group consisting of A1, A2, A3, A4, B, AD1, AD2,C and D. In certain embodiments an antigen binding moleculespecific for the TnC A1 and TnC A4 domains is provided. In certainembodiments an antigen binding molecule specific for the TnC Cdomain is provided. By "specific binding" is meant that the bindingis selective for the antigen and can be discriminated from unwantedor non-specific interactions. The ability of an antigen bindingmolecule to bind to a specific antigen can be measured eitherthrough an enzyme-linked immunosorbent assay (ELISA) or othertechniques familiar to one of skill in the art, e.g., SurfacePlasmon Resonance (SPR) technique (analyzed on a BIAcoreinstrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), andtraditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).In one embodiment, the extent of binding of an antigen bindingmolecule to an unrelated protein is less than about 10% of thebinding of the antigen binding molecule to the antigen as measured,e.g., by SPR. In certain embodiments, a molecule that binds to theantigen has a dissociation constant (Kd) of .ltoreq.1 .mu.M,.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.5 nM, .ltoreq.2 nM,.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM(e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M to 10.sup.-13 M,e.g. from 10.sup.-9 M to 10.sup.-13 M).

"Affinity" or "binding affinity" refers to the strength of the sumtotal of non-covalent interactions between a single binding site ofa molecule (e.g., an antibody) and its binding partner (e.g., anantigen). Unless indicated otherwise, as used herein, "bindingaffinity" refers to intrinsic binding affinity which reflects a 1:1interaction between members of a binding pair (e.g., antibody andantigen). The affinity of a molecule X for its partner Y cangenerally be represented by the dissociation constant (Kd), whichis the ratio of dissociation and association rate constants(k.sub.off and k.sub.on, respectively). Thus, equivalent affinitiesmay comprise different rate constants, as long as the ratio of therate constants remains the same. Affinity can be measured by commonmethods known in the art, including those described herein. Aparticular method for measuring affinity is Surface PlasmonResonance (SPR).

A "target cell antigen" as used herein refers to an antigenicdeterminant presented on the surface of a target cell or in theenvironment of a target cell, for example a cell in a tumor such asa cancer cell or a cell of the tumor stroma. In certainembodiments, the target cell antigen is expressed in theextracellular matrix in or around tumor tissue. In particular, thetarget cell antigen is Tenascin-C (TnC). The term "Tenascin-C" or"TnC" as used herein, refers to any native TnC from any vertebratesource, including mammals such as primates (e.g., humans andcynomolgus monkey) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses "full-length,"unprocessed TnC as well as any form of TnC that results fromprocessing in the cell. The term also encompasses naturallyoccurring variants of TnC, e.g., splice variants or allelicvariants. The amino acid sequence of an exemplary human TnC antigensequence (with N-terminal GST and 6.times.His-tag; and C-terminalavi-tag and 6.times.His-tag) is shown in SEQ ID NO: 4. The aminoacid sequence of an exemplary mouse TnC antigen sequence (withN-terminal GST and 6.times.His-tag; and C-terminal avi-tag and6.times.His-tag) is shown in SEQ ID NO: 5. The amino acid sequenceof an exemplary cynomolgus TnC antigen sequence (with N-terminalGST and 6.times.His-tag (SEQ ID NO: 221); and C-terminal avi-tagand 6.times.His-tag) is shown in SEQ ID NO: 6. In the human TnCmolecule, up to nine alternatively spliced fibronectin-type IIIdomains, which may be inserted between the fifth and the sixth ofthe constant fibronectin-type III domains are known (for aschematic representation of the domain structure of TnC, see e.g.,Orend and Chiquet-Ehrismann, Cancer Letters 244, 143-163 (2006).Similarly, in the mouse TnC molecule, six alternatively splicedfibronectin-type III domains are described (e.g., in Joestner andFaissner, J Biol Chem 274, 17144-17151 (1999)).

The term "variable region" or "variable domain" refers to thedomain of an antibody heavy or light chain that is involved inbinding the antigen binding molecule to antigen. The variabledomains of the heavy chain and light chain (VH and VL,respectively) of a native antibody generally have similarstructures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). See, e.g.,Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page91 (2007). A single VH or VL domain may be sufficient to conferantigen-binding specificity.

The term "hypervariable region" or "HVR," as used herein refers toeach of the regions of an antibody variable domain which arehypervariable in sequence and/or form structurally defined loops("hypervariable loops"). Generally, native four-chain antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in theVL (L1, L2, L3). HVRs generally comprise amino acid residues fromthe hypervariable loops and/or from the "complementaritydetermining regions" (CDRs), the latter being of highest sequencevariability and/or involved in antigen recognition. Exemplaryhypervariable loops occur at amino acid residues 26-32 (L1), 50-52(L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs(CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at aminoacid residues 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1,50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991).) Hypervariable regions(HVRs) are also referred to as complementarity determining regions(CDRs), and these terms are used herein interchangeably inreference to portions of the variable region that form the antigenbinding regions. This particular region has been described by Kabatet al., U.S. Dept. of Health and Human Services, "Sequences ofProteins of Immunological Interest" (1983) and by Chothia et al.,J. Mol. Biol. 196:901-917 (1987), where the definitions includeoverlapping or subsets of amino acid residues when compared againsteach other. Nevertheless, application of either definition to referto a CDR of an antibody or variants thereof is intended to bewithin the scope of the term as defined and used herein. Theappropriate amino acid residues which encompass the CDRs as definedby each of the above cited references are set forth below in TableA as a comparison. The exact residue numbers which encompass aparticular CDR will vary depending on the sequence and size of theCDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region aminoacid sequence of the antibody.

TABLE-US-00001 TABLE A CDR Definitions.sup.1 CDR Kabat ChothiaAbM.sup.2 V.sub.H CDR1 31-35 26-32 26-35 V.sub.H CDR2 50-65 52-5850-58 V.sub.H CDR3 95-102 95-102 95-102 V.sub.L CDR1 24-34 26-3224-34 V.sub.L CDR2 50-56 50-52 50-56 V.sub.L CDR3 89-97 91-96 89-97.sup.1Numbering of all CDR definitions in Table A is according tothe numbering conventions set forth by Kabat et al. (see below)..sup.2"AbM" with a lowercase "b" as used in Table A refers to theCDRs as defined by Oxford Molecular's "AbM" antibody modelingsoftware.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skillin the art can unambiguously assign this system of "Kabatnumbering" to any variable region sequence, without reliance on anyexperimental data beyond the sequence itself. As used herein,"Kabat numbering" refers to the numbering system set forth by Kabatet al., U.S. Dept. of Health and Human Services, "Sequence ofProteins of Immunological Interest" (1983). Unless otherwisespecified, references to the numbering of specific amino acidresidue positions in an antibody variable region are according tothe Kabat numbering system.

With the exception of CDR1 in VH, CDRs generally comprise the aminoacid residues that form the hypervariable loops. CDRs also comprise"specificity determining residues," or "SDRs," which are residuesthat contact antigen. SDRs are contained within regions of the CDRscalled abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1,a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur atamino acid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35Bof H1, 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson,Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated,HVR residues and other residues in the variable domain (e.g., FRresidues) are numbered herein according to Kabat et al., supra.

As used herein, the term "affinity matured" in the context ofantigen binding molecules (e.g., antibodies) refers to an antigenbinding molecule that is derived from a parent antigen bindingmolecule, e.g., by mutation, binds to the same antigen, preferablybinds to the same epitope, as the parent antibody; and has a higheraffinity for the antigen than that of the reference antigen bindingmolecule. Affinity maturation generally involves modification ofone or more amino acid residues in one or more CDRs of the antigenbinding molecule. Typically, the affinity matured antigen bindingmolecule binds to the same epitope as the initial parent antigenbinding molecule.

"Framework" or "FR" refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in thefollowing sequence in VH (or VL):FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

An "acceptor human framework" for the purposes herein is aframework comprising the amino acid sequence of a light chainvariable domain (VL) framework or a heavy chain variable domain(VH) framework derived from a human immunoglobulin framework or ahuman consensus framework, as defined below. An acceptor humanframework "derived from" a human immunoglobulin framework or ahuman consensus framework may comprise the same amino acid sequencethereof, or it may contain amino acid sequence changes. In someembodiments, the number of amino acid changes are 10 or less, 9 orless, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 orless, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

The term "chimeric" antibody refers to an antibody in which aportion of the heavy and/or light chain is derived from aparticular source or species, while the remainder of the heavyand/or light chain is derived from a different source orspecies.

The "class" of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several ofthese may be further divided into subclasses (isotypes), e.g.,IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constantdomains that correspond to the different classes of immunoglobulinsare called .alpha., .delta., .epsilon., .gamma., and .mu.respectively.

A "humanized" antibody refers to a chimeric antibody comprisingamino acid residues from non-human HVRs and amino acid residuesfrom human FRs. In certain embodiments, a humanized antibody willcomprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the HVRs(e.g., CDRs) correspond to those of a non-human antibody, and allor substantially all of the FRs correspond to those of a humanantibody. A humanized antibody optionally may comprise at least aportion of an antibody constant region derived from a humanantibody. A "humanized form" of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization.Other forms of "humanized antibodies" encompassed by the presentinvention are those in which the constant region has beenadditionally modified or changed from that of the original antibodyto generate the properties according to the invention, especiallyin regard to C1q binding and/or Fc receptor (FcR) binding.

A "human" antibody is one which possesses an amino acid sequencewhich corresponds to that of an antibody produced by a human or ahuman cell or derived from a non-human source that utilizes humanantibody repertoires or other human antibody-encoding sequences.This definition of a human antibody specifically excludes ahumanized antibody comprising non-human antigen-bindingresidues.

The term "Fc domain" or "Fc region" herein is used to define aC-terminal region of an antibody heavy chain that contains at leasta portion of the constant region. The term includes native sequenceFc regions and variant Fc regions. An IgG Fc region comprises anIgG CH2 and an IgG CH3 domain. The "CH2 domain" of a human IgG Fcregion usually extends from an amino acid residue at about position231 to an amino acid residue at about position 340. In oneembodiment, a carbohydrate chain is attached to the CH2 domain. TheCH2 domain herein may be a native sequence CH2 domain or variantCH2 domain. The "CH3 domain" comprises the stretch of residuesC-terminal to a CH2 domain in an Fc region (i.e., from an aminoacid residue at about position 341 to an amino acid residue atabout position 447 of an IgG). The CH3 region herein may be anative sequence CH3 domain or a variant CH3 domain (e.g., a CH3domain with an introduced "protuberance" ("knob") in one chainthereof and a corresponding introduced "cavity" ("hole") in theother chain thereof; see U.S. Pat. No. 5,821,333, expresslyincorporated herein by reference). Such variant CH3 domains may beused to promote heterodimerization of two non-identical antibodyheavy chains as herein described. In one embodiment, a human IgGheavy chain Fc region extends from Cys226, or from Pro230, to thecarboxyl-terminus of the heavy chain. However, the C-terminallysine (Lys447) of the Fc region may or may not be present. Unlessotherwise specified herein, numbering of amino acid residues in theFc region or constant region is according to the EU numberingsystem, also called the EU index, as described in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md.,1991.

The "knob-into-hole" (kih) technology is described e.g., in U.S.Pat. Nos. 5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621(1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, themethod involves introducing a protuberance ("knob") at theinterface of a first polypeptide and a corresponding cavity("hole") in the interface of a second polypeptide, such that theprotuberance can be positioned in the cavity so as to promoteheterodimer formation and hinder hom*odimer formation. Protuberancesare constructed by replacing small amino acid side chains from theinterface of the first polypeptide with larger side chains (e.g.,tyrosine or tryptophan). Compensatory cavities of identical orsimilar size to the protuberances are created in the interface ofthe second polypeptide by replacing large amino acid side chainswith smaller ones (e.g., alanine or threonine). The protuberanceand cavity can be made by altering the nucleic acid encoding thepolypeptides, e.g., by site-specific mutagenesis, or by peptidesynthesis. In a specific embodiment a knob modification comprisesthe amino acid substitution T366W in one of the two subunits of theFc domain, and the hole modification comprises the amino acidsubstitutions T366S, L368A and Y407V in the other one of the twosubunits of the Fc domain. In a further specific embodiment, thesubunit of the Fc domain comprising the knob modificationadditionally comprises the amino acid substitution S354C, and thesubunit of the Fc domain comprising the hole modificationadditionally comprises the amino acid substitution Y349C.Introduction of these two cysteine residues results in theformation of a disulfide bridge between the two subunits of the Fcregion, thus further stabilizing the dimer (Carter, J ImmunolMethods 248, 7-15 (2001)). The numbering is according to EU indexof Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991.

A "region equivalent to the Fc region of an immunoglobulin" isintended to include naturally occurring allelic variants of the Fcregion of an immunoglobulin as well as variants having alterationswhich produce substitutions, additions, or deletions but which donot decrease substantially the ability of the immunoglobulin tomediate effector functions (such as antibody-dependent cellularcytotoxicity). For example, one or more amino acids can be deletedfrom the N-terminus or C-terminus of the Fc region of animmunoglobulin without substantial loss of biological function.Such variants can be selected according to general rules known inthe art so as to have minimal effect on activity (see, e.g., Bowie,J. U. et al., Science 247:1306-10 (1990)). The term "effectorfunctions" refers to those biological activities attributable tothe Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC), Fc receptor binding,antibody-dependent cell-mediated cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokinesecretion, immune complex-mediated antigen uptake by antigenpresenting cells, down regulation of cell surface receptors (e.g.,B cell receptor), and B cell activation.

An "activating Fc receptor" is an Fc receptor that followingengagement by an Fc region of an antibody elicits signaling eventsthat stimulate the receptor-bearing cell to perform effectorfunctions. Activating Fc receptors include Fc.gamma.RIIIa (CD16a),Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), and Fc.alpha.RI (CD89). Aparticular activating Fc receptor is human Fc.gamma.RIIIa (seeUniProt accession no. P08637, version 141).

The term "TNF ligand family member" or "TNF family ligand" refersto a proinflammatory cytokine. Cytokines in general, and inparticular the members of the TNF ligand family, play a crucialrole in the stimulation and coordination of the immune system. Atpresent, nineteen cytokines have been identified as members of theTNF (tumor necrosis factor) ligand superfamily on the basis ofsequence, functional, and structural similarities. All theseligands are type II transmembrane proteins with a C-terminalextracellular domain (ectodomain), N-terminal intracellular domainand a single transmembrane domain. The C-terminal extracellulardomain, known as TNF hom*ology domain (THD), has 20-30% amino acididentity between the superfamily members and is responsible forbinding to the receptor. The TNF ectodomain is also responsible forthe TNF ligands to form trimeric complexes that are recognized bytheir specific receptors.

Members of the TNF ligand family are selected from the groupconsisting of Lymphotoxin .alpha. (also known as LTA or TNFSF1),TNF (also known as TNFSF2), LT.beta. (also known as TNFSF3), OX40L(also known as TNFSF4), CD40L (also known as CD154 or TNFSF5), FasL(also known as CD95L, CD178 or TNFSF6), CD27L (also known as CD70or TNFSF7), CD30L (also known as CD153 or TNFSF8), 4-1BBL (alsoknown as TNFSF9), TRAIL (also known as APO2L, CD253 or TNFSF10),RANKL (also known as CD254 or TNFSF11), TWEAK (also known asTNFSF12), APRIL (also known as CD256 or TNFSF13), BAFF (also knownas CD257 or TNFSF13B), LIGHT (also known as CD258 or TNFSF14), TL1A(also known as VEGI or TNFSF15), GITRL (also known as TNFSF18),EDA-A1 (also known as ectodysplasin A1) and EDA-A2 (also known asectodysplasin A2). The term refers to any native TNF family ligandfrom any vertebrate source, including mammals such as primates(e.g., humans), non-human primates (e.g., cynomolgus monkeys) androdents (e.g., mice and rats), unless otherwise indicated. Inspecific embodiments of the invention, the TNF ligand family memberis selected from the group consisting of OX40L, FasL, CD27L, TRAIL,4-1BBL, CD40L and GITRL. In a particular embodiment, the TNF ligandfamily member is selected from 4-1BBL and OX40L.

Further information, in particular sequences, of the TNF ligandfamily members may be obtained from publically accessible databasessuch as Uniprot (www.uniprot.org). For instance, the human TNFligands have the following amino acid sequences: human Lymphotoxin.alpha. (UniProt accession no. P01374, SEQ ID NO: 203), human TNF(UniProt accession no. P01375, SEQ ID NO: 204), human Lymphotoxin.beta. (UniProt accession no. Q06643, SEQ ID NO: 205), human OX40L(UniProt accession no. P23510, SEQ ID NO: 206), human CD40L(UniProt accession no. P29965, SEQ ID NO: 207), human FasL (UniProtaccession no. P48023, SEQ ID NO: 208), human CD27L (UniProtaccession no. P32970, SEQ ID NO: 209), human CD30L (UniProtaccession no. P32971, SEQ ID NO: 210), 4-1BBL (UniProt accessionno. P41273, SEQ ID NO: 211), TRAIL (UniProt accession no. P50591,SEQ ID NO: 212), RANKL (UniProt accession no. 014788, SEQ ID NO:213), TWEAK (UniProt accession no. 043508, SEQ ID NO: 214), APRIL(UniProt accession no. 075888, SEQ ID NO: 215), BAH- (UniProtaccession no. Q9Y275, SEQ ID NO: 216), LIGHT (UniProt accession no.043557, SEQ ID NO: 217), TL1A (UniProt accession no. 095150, SEQ IDNO: 218), GITRL (UniProt accession no. Q9UNG2, SEQ ID NO: 219) andectodysplasin A (UniProt accession no. Q92838, SEQ ID NO: 220).

An "ectodomain" is the domain of a membrane protein that extendsinto the extracellular space (i.e., the space outside the targetcell). Ectodomains are usually the parts of proteins that initiatecontact with surfaces, which leads to signal transduction. Theectodomain of TNF ligand family member as defined herein thusrefers to the part of the TNF ligand protein that extends into theextracellular space (the extracellular domain), but also includesshorter parts or fragments thereof that are responsible for thetrimerization and for the binding to the corresponding TNFreceptor. The term "ectodomain of a TNF ligand family member or afragment thereof" thus refers to the extracellular domain of theTNF ligand family member that forms the extracellular domain or toparts thereof that are still able to bind to the receptor (receptorbinding domain).

The term "costimulatory TNF ligand family member" or "costimulatoryTNF family ligand" refers to a subgroup of TNF ligand familymembers, which are able to costimulate proliferation and cytokineproduction of T-cells. These TNF family ligands can costimulate TCRsignals upon interaction with their corresponding TNF receptors andthe interaction with their receptors leads to recruitment ofTNFR-associated factors (TRAF), which initiate signalling cascadesthat result in T-cell activation. Costimulatory TNF family ligandsare selected from the group consisting of 4-1BBL, OX40L, GITRL,CD70, CD30L and LIGHT, more particularly the costimulatory TNFligand family member is selected from 4-1BBL and OX40L.

As described herein before, 4-1BBL is a type II transmembraneprotein and one member of the TNF ligand family Complete or fulllength 4-1BBL having the amino acid sequence of SEQ ID NO: 212 hasbeen described to form trimers on the surface of cells. Theformation of trimers is enabled by specific motives of theectodomain of 4-1BBL. Said motives are designated herein as"trimerization region". The amino acids 50-254 of the human 4-1BBLsequence (SEQ ID NO: 180) form the extracellular domain of 4-1BBL,but even fragments thereof are able to form the trimers. Inspecific embodiments of the invention, the term "ectodomain of4-1BBL or a fragment thereof" refers to a polypeptide having anamino acid sequence selected from SEQ ID NO: 175 (amino acids52-254 of human 4-1BBL), SEQ ID NO: 172 (amino acids 71-254 ofhuman 4-1BBL), SEQ ID NO: 174 (amino acids 80-254 of human 4-1BBL)and SEQ ID NO: 173 (amino acids 85-254 of human 4-1BBL) or apolypeptide having an amino acid sequence selected from SEQ ID NO:183 (amino acids 71-248 of human 4-1BBL), SEQ ID NO: 194 (aminoacids 52-248 of human 4-1BBL), SEQ ID NO: 193 (amino acids 80-248of human 4-1BBL) and SEQ ID NO: 192 (amino acids 85-248 of human4-1BBL), but also other fragments of the ectodomain capable oftrimerization are included herein.

As described herein before, OX40L is another type II transmembraneprotein and a further member of the TNF ligand family Complete orfull length human OX40L has the amino acid sequence of SEQ ID NO:206. The amino acids 51-183 of the human OX40L sequence (SEQ ID NO:181) form the extracellular domain of OX40L, but even fragmentsthereof that are able to form the trimers. In specific embodimentsof the invention, the term "ectodomain of OX40L or a fragmentthereof" refers to a polypeptide having an amino acid sequenceselected from SEQ ID NO: 181 (amino acids 51-183 of human OX40L) orSEQ ID NO: 182 (amino acids 52-183 of human OX40L), but also otherfragments of the ectodomain capable of trimerization are includedherein.

The term "peptide linker" refers to a peptide comprising one ormore amino acids, typically about 2 to 20 amino acids. Peptidelinkers are known in the art or are described herein. Suitable,non-immunogenic linker peptides are, for example, (G.sub.4S).sub.n(SEQ ID NO: 222), (SG.sub.4).sub.n (SEQ ID NO: 223) orG.sub.4(SG.sub.4).sub.n (SEQ ID NO: 224) peptide linkers, wherein"n" is generally a number between 1 and 10, typically between 1 and4, in particular 2, i.e., the peptides selected from the groupconsisting of GGGGS (SEQ ID NO: 162), GGGGSGGGGS (SEQ ID NO: 150),SGGGGSGGGG (SEQ ID NO: 151) and GGGGSGGGGSGGGG (SEQ ID NO: 152),but also include the sequences GSPGSSSSGS (SEQ ID NO: 153),GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 154), GSGSGNGS (SEQ ID NO: 155),GGSGSGSG (SEQ ID NO: 156), GGSGSG (SEQ ID NO: 157), GGSG (SEQ IDNO: 158), GGSGNGSG (SEQ ID NO: 159), GGNGSGSG (SEQ ID NO: 160) andGGNGSG (SEQ ID NO: 161). Peptide linkers of particular interest are(G4S).sub.1 (SEQ ID NO: 162) or GGGGS (SEQ ID NO: 162),(G.sub.4S).sub.2 (SEQ ID NO: 150) or GGGGSGGGGS (SEQ ID NO: 150)and GSPGSSSSGS (SEQ ID NO: 153), more particularly (G.sub.4S).sub.2(SEQ ID NO: 150) or GGGGSGGGGS (SEQ ID NO: 150) and GSPGSSSSGS (SEQID NO: 153).

The term "amino acid" as used within this application denotes thegroup of naturally occurring carboxy .alpha.-amino acids comprisingalanine (three letter code: ala, one letter code: A), arginine(arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly,G), histidine (his, H), isoleucine (ile, I), leucine (leu, L),lysine (lys, K), methionine (met, M), phenylalanine (phe, F),proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan(trp, W), tyrosine (tyr, Y), and valine (val, V).

A "single chain fusion protein" as used herein refers to a singlechain polypeptide composed of one or two ectodomains of said TNFligand family member fused to a part of antigen binding moiety orFc part. The fusion may occur by directly linking the N orC-terminal amino acid of the antigen binding moiety via a peptidelinker to the C- or N-terminal amino acid of the ectodomain of saidTNF ligand family member.

By "fused" or "connected" is meant that the components (e.g., apolypeptide and an ectodomain of said TNF ligand family member) arelinked by peptide bonds, either directly or via one or more peptidelinkers.

"Percent (%) amino acid sequence identity" with respect to areference polypeptide (protein) sequence is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the reference polypeptidesequence, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity, andnot considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways thatare within the skill in the art, for instance, using publiclyavailable computer software such as BLAST, BLAST-2, ALIGN. SAWI orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for aligning sequences, including anyalgorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, %amino acid sequence identity values are generated using thesequence comparison computer program ALIGN-2. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc., andthe source code has been filed with user documentation in the U.S.Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2program is publicly available from Genentech, Inc., South SanFrancisco, Calif., or may be compiled from the source code. TheALIGN-2 program should be compiled for use on a UNIX operatingsystem, including digital UNIX V4.0D. All sequence comparisonparameters are set by the ALIGN-2 program and do not vary. Insituations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequenceA that has or comprises a certain % amino acid sequence identityto, with, or against a given amino acid sequence B) is calculatedas follows: 100 times the fraction X/Y

where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will notequal the % amino acid sequence identity of B to A. Unlessspecifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

In certain embodiments, "amino acid sequence variants" of the TNFligand trimer-containing antigen binding molecules provided hereinare contemplated. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the TNFligand trimer-containing antigen binding molecules Amino acidsequence variants of the TNF ligand trimer-containing antigenbinding molecules may be prepared by introducing appropriatemodifications into the nucleotide sequence encoding the molecules,or by peptide synthesis. Such modifications include, for example,deletions from, and/or insertions into and/or substitutions ofresidues within the amino acid sequences of the antibody. Anycombination of deletion, insertion, and substitution can be made toarrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., antigen-binding. Sitesof interest for substitutional mutagenesis include the HVRs andFramework (FRs). Conservative substitutions are provided in Table Bunder the heading "Preferred Substitutions" and further describedbelow in reference to amino acid side chain classes (1) to (6)Amino acid substitutions may be introduced into the molecule ofinterest and the products screened for a desired activity, e.g.,retained/improved antigen binding, decreased immunogenicity, orimproved ADCC or CDC.

TABLE-US-00002 TABLE B Original Exemplary Preferred ResidueSubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; AsnGlu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln AspGly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile LeuPhe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chainproperties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp,Glu; (4) basic: His, Lys, Arg; (5) residues that influence chainorientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member ofone of these classes for another class.

The term "amino acid sequence variants" includes substantialvariants wherein there are amino acid substitutions in one or morehypervariable region residues of a parent antigen binding molecule(e.g., a humanized or human antibody). Generally, the resultingvariant(s) selected for further study will have modifications(e.g., improvements) in certain biological properties (e.g.,increased affinity, reduced immunogenicity) relative to the parentantigen binding molecule and/or will have substantially retainedcertain biological properties of the parent antigen bindingmolecule. An exemplary substitutional variant is an affinitymatured antibody, which may be generated, e.g., using phagedisplay-based affinity maturation techniques such as thosedescribed herein. Briefly, one or more HVR residues are mutated andthe variant antigen binding molecules displayed on phage andscreened for a particular biological activity (e.g., bindingaffinity). In certain embodiments, substitutions, insertions, ordeletions may occur within one or more HVRs so long as suchalterations do not substantially reduce the ability of the antigenbinding molecule to bind antigen. For example, conservativealterations (e.g., conservative substitutions as provided herein)that do not substantially reduce binding affinity may be made inHVRs. A useful method for identification of residues or regions ofan antibody that may be targeted for mutagenesis is called "alaninescanning mutagenesis" as described by Cunningham and Wells (1989)Science, 244:1081-1085. In this method, a residue or group oftarget residues (e.g., charged residues such as Arg, Asp, His, Lys,and Glu) are identified and replaced by a neutral or negativelycharged amino acid (e.g., alanine or polyalanine) to determinewhether the interaction of the antibody with antigen is affected.Further substitutions may be introduced at the amino acid locationsdemonstrating functional sensitivity to the initial substitutions.Alternatively, or additionally, a crystal structure of anantigen-antigen binding molecule complex to identify contact pointsbetween the antibody and antigen. Such contact residues andneighboring residues may be targeted or eliminated as candidatesfor substitution. Variants may be screened to determine whetherthey contain the desired properties.

Amino acid sequence insertions include amino- and/orcarboxyl-terminal fusions ranging in length from one residue topolypeptides containing a hundred or more residues, as well asintrasequence insertions of single or multiple amino acid residues.Examples of terminal insertions include TNF family ligandtrimer-containing antigen binding molecule with an N-terminalmethionyl residue. Other insertional variants of the moleculeinclude the fusion of the N- or C-terminus to a polypeptide whichincreases the serum half-life of the TNF ligand trimer-containingantigen binding molecules.

In certain embodiments, the TNF family ligand trimer-containingantigen binding molecules provided herein are altered to increaseor decrease the extent to which the antibody is glycosylated.Glycosylation variants of the molecules may be obtained by alteringthe amino acid sequence such that one or more glycosylation sitesis created or removed. Where the TNF ligand trimer-containingantigen binding molecule comprises an Fc region, the carbohydrateattached thereto may be altered. Native antibodies produced bymammalian cells typically comprise a branched, biantennaryoligosaccharide that is generally attached by an N-linkage toAsn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.TIBTECH 15:26-32 (1997). The oligosaccharide may include variouscarbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),galactose, and sialic acid, as well as a fucose attached to aGlcNAc in the "stem" of the biantennary oligosaccharide structure.In some embodiments, modifications of the oligosaccharide in TNFfamily ligand trimer-containing antigen binding molecule may bemade in order to create variants with certain improved properties.In one aspect, variants of TNF family ligand trimer-containingantigen binding molecules are provided having a carbohydratestructure that lacks fucose attached (directly or indirectly) to anFc region. Such fucosylation variants may have improved ADCCfunction, see e.g., US Patent Publication Nos. US 2003/0157108(Presta, L.) or US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).Further variants of the TNF family ligand trimer-containing antigenbinding molecules of the invention include those with bisectedoligosaccharides, e.g., in which a biantennary oligosaccharideattached to the Fc region is bisected by GlcNAc. Such variants mayhave reduced fucosylation and/or improved ADCC function, see forexample WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).Variants with at least one galactose residue in the oligosaccharideattached to the Fc region are also provided. Such antibody variantsmay have improved CDC function and are described, e.g., in WO1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO1999/22764 (Raju, S.).

In certain embodiments, it may be desirable to create cysteineengineered variants of the TNF family ligand trimer-containingantigen binding molecule of the invention, e.g., "thioMAbs," inwhich one or more residues of the molecule are substituted withcysteine residues. In particular embodiments, the substitutedresidues occur at accessible sites of the molecule. By substitutingthose residues with cysteine, reactive thiol groups are therebypositioned at accessible sites of the antibody and may be used toconjugate the antibody to other moieties, such as drug moieties orlinker-drug moieties, to create an immunoconjugate. In certainembodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the lightchain; A118 (EU numbering) of the heavy chain; and S400 (EUnumbering) of the heavy chain Fc region. Cysteine engineeredantigen binding molecules may be generated as described, e.g., inU.S. Pat. No. 7,521,541.

In certain aspects, the TNF family ligand trimer-containing antigenbinding molecules provided herein may be further modified tocontain additional non-proteinaceous moieties that are known in theart and readily available. The moieties suitable for derivatizationof the antibody include but are not limited to water solublepolymers. Non-limiting examples of water soluble polymers include,but are not limited to, polyethylene glycol (PEG), copolymers ofethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer,polyaminoacids (either hom*opolymers or random copolymers), anddextran or poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol hom*opolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol),polyvinyl alcohol, and mixtures thereof. Polyethylene glycolpropionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, andmay be branched or unbranched. The number of polymers attached tothe antibody may vary, and if more than one polymer is attached,they can be the same or different molecules. In general, the numberand/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, theparticular properties or functions of the antibody to be improved,whether the antibody derivative will be used in a therapy underdefined conditions, etc. In another aspect, conjugates of anantibody and non-proteinaceous moiety that may be selectivelyheated by exposure to radiation are provided. In one embodiment,the non-proteinaceous moiety is a carbon nanotube (Kam, N. W. etal., Proc. Natl. Acad. Sci. USA 102 (2005) 11600-11605). Theradiation may be of any wavelength, and includes, but is notlimited to, wavelengths that do not harm ordinary cells, but whichheat the non-proteinaceous moiety to a temperature at which cellsproximal to the antibody-non-proteinaceous moiety are killed.

In another aspect, immunoconjugates of the TNF family ligandtrimer-containing antigen binding molecules provided herein may beobtained. An "immunoconjugate" is an antibody conjugated to one ormore heterologous molecule(s), including but not limited to acytotoxic agent.

"Cross-species reactivity" refers to the ability of certainantibodies to specifically bind to their respective target antigenwherein said target antigen may derive from different species(e.g., human, mouse, cynomolgus, etc.). A cross-species reactiveantibody binds to its respective target antigen derived from atleast two different species with a K.sub.D value lower than about 1.mu.M, preferably lower than about 100 nM, more preferably lowerthan about 10 nM, more preferably lower than about 5 nM, mostpreferably lower than about 2 nM. The term "binds to its respectivetarget antigen derived from at least two different species with aK.sub.D value lower than" means that the respective antibody bindsto the target antigen deriving from each of the indicated specieswith a dissociation constant K.sub.D lower than the indicatedK.sub.D value. In preferred embodiments a cross-species reactiveantibody binds to the target antigen from all indicated specieswith similar affinity, preferably within a K.sub.D range of 10 nMto 0.1 nM, more preferably 5 nM to 0.1 nM, most preferably 2 nM to0.1 nM. In some embodiment, similar affinity for the antigenderived from several species, which means binding of the targetantigen within a narrow K.sub.D range (e.g., within a range of 10nM to 0.1 nM or narrower) for all species of interest, isadvantageous, e.g., for diagnostic assays or animal models of humandiseases. In further preferred embodiment, a cross-species reactiveantibody binds to the target antigen from all indicated species(e.g., human, mouse and cynomolgus monkey) with similar affinity,in particular within a K.sub.D range of a factor of 100, within aK.sub.D range of a factor of 50, within a K.sub.D range of a factorof 20, within a K.sub.D range of a factor of 10, within a K.sub.Drange of a factor of 5. In a preferred embodiment, a cross-speciesreactive antibody binds to the target antigen from human, mouse andcynomolgus monkey with similar affinity, in particular within aK.sub.D range of a factor of 10. For clarity, the cross-speciesreactive antibody binds to one of the indicated species withhighest affinity compared to the other indicated species.Accordingly, the cross-species reactive antibody binds to one ofthe indicated species with lowest affinity compared to the otherindicated species. Within a K.sub.D range of a defined factor Xmeans that the affinity for the indicated species with hightestaffinity is not more than X-times higher than the affinity for theindicated species with lowest affinity. In other words, the K.sub.Dvalue for the indicated species with lowest affinity is not morethan X-times the K.sub.D value for the indicated species withhighest affinity. It is clear to the field that any method formeasuring affinity or avidity can be used to verify that across-species reactive antibody binds to the target antigen fromall indicated species within a given K.sub.D factor range asdescribed herein as long as the same conditions are applied to theK.sub.D measurement for all indicated species. Preferably, theK.sub.D values are measured using SPR, in particular at 25.degree.C. Preferably, the affinities are measured using the cross-speciesreactive antibody as Fab fragment.

The term "polynucleotide" refers to an isolated nucleic acidmolecule or construct, e.g., messenger RNA (mRNA), virally-derivedRNA, or plasmid DNA (pDNA). A polynucleotide may comprise aconventional phosphodiester bond or a non-conventional bond (e.g.,an amide bond, such as found in peptide nucleic acids (PNA). Theterm "nucleic acid molecule" refers to any one or more nucleic acidsegments, e.g. DNA or RNA fragments, present in apolynucleotide.

By "isolated" nucleic acid molecule or polynucleotide is intended anucleic acid molecule, DNA or RNA, which has been removed from itsnative environment. For example, a recombinant polynucleotideencoding a polypeptide contained in a vector is considered isolatedfor the purposes of the present invention. Further examples of anisolated polynucleotide include recombinant polynucleotidesmaintained in heterologous host cells or purified (partially orsubstantially) polynucleotides in solution. An isolatedpolynucleotide includes a polynucleotide molecule contained incells that ordinarily contain the polynucleotide molecule, but thepolynucleotide molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation. Isolated RNA molecules include in vivo or in vitro RNAtranscripts of the present invention, as well as positive andnegative strand forms, and double-stranded forms. Isolatedpolynucleotides or nucleic acids according to the present inventionfurther include such molecules produced synthetically. In addition,a polynucleotide or a nucleic acid may be or may include aregulatory element such as a promoter, ribosome binding site, or atranscription terminator.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% "identical" to a reference nucleotidesequence of the present invention, it is intended that thenucleotide sequence of the polynucleotide is identical to thereference sequence except that the polynucleotide sequence mayinclude up to five point mutations per each 100 nucleotides of thereference nucleotide sequence. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identicalto a reference nucleotide sequence, up to 5% of the nucleotides inthe reference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the 5' or 3' terminal positions of the referencenucleotide sequence or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence or in one or more contiguous groups within the referencesequence. As a practical matter, whether any particularpolynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% identical to a nucleotide sequence of the presentinvention can be determined conventionally using known computerprograms, such as the ones discussed above for polypeptides (e.g.,ALIGN-2).

The term "expression cassette" refers to a polynucleotide generatedrecombinantly or synthetically, with a series of specified nucleicacid elements that permit transcription of a particular nucleicacid in a target cell. The recombinant expression cassette can beincorporated into a plasmid, chromosome, mitochondrial DNA, plastidDNA, virus, or nucleic acid fragment. Typically, the recombinantexpression cassette portion of an expression vector includes, amongother sequences, a nucleic acid sequence to be transcribed and apromoter. In certain embodiments, the expression cassette of theinvention comprises polynucleotide sequences that encode bispecificantigen binding molecules of the invention or fragmentsthereof.

The term "vector" or "expression vector" is synonymous with"expression construct" and refers to a DNA molecule that is used tointroduce and direct the expression of a specific gene to which itis operably associated in a target cell. The term includes thevector as a self-replicating nucleic acid structure as well as thevector incorporated into the genome of a host cell into which ithas been introduced. The expression vector of the present inventioncomprises an expression cassette. Expression vectors allowtranscription of large amounts of stable mRNA. Once the expressionvector is inside the target cell, the ribonucleic acid molecule orprotein that is encoded by the gene is produced by the cellulartranscription and/or translation machinery. In one embodiment, theexpression vector of the invention comprises an expression cassettethat comprises polynucleotide sequences that encode bispecificantigen binding molecules of the invention or fragmentsthereof.

The terms "host cell", "host cell line," and "host cell culture"are used interchangeably and refer to cells into which exogenousnucleic acid has been introduced, including the progeny of suchcells. Host cells include "transformants" and "transformed cells,"which include the primary transformed cell and progeny derivedtherefrom without regard to the number of passages. Progeny may notbe completely identical in nucleic acid content to a parent cell,but may contain mutations. Mutant progeny that have the samefunction or biological activity as screened or selected for in theoriginally transformed cell are included herein. A host cell is anytype of cellular system that can be used to generate the bispecificantigen binding molecules of the present invention. Host cellsinclude cultured cells, e.g., mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63mouse myeloma cells, PER cells, PER. C6 cells or hybridoma cells,yeast cells, insect cells, and plant cells, to name only a few, butalso cells comprised within a transgenic animal, transgenic plantor cultured plant or animal tissue.

An "effective amount" of an agent refers to the amount that isnecessary to result in a physiological change in the cell or tissueto which it is administered.

A "therapeutically effective amount" of an agent, e.g., apharmaceutical composition, refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic or prophylactic result. A therapeutically effectiveamount of an agent for example eliminates, decreases, delays,minimizes or prevents adverse effects of a disease.

An "individual" or "subject" is a mammal. Mammals include, but arenot limited to, domesticated animals (e.g., cows, sheep, cats,dogs, and horses), primates (e.g., humans and non-human primatessuch as monkeys), rabbits, and rodents (e.g., mice and rats).Particularly, the individual or subject is a human.

The term "pharmaceutical composition" refers to a preparation whichis in such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject towhich the formulation would be administered.

A "pharmaceutically acceptable excipient" refers to an ingredientin a pharmaceutical composition, other than an active ingredient,which is nontoxic to a subject. A pharmaceutically acceptableexcipient includes, but is not limited to, a buffer, a stabilizer,or a preservative.

The term "package insert" is used to refer to instructionscustomarily included in commercial packages of therapeuticproducts, that contain information about the indications, usage,dosage, administration, combination therapy, contraindicationsand/or warnings concerning the use of such therapeuticproducts.

As used herein, "treatment" (and grammatical variations thereofsuch as "treat" or "treating") refers to clinical intervention inan attempt to alter the natural course of the individual beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatmentinclude, but are not limited to, preventing occurrence orrecurrence of disease, alleviation of symptoms, diminishment of anydirect or indirect pathological consequences of the disease,preventing metastasis, decreasing the rate of disease progression,amelioration or palliation of the disease state, and remission orimproved prognosis. In some embodiments, the molecules of theinvention are used to delay development of a disease or to slow theprogression of a disease.

The term "cancer" as used herein refers to proliferative diseases,such as lymphomas, carcinoma, lymphoma, blastoma, sarcoma,leukemia, lymphocytic leukemias, lung cancer, non-small cell lung(NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck,cutaneous or intraocular melanoma, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, gastriccancer, colorectal cancer (CRC), pancreatic cancer, breast cancer,triple-negative breast cancer, uterine cancer, carcinoma of thefallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagin*, carcinoma of the vulva, Hodgkin'sDisease, cancer of the esophagus, cancer of the small intestine,cancer of the endocrine system, cancer of the thyroid gland, cancerof the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, prostatecancer, cancer of the bladder, cancer of the kidney or ureter,renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma,hepatocellular cancer, biliary cancer, neoplasms of the centralnervous system (CNS), spinal axis tumors, brain stem glioma,glioblastoma multiforme, astrocytomas, schwanomas, ependymonas,medulloblastomas, meningiomas, squamous cell carcinomas, pituitaryadenoma and Ewings sarcoma, melanoma, multiple myeloma, B-cellcancer (lymphoma), chronic lymphocytic leukemia (CLL), acutelymphoblastic leukemia (ALL), hairy cell leukemia, chronicmyeloblastic leukemia, including refractory versions of any of theabove cancers, or a combination of one or more of the abovecancers.

Compositions and Methods

Distinct alternatively spliced isoforms of Tenascin-C (TnC), arespecifically expressed in certain pathological conditions butessentially absent from healthy adult tissues, thus antigen bindingmolecules targeting TnC have great therapeutic potential. Thepresent invention provides novel TNF family ligandtrimer-containing antigen binding molecules with particularlyadvantageous properties such as producibility, stability, bindingaffinity, biological activity, targeting efficiency and reducedtoxicity. The invention further provides antigen binding moleculesthat bind to TnC, in particular antigen binding molecules withimproved affinity and cross-species reactivity. Antigen bindingmolecules of the invention are useful, e.g., for the diagnosis ortreatment of diseases characterized by expression of TnC, such ascancer.

In a one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule, wherein the target cell antigen isTenascin-C (TnC). In a further aspect, the invention provides a TNFfamily ligand trimer-containing antigen binding moleculecomprising

(a) at least one moiety capable of specific binding to Tenascin-C(TnC) and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, and (c) an Fc domain composed of a first and a secondsubunit capable of stable association.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, wherein the TNF ligand family member costimulates humanT-cell activation.

In another particular aspect, the TNF family ligandtrimer-containing antigen binding molecule comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, wherein the ectodomains of a TNF ligand family member areidentical in all instances.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule as described herein,comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that

(i) the first polypeptide contains a CH1 or CL domain and thesecond polypeptide contains a CL or CH1 domain, respectively,wherein the second polypeptide is linked to the first polypeptideby a disulfide bond between the CH1 and CL domain, and wherein thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or fragments thereof that are connected to each other and tothe CH1 or CL domain by a peptide linker and wherein the secondpolypeptide comprises one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCL or CH1 domain of said polypeptide, or (ii) the first polypeptidecontains a CH3 domain and the second polypeptide contains a CH3domain, respectively, and wherein the first polypeptide comprisestwo ectodomains of a TNF ligand family member or fragments thereofthat are connected to each other and to the C-terminus of the CH3domain by a peptide linker and wherein the second polypeptidecomprises only one ectodomain of said TNF ligand family member or afragment thereof connected via a peptide linker to C-terminus ofthe CH3 domain of said polypeptide, or (iii) the first polypeptidecontains a VH-CL or a VL-CH1 domain and the second polypeptidecontains a VL-CH1 domain or a VH-CL domain, respectively, whereinthe second polypeptide is linked to the first polypeptide by adisulfide bond between the CH1 and CL domain, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to VH orVL by a peptide linker and wherein the second polypeptide comprisesone ectodomain of said TNF ligand family member or a fragmentthereof connected via a peptide linker to VL or VH of saidpolypeptide.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule of claim 1,comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that

(i) the first polypeptide contains a CH1 or CL domain and thesecond polypeptide contains a CL or CH1 domain, respectively,wherein the second polypeptide is linked to the first polypeptideby a disulfide bond between the CH1 and CL domain, and wherein thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or fragments thereof that are connected to each other and tothe CH1 or CL domain by a peptide linker and wherein the secondpolypeptide comprises one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCL or CH1 domain of said polypeptide, or (ii) the first polypeptidecontains a CH3 domain and the second polypeptide contains a CH3domain, respectively, and wherein the first polypeptide comprisestwo ectodomains of a TNF ligand family member or fragments thereofthat are connected to each other and to the C-terminus of the CH3domain by a peptide linker and wherein the second polypeptidecomprises only one ectodomain of said TNF ligand family member or afragment thereof connected via a peptide linker to C-terminus ofthe CH3 domain of said polypeptide.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule comprises a TNF ligand family member thatcostimulates human T-cell activation which is selected from 4-1BBLand OX40L. More particularly, the TNF ligand family member is4-1BBL.

In another aspect, wherein the ectodomain of a TNF ligand familymember comprises the amino acid sequence selected from the groupconsisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO: 193 and SEQID NO: 194, particularly the amino acid sequence of SEQ ID NO: 172or SEQ ID NO: 183. In one aspect, the ectodomain of a TNF ligandfamily member or fragment thereof comprises the amino acid sequenceselected from the group consisting of SEQ ID NO: 172, SEQ ID NO:173, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183,particularly the amino acid sequence of SEQ ID NO: 172 or SEQ IDNO: 183. In a particular aspect, the ectodomain of a TNF ligandfamily member or fragment thereof comprises the amino acid sequenceof SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO:185 and SEQ ID NO: 186 and in that the second polypeptide comprisesthe amino acid sequence selected from the group consisting of SEQID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183. In aparticular aspect, the first polypeptide comprises the amino acidsequence of SEQ ID NO: 184 and the second polypeptide comprises theamino acid sequence of SEQ ID NO: 183.

In one aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:176 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 177.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:176 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 187.

In yet a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:184 and in that the second polypeptide comprises the amino acidsequence of SEQ ID NO: 188 or SEQ ID NO: 189.

In another aspect, the TNF ligand family member is OX40L. In aparticular aspect, provided is TNF family ligand trimer-containingantigen binding molecule, wherein the ectodomain of a TNF ligandfamily member comprises the amino acid sequence of SEQ ID NO: 181or SEQ ID NO: 182, particularly the amino acid sequence of SEQ IDNO: 181.

In one aspect, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence of SEQ ID NO:190 or SEQ ID: 191 and in that the second polypeptide comprises theamino acid sequence of SEQ ID NO: 181 or SEQ ID NO: 182,respectively.

In one aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 or CL domain and a secondpolypeptide containing a CL or CH1 domain, respectively, whereinthe second polypeptide is linked to the first polypeptide by adisulfide bond between the CH1 and CL domain, and wherein theantigen binding molecule is characterized in that the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to theCH1 or CL domain by a peptide linker and in that the secondpolypeptide comprises only one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCL or CH1 domain of said polypeptide.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CH1 domain and a secondpolypeptide containing a CL domain, wherein the second polypeptideis linked to the first polypeptide by a disulfide bond between theCH1 and CL domain,

and wherein the antigen binding molecule is characterized in thatthe first polypeptide comprises two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherand to the CH1 domain by a peptide linker and in that the secondpolypeptide comprises one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCL domain of said polypeptide.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first polypeptide containing a CL domain and a secondpolypeptide containing a CH1 domain, wherein the second polypeptideis linked to the first polypeptide by a disulfide bond between theCH1 and CL domain,

and wherein the antigen binding molecule is characterized in thatthe first polypeptide comprises two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherand to the CL domain by a peptide linker and in that the secondpolypeptide comprises one ectodomain of said TNF ligand familymember or a fragment thereof connected via a peptide linker to theCH1 domain of said polypeptide.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof.

In yet another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) more than one moiety capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof connected via a peptide linker to said polypeptide.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) two moities capable of specific binding to TnC and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide contains a CH3 domain and the second polypeptidecontains a CH3 domain, respectively, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to theC-terminus of the CH3 domain by a peptide linker and wherein thesecond polypeptide comprises one ectodomain of said TNF ligandfamily member or a fragment thereof connected via a peptide linkerto C-terminus of the CH3 domain of said polypeptide. Particularly,such TNF family ligand trimer-containing antigen binding moleculecomprises two moieties capable of specific binding to TnC.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined hereinbefore, wherein the moiety capable of specific binding to TnC isselected from the group consisting of an antibody, an antibodyfragment and a scaffold antigen binding protein.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before, wherein themoiety capable of specific binding to TnC is selected from thegroup consisting of an antibody fragment, a Fab molecule, acrossover Fab molecule, a single chain Fab molecule, a Fv molecule,a scFv molecule, a single domain antibody, an aVH and a scaffoldantigen binding protein. In one aspect, the moiety capable ofspecific binding to TnC is an aVH or a scaffold antigen bindingprotein. In one aspect, the moiety capable of specific binding to atarget cell antigen is a scaffold antigen binding protein capableof specific binding to a target cell antigen.

In particular, the TNF family ligand trimer-containing antigenbinding molecule comprises one or two moieties capable of specificbinding to TnC.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the moietycapable of specific binding to TnC is a Fab molecule or a crossoverFab molecule capable of specific binding to a target cell antigen.In particular, the moiety capable of specific binding to TnC is aFab molecule.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein the antigen binding molecule comprises a firstheavy chain and a first light chain, both comprising a Fab moleculecapable of specific binding to TnC, a first peptide comprising twoectodomains of a TNF ligand family member or fragments thereofconnected to each other by a first peptide linker fused at itsC-terminus by a second peptide linker to a second heavy or lightchain, and a second peptide comprising one ectodomain of said TNFligand family member fused at its C-terminus by a third peptidelinker to a second light or heavy chain, respectively.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein a peptide comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker is fused at its C-terminus to the CH1domain of a heavy chain by a second peptide linker and wherein oneectodomain of said TNF ligand family member or a fragment thereofis fused at the its C-terminus to the CL domain on a light chain bya third peptide linker.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein the first peptide comprising two ectodomains ofa TNF ligand family member or fragments thereof connected to eachother by a first peptide linker is fused at its C-terminus by asecond peptide linker to a CH1 domain that is part of a heavychain, and the second peptide comprising one ectodomain of said TNFligand family member or a fragment thereof is fused at itsC-terminus by a third peptide linker to a CL domain that is part ofa light chain.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein a peptide comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker is fused at its C-terminus to the CLdomain of a heavy chain by a second peptide linker and wherein oneectodomain of said TNF ligand family member or a fragment thereofis fused at the its C-terminus to the CH1 domain on a light chainby a third peptide linker.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein the first peptide comprising two ectodomains ofa TNF ligand family member or fragments thereof connected to eachother by a first peptide linker is fused at its C-terminus by asecond peptide linker to a CL domain that is part of a heavy chain,and the second peptide comprising one ectodomain of said TNF ligandfamily member or a fragment thereof is fused at its C-terminus by athird peptide linker to a CH1 domain that is part of a lightchain.

In a further aspect, the invention is concerned with a TNF familyligand trimer-containing antigen binding molecule according to theinvention, wherein a peptide comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker is fused at its C-terminus to the CLdomain of a light chain by a second peptide linker and wherein oneectodomain of said TNF ligand family member or a fragment thereofis fused at the its C-terminus to the CH1 domain of the heavy chainby a third peptide linker.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule according to theinvention, wherein the first peptide comprising two ectodomains ofa TNF ligand family member or fragments thereof connected to eachother by a first peptide linker is fused at its C-terminus by asecond peptide linker to a VH domain that is part of a heavy chain,and the second peptide comprising one ectodomain of said TNF ligandfamily member or a fragment thereof is fused at its C-terminus by athird peptide linker to a VL domain that is part of a lightchain.

In a particular aspect, the invention relates to a TNF familyligand trimer-containing antigen binding molecule as defined above,wherein the peptide linker is (G4S).sub.2 (SEQ ID NO: 150). In oneaspect, the first peptide linker is (G4S).sub.2 (SEQ ID NO: 150),the second peptide linker is GSPGSSSSGS (SEQ ID NO: 153) and thethird peptide linker is (G4S).sub.2 (SEQ ID NO: 150). Inparticular, the invention relates to a TNF ligand trimer-containingantigen binding molecule as defined above, wherein the firstpeptide linker is (G4S).sub.2 (SEQ ID NO: 150), the second peptidelinker is (G4S).sub.2 (SEQ ID NO: 150), and the third peptidelinker is (G4S).sub.2 (SEQ ID NO: 150).

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule as defined herein before comprises an Fc domaincomposed of a first and a second subunit capable of stableassociation.

In particular, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises (a) a Fab moleculecapable of specific binding to TnC, wherein the Fab heavy chain isfused at the C-terminus to the N-terminus of a CH2 domain in the Fcdomain and (c) an Fc domain composed of a first and a secondsubunit capable of stable association.

In a further aspect, the Fc domain is an IgG, particularly an IgG1Fc domain or an IgG4 Fc domain. More particularly, the Fc domain isan IgG1 Fc domain. In a particular aspect, the Fc domain comprisesa modification promoting the association of the first and secondsubunit of the Fc domain.

Fc Domain Modifications Reducing Fc Receptor Binding and/orEffector Function

The Fc domain of the TNF family ligand trimer-containing antigenbinding molecules of the invention consists of a pair ofpolypeptide chains comprising heavy chain domains of animmunoglobulin molecule. For example, the Fc domain of animmunoglobulin G (IgG) molecule is a dimer, each subunit of whichcomprises the CH2 and CH3 IgG heavy chain constant domains. The twosubunits of the Fc domain are capable of stable association witheach other.

The Fc domain confers favorable pharmaco*kinetic properties to theantigen binding molecules of the invention, including a long serumhalf-life which contributes to good accumulation in the targettissue and a favorable tissue-blood distribution ratio. At the sametime it may, however, lead to undesirable targeting of thebispecific antibodies of the invention to cells expressing Fcreceptors rather than to the preferred antigen-bearing cells.Accordingly, in particular aspects, the Fc domain of the TNF familyligand trimer-containing antigen binding molecule of the inventionexhibits reduced binding affinity to an Fc receptor and/or reducedeffector function, as compared to a native IgG1 Fc domain. In oneaspect, the Fc does not substantially bind to an Fc receptor and/ordoes not induce effector function. In a particular aspect the Fcreceptor is an Fc.gamma. receptor. In one aspect, the Fc receptoris a human Fc receptor. In a specific aspect, the Fc receptor is anactivating human Fc.gamma. receptor, more specifically humanFc.gamma.RIIIa, Fc.gamma.RI or Fc.gamma.RIIa, most specificallyhuman Fc.gamma.RIIIa. In one aspect, the Fc domain does not induceeffector function. The reduced effector function can include, butis not limited to, one or more of the following: reduced complementdependent cytotoxicity (CDC), reduced antibody-dependentcell-mediated cytotoxicity (ADCC), reduced antibody-dependentcellular phagocytosis (ADCP), reduced cytokine secretion, reducedimmune complex-mediated antigen uptake by antigen-presenting cells,reduced binding to NK cells, reduced binding to macrophages,reduced binding to monocytes, reduced binding to polymorphonuclearcells, reduced direct signaling inducing apoptosis, reduceddendritic cell maturation, or reduced T cell priming.

In certain aspects, one or more amino acid modifications may beintroduced into the Fc region of a antigen binding moleculeprovided herein, thereby generating an Fc region variant. The Fcregion variant may comprise a human Fc region sequence (e.g., ahuman IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acidmodification (e.g., a substitution) at one or more amino acidpositions.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, and (c) an Fc domain composed of a first and a secondsubunit capable of stable association, wherein the Fc domaincomprises one or more amino acid substitution that reduces bindingto an Fc receptor, in particular towards Fc.gamma. receptor.

In one aspect, the Fc domain of the antigen binding molecule of theinvention comprises one or more amino acid mutation that reducesthe binding affinity of the Fc domain to an Fc receptor and/oreffector function. Typically, the same one or more amino acidmutation is present in each of the two subunits of the Fc domain.In particular, the Fc domain comprises an amino acid substitutionat a position of E233, L234, L235, N297, P331 and P329 (EUnumbering). In particular, the Fc domain comprises amino acidsubstitutions at positions 234 and 235 (EU numbering) and/or 329(EU numbering) of the IgG heavy chains. More particularly, providedis an antigen binding molecule according to the invention whichcomprises an Fc domain with the amino acid substitutions L234A,L235A and P329G ("PGLALA", EU numbering) in the IgG heavy chains.The amino acid substitutions L234A and L235A refer to the so-calledLALA mutation. The "PGLALA" combination of amino acid substitutionsalmost completely abolishes Fc.gamma. receptor binding of a humanIgG1 Fc domain and is described in International Patent Appl. Publ.No. WO 2012/130831 A1 which also describes methods of preparingsuch mutant Fc domains and methods for determining its propertiessuch as Fc receptor binding or effector functions. "EU numbering"refers to the numbering according to EU index of Kabat et al,Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md.,1991.

Fc domains with reduced Fc receptor binding and/or effectorfunction also include those with substitution of one or more of Fcdomain residues 238, 265, 269, 270, 297, 327 and 329 (U.S. Pat. No.6,737,056). Such Fc mutants include Fc mutants with substitutionsat two or more of amino acid positions 265, 269, 270, 297 and 327,including the so-called "DANA" Fc mutant with substitution ofresidues 265 and 297 to alanine (U.S. Pat. No. 7,332,581).

In another aspect, the Fc domain is an IgG4 Fc domain. IgG4antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG1 antibodies. In amore specific aspect, the Fc domain is an IgG4 Fc domain comprisingan amino acid substitution at position 5228 (Kabat numbering),particularly the amino acid substitution S228P. In a more specificaspect, the Fc domain is an IgG4 Fc domain comprising amino acidsubstitutions L235E and S228P and P329G (EU numbering). Such IgG4Fc domain mutants and their Fc.gamma. receptor binding propertiesare also described in WO 2012/130831.

Mutant Fc domains can be prepared by amino acid deletion,substitution, insertion or modification using genetic or chemicalmethods well known in the art. Genetic methods may includesite-specific mutagenesis of the encoding DNA sequence, PCR, genesynthesis, and the like. The correct nucleotide changes can beverified for example by sequencing.

Binding to Fc receptors can be easily determined e.g., by ELISA, orby Surface Plasmon Resonance (SPR) using standard instrumentationsuch as a BIAcore instrument (GE Healthcare), and Fc receptors suchas may be obtained by recombinant expression. A suitable suchbinding assay is described herein. Alternatively, binding affinityof Fc domains or cell activating bispecific antigen bindingmolecules comprising an Fc domain for Fc receptors may be evaluatedusing cell lines known to express particular Fc receptors, such ashuman NK cells expressing Fc.gamma.IIIa receptor.

Effector function of an Fc domain, or antigen binding molecule ofthe invention comprising an Fc domain, can be measured by methodsknown in the art. A suitable assay for measuring ADCC is describedherein. Other examples of in vitro assays to assess ADCC activityof a molecule of interest are described in U.S. Pat. No. 5,500,362;Hellstrom et al. Proc Natl Acad Sci USA 83, 7059-7063 (1986) andHellstrom et al., Proc Natl Acad Sci USA 82, 1499-1502 (1985); U.S.Pat. No. 5,821,337; Bruggemann et al., J Exp Med 166, 1351-1361(1987). Alternatively, non-radioactive assays methods may beemployed (see, for example, ACTI.TM. non-radioactive cytotoxicityassay for flow cytometry (CellTechnology, Inc. Mountain View,Calif.); and CytoTox 96.RTM. non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and NaturalKiller (NK) cells. Alternatively, or additionally, ADCC activity ofthe molecule of interest may be assessed in vivo, e.g., in a animalmodel such as that disclosed in Clynes et al., Proc Natl Acad SciUSA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complementcomponent, specifically to C1q, is reduced. Accordingly, in someembodiments wherein the Fc domain is engineered to have reducedeffector function, said reduced effector function includes reducedCDC. C1q binding assays may be carried out to determine whether theantigen binding molecule of the invention is able to bind C1q andhence has CDC activity. See e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, aCDC assay may be performed (see, for example, Gazzano-Santoro etal., J Immunol Methods 202, 163 (1996); Cragg et al., Blood 101,1045-1052 (2003); and Cragg and Glennie, Blood 103, 2738-2743(2004)).

In a particular aspect, the Fc domain comprises a modificationpromoting the association of the first and second subunit of the Fcdomain.

Fc Domain Modifications Promoting Heterodimerization

In one aspect, the TNF family ligand trimer-containing antigenbinding molecules of the invention comprise

(a) at least one moiety capable of specific binding to TnC,

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond,

wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or two fragments thereof that are connected to each other bya peptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof, and (c) an Fc domain composed of a first and a secondsubunit capable of stable association, wherein the Fc domaincomprises one or more amino acid substitution that reduces bindingto an Fc receptor, in particular to Fc.gamma. receptor. Thus, theycomprise different moieties, fused to one or the other of the twosubunits of the Fc domain that are typically comprised in twonon-identical polypetide chains ("heavy chains"). Recombinantco-expression of these polypeptides and subsequent dimerizationleads to several possible combinations of the two polypeptides. Toimprove the yield and purity of the TNF family ligandtrimer-containing antigen binding molecules in recombinantproduction, it will thus be advantageous to introduce in the Fcdomain of the TNF family ligand trimer-containing antigen bindingmolecules of the invention a modification promoting the associationof the desired polypeptides.

Accordingly, the Fc domain of the TNF family ligandtrimer-containing antigen binding molecules of the inventioncomprises a modification promoting the association of the first andthe second subunit of the Fc domain. The site of most extensiveprotein-protein interaction between the two subunits of a human IgGFc domain is in the CH3 domain of the Fc domain. Thus, saidmodification is particularly in the CH3 domain of the Fc domain

In a specific aspect, said modification is a so-called"knob-into-hole" modification, comprising a "knob" modification inone of the two subunits of the Fc domain and a "hole" modificationin the other one of the two subunits of the Fc domain. Thus, in aparticular aspect, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule as described hereinbefore which comprises an IgG molecule, wherein the Fc part of thefirst heavy chain comprises a first dimerization module and the Fcpart of the second heavy chain comprises a second dimerizationmodule allowing a heterodimerization of the two heavy chains of theIgG molecule and the first dimerization module comprises knobs andthe second dimerization module comprises holes according to theknob into hole technology.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996)and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the methodinvolves introducing a protuberance ("knob") at the interface of afirst polypeptide and a corresponding cavity ("hole") in theinterface of a second polypeptide, such that the protuberance canbe positioned in the cavity so as to promote heterodimer formationand hinder hom*odimer formation. Protuberances are constructed byreplacing small amino acid side chains from the interface of thefirst polypeptide with larger side chains (e.g., tyrosine ortryptophan). Compensatory cavities of identical or similar size tothe protuberances are created in the interface of the secondpolypeptide by replacing large amino acid side chains with smallerones (e.g., alanine or threonine).

Accordingly, in a particular aspect, in the CH3 domain of the firstsubunit of the Fc domain of the TNF family ligand trimer-containingantigen binding molecules of the invention an amino acid residue isreplaced with an amino acid residue having a larger side chainvolume, thereby generating a protuberance within the CH3 domain ofthe first subunit which is positionable in a cavity within the CH3domain of the second subunit, and in the CH3 domain of the secondsubunit of the Fc domain an amino acid residue is replaced with anamino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunitwithin which the protuberance within the CH3 domain of the firstsubunit is positionable.

The protuberance and cavity can be made by altering the nucleicacid encoding the polypeptides, e.g., by site-specific mutagenesis,or by peptide synthesis.

In a specific aspect, in the CH3 domain of the first subunit of theFc domain the threonine residue at position 366 is replaced with atryptophan residue (T366W), and in the CH3 domain of the secondsubunit of the Fc domain the tyrosine residue at position 407 isreplaced with a valine residue (Y407V). More particularly, in thesecond subunit of the Fc domain additionally the threonine residueat position 366 is replaced with a serine residue (T366S) and theleucine residue at position 368 is replaced with an alanine residue(L368A). More particularly, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C), and in the second subunit of the Fcdomain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C). The introduction of thesetwo cysteine residues results in the formation of a disulfidebridge between the two subunits of the Fc domain. The disulfidebridge further stabilizes the dimer (Carter, J Immunol Methods 248,7-15 (2001)).

In an alternative aspect, a modification promoting association ofthe first and the second subunit of the Fc domain comprises amodification mediating electrostatic steering effects, e.g., asdescribed in PCT publication WO 2009/089004. Generally, this methodinvolves replacement of one or more amino acid residues at theinterface of the two Fc domain subunits by charged amino acidresidues so that hom*odimer formation becomes electrostaticallyunfavorable but heterodimerization electrostatically favorable.

Modifications in the CH1/CL Domains

To further improve correct pairing, the TNF family ligandtrimer-containing antigen binding molecules can contain differentcharged amino acid substitutions (so-called "charged residues").These modifications are introduced in the crossed or non-crossedCH1 and CL domains. In a particular aspect, the invention relatesto a TNF family ligand trimer-containing antigen binding molecule,wherein in one of CL domains the amino acid at position 123 (EUnumbering) has been replaced by arginine (R) and the amino acid atposition 124 (EU numbering) has been substituted by lysine (K) andwherein in one of the CH1 domains the amino acids at position 147(EU numbering) and at position 213 (EU numbering) have beensubstituted by glutamic acid (E).

More particularly, the invention relates to a TNF family ligandtrimer-containing antigen binding molecule, wherein in the CLdomain adjacent to the TNF ligand family member the amino acid atposition 123 (EU numbering) has been replaced by arginine (R) andthe amino acid at position 124 (EU numbering) has been substitutedby lysine (K), and wherein in the CH1 domain adjacent to the TNFligand family member the amino acids at position 147 (EU numbering)and at position 213 (EU numbering) have been substituted byglutamic acid (E).

Particular TNF Family Ligand Trimer-Containing Antigen BindingMolecules

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises a first heavy chain and a first lightchain, both comprising a Fab molecule capable of specific bindingto TnC, a first peptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof connected to each other by afirst peptide linker fused at its C-terminus by a second peptidelinker to a second heavy or light chain, and a second peptidecomprising one ectodomain of said TNF ligand family member fused atit* C-terminus by a third peptide linker to a second light or heavychain, respectively.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a CH1domain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a CL domain that is part of a light chain.

In yet another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a CLdomain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof that is fused at its C-terminus by a third peptidelinker to a CH1 domain that is part of a light chain.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the firstpeptide comprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a VHdomain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a VL domain that is part of a light chain.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as described herein,wherein in the CL domain adjacent to the TNF ligand family memberthe amino acid at position 123 (EU numbering) has been replaced byarginine (R) and the amino acid at position 124 (EU numbering) hasbeen substituted by lysine (K), and wherein in the CH1 domainadjacent to the TNF ligand family member the amino acids atposition 147 (EU numbering) and at position 213 (EU numbering) havebeen substituted by glutamic acid (E). These modifications lead toso-called charged residues with advantageous properties that avoidundesired effects such as for example mispairing.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the second subunit of the Fc domain comprising theknob mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domaincomprising the hole mutations.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the second subunit of the Fc domain comprising theknob mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domaincomprising the hole mutations and wherein the Fab heavy chain isfused at the C-terminus to the N-terminus of the second subunit ofthe Fc domain comprising the knob mutations.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the second subunit of the Fc domain comprising theknob mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domaincomprising the hole mutations and wherein the Fab heavy chain isfused at the C-terminus to the N-terminus of the first subunit ofthe Fc domain comprising the hole mutations.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the first subunit of the Fc domain comprising thehole mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the second subunit of the Fc domaincomprising the knob mutations.

In a particular aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the first subunit of the Fc domain comprising thehole mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the second subunit of the Fc domaincomprising the knob mutations and wherein the Fab heavy chain isfused at the C-terminus to the N-terminus of the second subunit ofthe Fc domain comprising the knob mutations.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to the first subunit of the Fc domain comprising thehole mutations and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the second subunit of the Fc domaincomprising the knob mutations and wherein the Fab heavy chain isfused at the C-terminus to the N-terminus of the first subunit ofthe Fc domain comprising the hole mutations.

Particular TnC Binding Moieties

In one aspect, the present invention provides for TnC bindingmoieties. In another aspect, TNC binding moieties of the presentinvention can be included in bivalent or multivalent bindingmolecules as described herein. In one embodiment the moiety capableof specific binding to TnC is selected from the group consisting ofan antibody fragment, a Fab molecule, a crossover Fab molecule, asingle chain Fab molecule, a Fv molecule, a scFv molecule, a singledomain antibody, and aVH and a scaffold antigen binding protein. Inanother aspect, the present invention provides for antigen bindingmolecules comprising one or more moiety capable of specific bindingto TnC. The molecules of the invention comprising a TnC bindingmoiety as described herein have a high affinity for one ore moreTnC domains and/or cross-species reactivity.

In one embodiment, an anti-TnC antigen binding molecule of theinvention comprises at least one (e.g., one, two, three, four,five, or six) heavy or light chain complementarity determiningregion (CDR) selected from the group consisting of SEQ ID NO: 55,SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ IDNO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70,SEQ ID NO: 71 and SEQ ID NO: 72, or a variant or truncated formthereof containing at least the specificity-determining residues(SDRs) for said CDR.

In one embodiment, an antigen binding molecule of the inventioncomprises at least one, at least two, or all three heavy chain CDR(HCDR) sequences selected from (a) HCDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 67 andSEQ ID NO: 70; (b) HCDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 68 and SEQ ID NO: 71; and(c) HCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 69 and SEQ ID NO: 72. In a furtherembodiment, the antigen binding molecule comprises a heavy chainvariable region comprising (a) a heavy chain CDR1 selected from thegroup consisting of SEQ ID NO: 67 and SEQ ID NO: 70; (b) a heavychain CDR2 selected from the group consisting of SEQ ID NO: 68 andSEQ ID NO: 71; and (c) a heavy chain CDR3 selected from the groupconsisting of SEQ ID NO: 69 and SEQ ID NO: 72, or variants ortruncated forms thereof containing at least the SDRs for saidCDRs.

In one embodiment, an antigen binding molecule of the inventioncomprises at least one, at least two, or all three light chain CDR(LCDR) sequences selected from (a) LCDR1 comprising an amino acidsequence selected from the group consisting of SEQ ID NO: 55 andSEQ ID NO: 58; (b) LCDR2 comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 56 and SEQ ID NO: 59; and(c) LCDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 57 and SEQ ID NO: 60. In a furtherembodiment, the antigen binding molecule comprises a light chainvariable region comprising (a) a light chain CDR1 selected from thegroup consisting of SEQ ID NO: 55 and SEQ ID NO: 58 (b) a lightchain CDR2 selected from the group consisting of SEQ ID NO: 56 andSEQ ID NO: 59; and (c) a light chain CDR3 selected from the groupconsisting of SEQ ID NO: 57 and SEQ ID NO: 60, or variants ortruncated forms thereof containing at least the SDRs for saidCDRs.

In one embodiment, an antigen binding molecule of the inventioncomprises a heavy chain variable region comprising a heavy chainCDR1 selected from the group consisting of SEQ ID NO: 67 and SEQ IDNO: 70; a heavy chain CDR2 selected from the group consisting ofSEQ ID NO: 68 and SEQ ID NO: 71; and a heavy chain CDR3 selectedfrom the group consisting of SEQ ID NO: 69 and SEQ ID NO: 72, and alight chain variable region comprising a light chain CDR1 selectedfrom the group consisting of SEQ ID NO: 55 and SEQ ID NO: 58; alight chain CDR2 selected from the group consisting of SEQ ID NO:56 and SEQ ID NO: 59; and a light chain CDR3 selected from thegroup consisting of SEQ ID NO: 57 and SEQ ID NO: 60, or variants ortruncated forms thereof containing at least the SDRs for saidCDRs.

In yet another specific embodiment, an antigen binding molecule ofthe invention comprises a heavy chain variable region comprisingthe heavy chain CDR1 of SEQ ID NO: 67; the heavy chain CDR2 of SEQID NO: 68; and the heavy chain CDR3 of SEQ ID NO: 69, and a lightchain variable region comprising the light chain CDR1 of SEQ ID NO:55; the light chain CDR2 of SEQ ID NO: 56; and the light chain CDR3of SEQ ID NO: 57.

In yet another specific embodiment, an antigen binding molecule ofthe invention comprises a heavy chain variable region comprisingthe heavy chain CDR1 of SEQ ID NO: 70; the heavy chain CDR2 of SEQID NO: 71; and the heavy chain CDR3 of SEQ ID NO: 72, and a lightchain variable region comprising the light chain CDR1 of SEQ ID NO:58, the light chain CDR2 of SEQ ID NO: 59; and the light chain CDR3of SEQ ID NO: 60.

In one embodiment, an antigen binding molecule of the inventioncomprises a heavy chain variable region (VH) comprising an aminoacid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% identity to a sequence selected from the groupconsisting of SEQ ID NO: 46 and SEQ ID NO: 48. In one embodiment,the antigen binding molecule comprises a heavy chain variableregion comprising an amino acid sequence selected from the groupconsisting of: SEQ ID NO: 46 and SEQ ID NO: 48.

In certain embodiments, a VH sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-TnCantigen binding molecule comprising that sequence retains theability to bind to TnC. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQID NO: 46 or SEQ ID NO: 48. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs or CDRs(i.e., in the FRs). Optionally, an anti-TnC antigen bindingmolecule according to the invention comprises the VH sequence inSEQ ID NO: 46 or SEQ ID NO: 48, including post-translationalmodifications of that sequence. In a particular embodiment, the VHcomprises one, two or three heavy chain CDRs selected from thesequences set forth in SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69,SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72 for the HCDR1, HCDR2and HCDR3.

In another embodiment, an antigen binding molecule of the inventioncomprises a light chain variable region comprising an amino acidsequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to a sequence selected from the groupconsisting of SEQ ID NO: 45 and SEQ ID NO: 47. In yet anotherembodiment, the antigen binding molecule comprises a light chainvariable region comprising an amino acid sequence selected from thegroup consisting of: SEQ ID NO: 45 and SEQ ID NO: 47.

In certain embodiments, a VL sequence having at least 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity containssubstitutions (e.g., conservative substitutions), insertions, ordeletions relative to the reference sequence, but an anti-TnCantigen binding molecule comprising that sequence retains theability to bind to TnC. In certain embodiments, a total of 1 to 10amino acids have been substituted, inserted and/or deleted in SEQID NO: 45 or SEQ ID NO: 47. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outsidethe HVRs or CDRs (i.e., in the FRs). Optionally, an anti-TnCantigen binding molecule of the invention comprises the VL sequencein SEQ ID NO: 45 or SEQ ID NO: 47, including post-translationalmodifications of that sequence. In a particular embodiment, the VLcomprises one, two or three light chain CDRs selected fromsequences set forth in SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57,SEQ ID NO: 58, SEQ ID NO: 59 and SEQ ID NO: 60 for the LCDR1, LCDR2and LCDR3.

In another aspect, an anti-TnC antigen binding molecule isprovided, wherein the antigen binding molecule comprises a VH as inany of the embodiments provided above, and a VL as in any of theembodiments provided above. In one embodiment, the antigen bindingmolecule comprises a heavy chain variable region comprising anamino acid sequence that is at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or 100% identical to a sequence selectedfrom the group of: SEQ ID NO: 46 and SEQ ID NO: 48, and a lightchain variable region comprising an amino acid sequence that is atleast about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a sequence selected from the group of: SEQ ID NO:45 and SEQ ID NO: 47. In one embodiment, the antigen bindingmolecule comprises the VH and VL sequences in SEQ ID NO: 46 or SEQID NO: 48 and SEQ ID NO: 45 or SEQ ID NO: 47, respectively,including post-translational modifications of those sequences.

In a specific embodiment, an antigen binding molecule of theinvention comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO: 46 and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 45. In aspecific embodiment, an antigen binding molecule of the inventioncomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 48 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47. In aparticular embodiment, the antigen binding molecule according toany of the above embodiments additionally comprises an Fc region ora region equivalent to the Fc region of an immunoglobulin. In oneembodiment an antigen binding molecule of the invention comprisesan Fc region, particularly a IgG Fc region, most particularly aIgG1 Fc region. In a particular embodiment, the antigen bindingmolecule of the invention is a full length antibody, particularlyan IgG class antibody, most particularly an IgG1 isotype antibody.In another embodiment, the TnC binding moiety of the antigenbinding molecule of the invention is an antibody fragment, selectedfrom the group of: an scFv fragment, an Fv fragment, a Fabfragment, and a F(ab')2 fragment. In a further embodiment, theantigen binding molecule of the invention is an antibody fragmenthaving an Fc region, or a fusion protein that comprises a regionequivalent to the Fc region of an immunoglobulin. In oneembodiment, the antigen binding molecule of the invention is amonoclonal antibody. In one embodiment, an antigen binding moleculeof the invention is chimeric, more specifically humanized. In aparticular embodiment, an antigen binding molecule of the inventionis human. In another embodiment, an antigen binding molecule of theinvention comprises a human constant region. In one embodiment theantigen binding molecule of the invention comprises a human Fcregion, preferably a human IgG Fc region, most particularly a humanIgG1 Fc region.

In one embodiment, an antigen binding molecule of the inventioncomprises a heavy chain constant region, wherein said heavy chainconstant region is a human IgG constant region, particularly ahuman IgG1 constant region, comprising an Fc region. In oneembodiment, an antigen binding molecule of the invention comprisesa heavy chain region, wherein said heavy chain region is a humanIgG heavy chain region, particularly a human IgG1 heavy chainregion, comprising an Fc region. In a specific embodiment, theantigen binding molecule comprises a heavy chain region comprisingan amino acid sequence selected from the group consisting of: SEQID NO: 78, SEQ ID NO: 80, SEQ ID NO: 83, and SEQ ID NO: 84. Inanother specific embodiment an antigen binding molecule of theinvention comprises a light chain region comprising an amino acidsequence selected from the group consisting of: SEQ ID NO: 77 andSEQ ID NO: 79. In yet another specific embodiment, an antigenbinding molecule of the invention comprises a heavy chain regioncomprising the amino acid sequence of SEQ ID NO: 78, and a lightchain region comprising the amino acid sequence of SEQ ID NO: 77.In yet another specific embodiment, an antigen binding molecule ofthe invention comprises a heavy chain region comprising the aminoacid sequence of SEQ ID NO: 80, and a light chain region comprisingthe amino acid sequence of SEQ ID NO: 79.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to TnC, wherein saidantigen binding molecule comprises a) a heavy chain regioncomprising an amino acid sequence that is at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to asequence selected from the group consisting of: SEQ ID NO: 78 andSEQ ID NO: 80, or a light chain region comprising an amino acidsequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from thegroup consisting of SEQ ID NO: 77 and SEQ ID NO: 79, or acombination thereof, comprising an Fc region or a region equivalentto the Fc region of an immunoglobulin.

In one embodiment, an antigen binding molecule of the inventionbinds to Tenascin-C (TnC) with a dissociation constant (K.sub.D)value lower than about 1 .mu.M to about 0.001 nM, particularly aK.sub.D value lower than about 100 nM, lower than about 10 nM, orlower than about 1 nM. In a specific embodiment, an antigen bindingmolecule of the invention binds to human Tenascin-C (TnC) with adissociation constant (K.sub.D) value lower than about 1 nM. In oneembodiment, an antigen binding molecule of the invention binds tohuman, mouse, and cynomolgus TnC. In one embodiment, an antigenbinding molecule of the invention has cross-species reactivity. Inanother specific embodiment, an antigen binding molecule of theinvention binds to the C domain of human, mouse, and cynomolgusTnC. In one embodiment the antigen binding molecule of theinvention has cross-species reactivity. In one embodiment theantigen binding molecule of the present invention binds to at leastone of human, mouse and cynomolgus TnC with a K.sub.D value lowerthan about 100 nM, lower than 10 nM, lower than 5 nM or lower than2 nM. In a specific embodiment the antigen binding molecule of thepresent invention binds to at least one of human, mouse andcynomolgus TnC with a K.sub.D value lower than about 2 nM. Infurther embodiments, the antigen binding molecule of the presentinvention binds to human TnC with a first K.sub.D value K.sub.D1,wherein said antigen binding molecule binds to mouse TnC with asecond K.sub.D value K.sub.D2, and wherein said antigen bindingmolecule binds to cynomolgus TnC with a third K.sub.D valueK.sub.D3, wherein all of the K.sub.D values selected from the groupconsisting of K.sub.D1, K.sub.D2 and K.sub.D3 are lower than about10 nM, lower than about 5 nM or lower than about 2 nM. In yetfurther embodiments, the antigen binding molecule of the presentinvention binds to human TnC with a first K.sub.D value K.sub.D1,wherein said antigen binding molecule binds to mouse TnC with asecond K.sub.D value K.sub.D2, and wherein said antigen bindingmolecule binds to cynomolgus TnC with a third K.sub.D valueK.sub.D3, wherein all of the K.sub.D values selected from the groupconsisting of K.sub.D1, K.sub.D2 and K.sub.D3 are in the range of10 nM to 0.1 nM, in the range of 5 nM to 0.1 nM or in the range of2 nM to 0.1 nM. In a further embodiment, the antibody of thepresent invention binds to human TnC with a first K.sub.D valueK.sub.D1, wherein said antibody binds to mouse TnC with a secondK.sub.D value K.sub.D2, and wherein said antibody binds tocynomolgus TnC with a third K.sub.D value K.sub.D3, wherein all ofthe K.sub.D values selected from the group consisting of K.sub.D1,K.sub.D2 and K.sub.D3 are within a K.sub.D range of a factor of 20.In a further embodiment, the antibody of the present inventionbinds to human TnC with a first K.sub.D value K.sub.D1, whereinsaid antibody binds to mouse TnC with a second K.sub.D valueK.sub.D2, and wherein said antibody binds to cynomolgus TnC with athird K.sub.D value K.sub.D3, wherein all of the K.sub.D valuesselected from the group consisting of K.sub.D1, K.sub.D2 andK.sub.D3 are within a K.sub.D range of a factor of 10. In oneembodiment an antigen binding molecule of the invention is specificfor at least one of the TnC domain selected from the groupconsisting of A1, A2, A3, A4, B, AD1, AD2, C and D. In oneembodiment, an antigen binding molecule is provided, wherein saidantigen binding molecule is able to bind to at least one of the TnCdomain selected from the group consisting of A1, A4 and C. In oneembodiment, an antigen binding molecule of the invention isspecific for the TnC domains A1 and A4. In one embodiment, anantigen binding molecule of the invention is specific for the TnCdomain C. In a specific embodiment, an antigen binding molecule ofthe invention binds to the A1 and to the A4 domain of human, mouse,and cynomolgus TnC. In another specific embodiment, an antigenbinding molecule of the invention binds to the C domain of human,mouse, and cynomolgus TnC. In one embodiment the antigen bindingmolecule of the invention has cross-species reactivity. In a morespecific embodiment, an antigen binding molecule of the inventionbinds to the A1 domain of human, mouse and cynomolgus TnC A1 and tothe A4 domain of human, mouse and cynomolgus TnC A4 with a K.sub.Dvalue lower than about 100 nM, lower than about 10 nM, lower thanabout 5 nM or lower than about 2 nM. K.sub.D values are determinedby Surface Plasmon Resonance, using the antibodies as Fab or IgG.In one embodiment, an anti-TnC antigen binding molecule of theinvention binds TnC in human tissues.

In one embodiment, an antigen binding molecule of the inventioncomprises an Fc region, wherein said antigen binding moleculecomprises at least one amino acid substitution in the Fc region. Inone embodiment, an antigen binding molecule of the inventioncomprising at least one amino acid substitution in the Fc regionhas decreased effector function and/or decreased Fc receptorbinding affinity compared to an antigen binding molecule comprisingthe parent non-substituted Fc region. In a specific embodiment saidparent non-substituted Fc region comprises the amino acid residuesLeu234, Leu235 and Pro329, wherein the substituted Fc regioncomprises at least one of the amino acid substitutions selectedfrom the group consisting of Leu234Ala, Leu235Ala and Pro329Glyrelative to the parent non-substituted Fc region. In a particularembodiment, the invention provides an antigen binding molecule thatspecifically binds to TnC, wherein said antigen binding moleculecomprises a) a heavy chain region comprising an amino acid sequencethat is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 83 and SEQ ID NO: 84, and a light chainregion comprising an amino acid sequence that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group consisting of SEQ ID NO: 77and SEQ ID NO: 79. In a specific embodiment, antibodies of theinvention comprise a heavy chain region comprising an amino acidsequence selected from the group of: SEQ ID NO: 83, and SEQ ID NO:84. In a further embodiment antigen binding molecule comprising thesubstituted Fc region has decreased effector function and/ordecreased Fc receptor binding affinity compared to the antigenbinding molecule comprising the parent non-substituted heavy chainregion. In a further specific embodiment, the antigen bindingmolecule of the invention, comprising said substituted Fc region,comprises the amino acid substitutions Leu234Ala, Leu235Ala andPro329Gly relative to the parent non-substituted Fc region, whereinbinding to Fc.gamma.R and C1q is abolished and/or whereinFc-mediated effector function is abolished. In a particularembodiment, the decreased effector function is decreased ADCC. Inanother particular embodiment, the decreased effector function isabolished ADCC. The decreased Fc receptor binding preferably isdecreased binding to an activating Fc receptor, most preferablyFc.gamma.RIIIa. In one embodiment, an antigen binding molecule ofthe invention does not cause a clinically significant level oftoxicity when administered to an individual in a therapeuticallyeffective amount.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to TnC, wherein saidantigen binding molecule comprises a) a heavy chain variable regioncomprising an amino acid sequence that is at least about 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to asequence selected from the group consisting of SEQ ID NO: 46 andSEQ ID NO: 48, or a light chain variable region comprising an aminoacid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or 100% identical to a sequence selected fromthe group consisting of SEQ ID NO: 45 and SEQ ID NO: 47, or acombination thereof, and b) an Fc region or a region equivalent tothe Fc region of an immunoglobulin. In one embodiment, an antigenbinding molecule of the invention comprises an Fc region, whereinsaid Fc region is a glycoengineered Fc region. In a furtherembodiment, an antigen binding molecule of the invention isglycoengineered to have modified oligosaccharides in the Fc region.In a specific embodiment, the antigen binding molecule has anincreased proportion of bisected oligosaccharides in the Fc region,compared to a non-glycoengineered antigen binding molecule. In amore specific embodiment, at least about 10%, about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%,about 55%, about 60%, about 65%, about 70%, about 75%, about 80%,about 85%, about 90%, about 95%, or about 100%, preferably at leastabout 50%, more preferably at least about 70%, of the N-linkedoligosaccharides in the Fc region of the antigen binding moleculeare bisected. The bisected oligosaccharides may be of the hybrid orcomplex type. In another specific embodiment, an antigen bindingmolecule of the invention has an increased proportion ofnon-fucosylated oligosaccharides in the Fc region, compared to anon-glycoengineered antigen binding molecule. In a more specificembodiment, at least about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 100%, preferably at least about 50%, more preferably atleast about 70%, of the N-linked oligosaccharides in the Fc regionof the antigen binding molecule are non-fucosylated. Thenon-fucosylated oligosaccharides may be of the hybrid or complextype. In a particular embodiment, an antigen binding molecule ofthe invention has an increased proportion of bisected,non-fucosylated oligosaccharides in the Fc region, compared to anon-glycoengineered antigen binding molecule. Specifically, theantigen binding molecule comprises an Fc region in which at leastabout 10%, about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%,or about 100%, preferably at least about 15%, more preferably atleast about 25%, at least about 35% or at least about 50%, of theN-linked oligosaccharides are bisected, non-fucosylated. Thebisected, non-fucosylated oligosaccharides may be of the hybrid orcomplex type. In one embodiment, an antigen binding molecule of theinvention has increased effector function and/or increased Fcreceptor binding affinity. Increased effector function and/orincreased Fc receptor binding can result e.g., fromglycoengineering and/or affinity maturation of antibodies. In oneembodiment, the increased effector function and/or increased Fcreceptor binding is a result of glycoengineering of the Fc regionof the antigen binding molecule. In another embodiment, theincreased effector function and/or increased Fc receptor binding isa result of a combination of increased affinity andglycoengineering. The increased effector function can include, butis not limited to, one or more of the following: increasedFc-mediated cellular cytotoxicity (including increasedantibody-dependent cell-mediated cytotoxicity (ADCC)), increasedantibody-dependent cellular phagocytosis (ADCP), increased cytokinesecretion, increased immune-complex-mediated antigen uptake byantigen-presenting cells, increased binding to NK cells, increasedbinding to macrophages, increased binding to monocytes, increasedbinding to polymorphonuclear cells, increased direct signalinginducing apoptosis, increased crosslinking of target-boundantibodies, increased dendritic cell maturation, or increased Tcell priming. In a particular embodiment, the increased effectorfunction is increased ADCC. The increased Fc receptor bindingpreferably is increased binding to an activating Fc receptor, mostpreferably Fc.gamma.RIIIa. In one embodiment, an antigen bindingmolecule of the invention does not cause a clinically significantlevel of toxicity when administered to an individual in atherapeutically effective amount.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 46, alight chain variable region comprising the amino acid sequence SEQID NO: 45, and a human IgG Fc region.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of TnC,wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 48, alight chain variable region comprising the amino acid sequence SEQID NO: 47, and a human IgG Fc region.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 46, alight chain variable region comprising the amino acid sequence SEQID NO: 45, and a human IgG Fc region, and wherein said antigenbinding molecule is glycoengineered to have increased effectorfunction and/or Fc receptor binding affinity.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of TnC,wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 48, alight chain variable region comprising the amino acid sequence SEQID NO: 47, and a human IgG Fc region, and wherein said antigenbinding molecule is glycoengineered to have increased effectorfunction and/or Fc receptor binding affinity.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 46, alight chain variable region comprising the amino acid sequence SEQID NO: 45, and a human IgG Fc region, wherein said antigen bindingmolecule comprises at least one amino acid substitution in the Fcregion, wherein the parent non-substituted Fc region comprises theamino acid residues Leu234, Leu235 and Pro329, wherein thesubstituted Fc region comprises at least one of the amino acidsubstitutions selected from the group consisting of Leu234Ala,Leu235Ala and Pro329Gly relative to the parent non-substituted Fcregion, wherein the antigen binding molecule comprising thesubstituted Fc region has decreased effector function and/ordecreased Fc receptor binding affinity compared to the antigenbinding molecule comprising the parent non-substituted Fcregion.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of TnC,wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 48, alight chain variable region comprising the amino acid sequence SEQID NO: 47, and a human IgG Fc region, wherein said antigen bindingmolecule comprises at least one amino acid substitution in the Fcregion, wherein the parent non-substituted Fc region comprises theamino acid residues Leu234, Leu235 and Pro329, wherein thesubstituted Fc region comprises at least one of the amino acidsubstitutions selected from the group consisting of Leu234Ala,Leu235Ala and Pro329Gly relative to the parent non-substituted Fcregion, wherein the antigen binding molecule comprising thesubstituted Fc region has decreased effector function and/ordecreased Fc receptor binding affinity compared to the antigenbinding molecule comprising the parent non-substituted Fcregion.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainvariable region comprising

(a) the heavy chain CDR1 of SEQ ID NO: 67;

(b) the heavy chain CDR2 of SEQ ID NO: 68;

(c) the heavy chain CDR3 of SEQ ID NO: 69,

and a light chain variable region comprising

(a) the light chain CDR1 of SEQ ID NO: 55;

(b) the light chain CDR2 of SEQ ID NO: 56, and

(c) the light chain CDR3 of SEQ ID NO: 57.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of TnC,wherein said antigen binding molecule comprises a heavy chainvariable region comprising

(a) the heavy chain CDR1 of SEQ ID NO: 70;

(b) the heavy chain CDR2 of SEQ ID NO: 71;

(c) the heavy chain CDR3 of SEQ ID NO: 72,

and a light chain variable region comprising

(a) the light chain CDR1 of SEQ ID NO: 58;

(b) the light chain CDR2 of SEQ ID NO: 59, and

(c) the light chain CDR3 of SEQ ID NO: 60.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainregion comprising the amino acid sequence SEQ ID NO: 78, and alight chain region comprising the amino acid sequence SEQ ID NO:77.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofhuman, mouse and cynomolgus TnC, wherein said antigen bindingmolecule comprises a heavy chain variable region comprising theamino acid sequence SEQ ID NO: 46, a light chain variable regioncomprising the amino acid sequence SEQ ID NO: 45, and a human IgGFc region.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of TnC,wherein said antigen binding molecule comprises a heavy chainregion comprising the amino acid sequence SEQ ID NO: 80, and alight chain region comprising an amino acid sequence selected fromthe group of SEQ ID NO: 79.

In a particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the C domain of human,mouse and cynomolgus TnC, wherein said antigen binding moleculecomprises a heavy chain variable region comprising the amino acidsequence SEQ ID NO: 48, a light chain variable region comprisingthe amino acid sequence SEQ ID NO: 47, and a human IgG Fcregion.

In another particular embodiment, the invention provides an antigenbinding molecule that specifically binds to the A1 and A4 domain ofTnC, wherein said antigen binding molecule comprises a heavy chainvariable region comprising the amino acid sequence SEQ ID NO: 46, alight chain variable region comprising the amino acid sequence SEQID NO: 45, and a human IgG Fc region, and wherein said antigenbinding molecule has an increased proportion of non-fucosylatedoligosaccharides and/or an increased proportion of bisectedoligosaccharides in said Fc region. In another particularembodiment, the invention provides an antigen binding molecule thatspecifically binds to the C domain of TnC, wherein said antigenbinding molecule comprises a heavy chain variable region comprisingthe amino acid sequence SEQ ID NO: 48, a light chain variableregion comprising the amino acid sequence SEQ ID NO: 47, and ahuman IgG Fc region, and wherein said antigen binding molecule hasan increased proportion of non-fucosylated oligosaccharides and/oran increased proportion of bisected oligosaccharides in said Fcregion.

In one aspect, the invention provides for an antigen bindingmolecule that specifically binds to TnC, wherein said antigenbinding molecule comprises at least one amino acid substitution inat least one heavy or light chain CDR of the parent antigen bindingmolecule. For example, the antigen binding molecule may comprise atleast one, e.g., from about one to about ten (i.e., about 1, 2, 3,4, 5, 6, 7, 8, 9, or 10), and particularly from about two to aboutfive, substitutions in one or more hypervariable regions or CDRs(i.e., 1, 2, 3, 4, 5, or 6 hypervariable regions or CDRs) of theparent antigen binding molecule.

Additionally, the antigen binding molecule may also comprise one ormore additions, deletions and/or substitutions in one or moreframework regions of either the heavy or the light chain, comparedto the parent antigen binding molecule. In one embodiment, said atleast one amino acid substitution in at least one CDR contributesto increased binding affinity of the antigen binding moleculecompared to its parent antigen binding molecule. In anotherembodiment said antigen binding molecule has at least about 2-foldto about 10-fold greater affinity for TnC than the parent antigenbinding molecule (when comparing the antigen binding molecule ofthe invention and the parent antigen binding molecule in the sameformat, e.g., the Fab format). Further, the antigen bindingmolecule derived from a parent antigen binding molecule mayincorporate any of the features, singly or in combination,described in the preceding paragraphs in relation to the antibodiesof the invention.

TNF Family Ligand Trimer-Containing Antigen Binding Molecules whichParticular TnC Binding Moieties

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the moietycapable of specific binding to TnC comprises a VH domain comprising(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 orSEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence ofSEQ ID NO: 68 or SEQ ID NO: 71, and (iii) CDR-H3 comprising theamino acid sequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VLdomain comprising (iv) CDR-L1 comprising the amino acid sequence ofSEQ ID NO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the aminoacid sequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:60.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule of the invention,wherein the moiety capable of specific binding to TnC is a Fabmolecule capable of specific binding to TnC and comprises a VHdomain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO: 67, (ii) CDR-H2 comprising the amino acid sequence ofSEQ ID NO: 68 and (iii) CDR-H3 comprising the amino acid sequenceof SEQ ID NO: 69, and a VL domain comprising (iv) CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 55, (v) CDR-L2 comprising theamino acid sequence of SEQ ID NO: 56 and (vi) CDR-L3 comprising theamino acid sequence of SEQ ID NO: 57.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule of the invention,wherein the moiety capable of specific binding to TnC is a Fabmolecule capable of specific binding to TnC and comprises a VHdomain comprising (i) CDR-H1 comprising the amino acid sequence ofSEQ ID NO: 70, (ii) CDR-H2 comprising the amino acid sequence ofSEQ ID NO: 71 and (iii) CDR-H3 comprising the amino acid sequenceof SEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 58, (v) CDR-L2 comprising theamino acid sequence of SEQ ID NO: 59 and (vi) CDR-L3 comprising theamino acid sequence of SEQ ID NO: 60.

In one aspect, the moiety capable of specific binding to TnCcomprises a variable heavy chain comprising an amino acid sequenceof SEQ ID NO: 46 and a variable light chain comprising an aminoacid sequence of SEQ ID NO: 45 or a variable heavy chain comprisingan amino acid sequence of SEQ ID NO: 48 and a variable light chaincomprising an amino acid sequence of SEQ ID NO: 47.

In a further aspect, the moiety capable of specific binding to TnCcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 46 and a lightchain variable region comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 45.

In another aspect, the moiety capable of specific binding to TnCcomprises a heavy chain variable region comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 48 and a lightchain variable region comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 47.

In a particular aspect, the moiety capable of specific binding toTnC comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 46 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 45. In anotherparticular aspect, the moiety capable of specific binding to TnCcomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 48 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47. In a specificaspect, the moiety capable of specific binding to TnC comprises aVH domain consisting of amino acid sequence of SEQ ID NO: 46 and aVL domain consisting of the amino acid sequence of SEQ ID NO:45.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) a first heavy chain and a first light chain, both comprising aFab molecule capable of specific binding to TnC,

(b) a second heavy chain comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ IDNO: 185 and SEQ ID NO: 186, and a second light chain comprising anamino acid sequence selected from the group consisting of SEQ IDNO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnCcomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 46 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 176,SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that thesecond polypeptide comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:175 and SEQ ID NO: 183.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprisinga heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 46 and a light chain variable region comprising theamino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the the aminoacid sequence of SEQ ID NO: 176 and the second polypeptidecomprises the amino acid sequence of SEQ ID NO: 172.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprisinga heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 46 and a light chain variable region comprising theamino acid sequence of SEQ ID NO: 45, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the the aminoacid sequence of SEQ ID NO: 184 and the second polypeptidecomprises the amino acid sequence of SEQ ID NO: 183.

In another aspect, the TNF family ligand trimer-containing antigenbinding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnCcomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 48 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47, and

a first and a second polypeptide that are linked to each other by adisulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 176,SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186 and in that thesecond polypeptide comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:175 and SEQ ID NO: 183.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding to TnCcomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO: 48 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 47, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the the aminoacid sequence of SEQ ID NO: 176 and the second polypeptidecomprises the amino acid sequence of SEQ ID NO: 172.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) at least one moiety capable of specific binding TnC comprisinga heavy chain variable region comprising the amino acid sequence ofSEQ ID NO: 48 and a light chain variable region comprising theamino acid sequence of SEQ ID NO: 47, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises the the aminoacid sequence of SEQ ID NO: 184 and the second polypeptidecomprises the amino acid sequence of SEQ ID NO: 183.

In one aspect, provided is a TNF family ligand trimer-containingantigen binding molecule, wherein the antigen binding moleculecomprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or

a first heavy chain comprising the VH domain comprising the aminoacid sequence of SEQ ID NO: 48 and a first light chain comprisingthe VL domain comprising the amino acid sequence of SEQ ID NO:47,

(ii) a second heavy chain comprising the amino acid sequence of SEQID NO: 178, and

(iii) a second light chain comprising the amino acid sequence ofSEQ ID NO: 179.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises a first heavy chain and a first lightchain, both comprising a Fab molecule capable of specific bindingto TnC, a second heavy chain comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker that is fused at its C-terminus by asecond peptide linker to a CH1 domain, and a second light chaincomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a CL domain, and wherein the antigen binding moleculecomprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 48 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 47, (ii) a second heavy chain comprising theamino acid sequence of SEQ ID NO: 178, and (iii) a second lightchain comprising the amino acid sequence of SEQ ID NO: 179.

In a further particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises a first heavy chain and a first lightchain, both comprising a Fab molecule capable of specific bindingto TnC, a second heavy chain comprising two ectodomains of a TNFligand family member or fragments thereof connected to each otherby a first peptide linker and fused at its C-terminus by a secondpeptide linker to a CL domain, and a second light chain comprisingone ectodomain of said TNF ligand family member or a fragmentthereof that is fused at its C-terminus by a third peptide linkerto a CH1 domain, and wherein the molecule comprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 48 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 47, (ii) a second heavy chain comprising theamino acid sequence selected from the group consisting of SEQ IDNO: 102, SEQ ID NO: 108, SEQ ID NO: 116 and SEQ ID NO: 120, and(iii) a second light chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 103, SEQ ID NO:109, SEQ ID NO: 117 and SEQ ID NO: 121.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or

a first heavy chain comprising the VH domain comprising the aminoacid sequence of SEQ ID NO: 48 and a first light chain comprisingthe VL domain comprising the amino acid sequence of SEQ ID NO:47,

(ii) a second heavy chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 102, SEQ ID NO:108, SEQ ID NO: 116 and SEQ ID NO: 120, and

(iii) a second light chain comprising the amino acid sequenceselected from the group consisting of SEQ ID NO: 103, SEQ ID NO:109, SEQ ID NO: 117 and SEQ ID NO: 121.

In yet another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide contains a CH3 domainand the second polypeptide contains a CH3 domain, respectively, andwherein the first polypeptide comprises two ectodomains of a TNFligand family member or fragments thereof that are connected toeach other and to the C-terminus of the CH3 domain by a peptidelinker and wherein the second polypeptide comprises one ectodomainof said TNF ligand family member or a fragment thereof connectedvia a peptide linker to C-terminus of the CH3 domain of saidpolypeptide.

In a further aspect, the TNF family ligand trimer-containingantigen binding molecule of the invention comprises

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain and wherein the firstpolypeptide comprising two ectodomains of a TNF ligand familymember or fragments thereof comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 176 and SEQ ID NO:184 and the second polypeptide comprising one ectodomain of saidTNF ligand family member or a fragment thereof comprises the aminoacid sequence selected from the group consisting of SEQ ID NO: 172and SEQ ID NO: 183. In one aspect, the first polypeptide comprisingtwo ectodomains of a TNF ligand family member or fragments thereofcomprises an amino acid sequence SEQ ID NO: 176 and the secondpolypeptide comprising one ectodomain of said TNF ligand familymember or a fragment thereof comprises the amino acid sequence ofSEQ ID NO: 172. In a particular aspect, the first polypeptidecomprising two ectodomains of a TNF ligand family member orfragments thereof comprises an amino acid sequence of SEQ ID NO:184 and the second polypeptide comprising one ectodomain of saidTNF ligand family member or a fragment thereof comprises the aminoacid sequence of SEQ ID NO: 183.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) one Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain. The invention thus relates to aTNF family ligand trimer-containing antigen binding molecule,wherein TNF family ligand trimer-containing antigen bindingmolecule is monovalent for the binding to the target cellantigen.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 46 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 45, or a VHdomain comprising an amino acid sequence of SEQ ID NO: 48 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 47, (b) a Fcdomain composed of a first and a second subunit capable of stableassociation, and (c) a first polypeptide comprising two ectodomainsof a TNF ligand family member or fragments thereof that areconnected to each other by a peptide linker and a secondpolypeptide comprising one ectodomain of said TNF ligand familymember or a fragment thereof, wherein the first polypeptide isfused at its N-terminus to the C-terminus to one of the subunits ofthe Fc domain and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the other subunit of the Fcdomain.

In another aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 46 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 45, or a VHdomain comprising an amino acid sequence of SEQ ID NO: 48 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 47, (b) a Fcdomain composed of a first and a second subunit capable of stableassociation, and (c) a first polypeptide comprising two ectodomainsof a TNF ligand family member or fragments thereof that areconnected to each other by a peptide linker and a secondpolypeptide comprising one ectodomain of said TNF ligand familymember or a fragment thereof, wherein the first polypeptide isfused at its N-terminus to the C-terminus to one of the subunits ofthe Fc domain and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the other subunit of the Fc domainand wherein the first polypeptide comprising two ectodomains of aTNF ligand family member or fragments thereof comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 176and SEQ ID NO: 184 and the second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereofcomprises the amino acid sequence selected from the groupconsisting of SEQ ID NO: 172 and SEQ ID NO: 183.

In a particular aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC comprising a VHdomain comprising an amino acid sequence of SEQ ID NO: 46 and a VLdomain comprising an amino acid sequence of SEQ ID NO: 45,

(b) a Fc domain composed of a first and a second subunit capable ofstable association, and

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain and wherein the firstpolypeptide comprising two ectodomains of a TNF ligand familymember comprising an amino acid sequence of SEQ ID NO: 184 and thesecond polypeptide comprising one ectodomain of said TNF ligandfamily member comprising the amino acid sequence of SEQ ID NO:183.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a light chain comprising the VL domain of the Fab domaincapable of specific binding to TnC,

(ii) a fusion polypeptide comprising a first subunit of a Fc domainand a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker, wherein the first polypeptide is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domain,and (iii) a heavy chain comprising the VH domain of the Fab domaincapable of specific binding to TnC, a second subunit of the Fcdomain and a second polypeptide comprising one ectodomain of saidTNF ligand family member or a fragment thereof, wherein thevariable heavy chain of the Fab domain capable of specific bindingto TnC is fused at its C-terminus to the N-terminus of the secondsubunit of the Fc domain and wherein the second polypeptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its N-terminus to the C-terminus ofthe second subunit of the Fc domain.

In a further aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule as defined beforefurther comprising (d) a Fab domain that is not capable of specificbinding to TnC. Thus, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising

(a) a Fab domain capable of specific binding to TnC,

(b) a Fc domain composed of a first and a second subunit capable ofstable association,

(c) a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker and a second polypeptide comprising oneectodomain of said TNF ligand family member or a fragment thereof,wherein the first polypeptide is fused at its N-terminus to theC-terminus to one of the subunits of the Fc domain and wherein thesecond polypeptide is fused at its N-terminus to the C-terminus ofthe other subunit of the Fc domain, and (d) a Fab domain that isnot capable of specific binding to a target cell antigen.

In a further particular aspect of the invention, provided is a TNFfamily ligand trimer-containing antigen binding moleculecomprising

(a) a first heavy chain and a first light chain, both comprising aFab molecule capable of specific binding to TnC, wherein the firstheavy chain comprises the VH domain comprising the amino acidsequence of SEQ ID NO: 46 and the first light chain comprises theVL domain comprising the amino acid sequence of SEQ ID NO: 45, and(b) a second heavy chain comprising two ectodomains of a TNF ligandfamily member or fragments thereof connected to each other by afirst peptide linker that is fused at its C-terminus by a secondpeptide linker to a CL domain, and a second light chain comprisingone ectodomain of said TNF ligand family member or a fragmentthereof that is fused at its C-terminus by a third peptide linkerto a CH1 domain, wherein the second heavy chain comprises the aminoacid sequence of SEQ ID NO: 116 or SEQ ID NO: 120, and the secondlight chain comprises the amino acid sequence of SEQ ID NO: 117 orSEQ ID NO: 121. In particular, the second heavy chain comprises theamino acid sequence of SEQ ID NO: 116 and the second light chaincomprises the amino acid sequence of SEQ ID NO: 117.

Furthermore, the invention provides a TNF family ligandtrimer-containing antigen binding molecule, comprising

(a) at least one moiety capable of specific binding to TnC, and

(b) a first and a second polypeptide that are linked to each otherby a disulfide bond, wherein the antigen binding molecule ischaracterized in that the first polypeptide contains a CH3 domainand the second polypeptide contains a CH3 domain, respectively, andwherein the first polypeptide comprises two ectodomains of a TNFligand family member or fragments thereof that are connected toeach other and to the C-terminus of the CH3 domain by a peptidelinker and wherein the second polypeptide comprises one ectodomainof said TNF ligand family member or a fragment thereof connectedvia a peptide linker to C-terminus of the CH3 domain of saidpolypeptide.

In particular, provided is a TNF family ligand trimer-containingantigen binding molecule as described herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQID NO: 127, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 125, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 130, a second heavy chain comprising the amino acid sequenceof SEQ ID NO:131, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO:124, a second heavy chain comprising the amino acid sequenceof SEQ ID NO:133, and one light chain comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 136, a second heavy chain comprising the amino acid sequenceof SEQ ID NO:137, and one light chain comprising the amino acidsequence of SEQ ID NO: 77.

In a particular aspect, such a TNF family ligand trimer-containingantigen binding molecule comprises two moieties capable of specificbinding to TnC.

In one aspect, the invention provides a TNF family ligandtrimer-containing antigen binding molecule comprising two moietiescapable of specific binding to TnC. In particular, provided is aTNF family ligand trimer-containing antigen binding molecule asdescribed herein before comprising

(i) a first heavy chain comprising the amino acid sequence of SEQID NO: 112, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 113, and two light chains comprising the amino acidsequence of SEQ ID NO: 77, or

(ii) a first heavy chain comprising the amino acid sequence of SEQID NO: 124, a second heavy chain comprising the amino acid sequenceof SEQ ID NO: 125, and two light chains comprising the amino acidsequence of SEQ ID NO: 77.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a light chain comprising the VL domain of the Fab domaincapable of specific binding to TNC,

(ii) a fusion polypeptide comprising a first subunit of a Fc domainand a second polypeptide comprising one ectodomain of said TNFligand family member or a fragment thereof, wherein the secondpolypeptide is fused at its N-terminus to the C-terminus of thefirst subunit of the Fc domain, and (iii) a heavy chain comprisingthe VH domain of the Fab domain capable of specific binding to TNC,a second subunit of the Fc domain and a first polypeptidecomprising two ectodomains of a TNF ligand family member orfragments thereof that are connected to each other by a peptidelinker, wherein the variable heavy chain of the Fab domain capableof specific binding to TNC is fused at its C-terminus to theN-terminus of the second subunit of the Fc domain and wherein thefirst polypeptide comprising two ectodomains of a TNF ligand familymember or fragments thereof is fused at its N-terminus to theC-terminus of the second subunit of the Fc domain.

In a further aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a first light chain comprising the VL domain of the Fab domaincapable of specific binding to TnC,

(ii) a first heavy chain comprising the VH domain of the Fab domaincapable of specific binding to TnC, a first subunit of a Fc domainand a second polypeptide comprising one ectodomain of said TNFligand family member or a fragment thereof, wherein the variableheavy chain of the Fab domain capable of specific binding to TnC isfused at its C-terminus to the N-terminus of the second subunit ofthe Fc domain and wherein the second polypeptide is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domain,(iii) a second heavy chain comprising the VH domain of a Fab domainthat is not capable of specific binding to a target cell antigen, asecond subunit of the Fc domain and a first polypeptide comprisingtwo ectodomains of a TNF ligand family member or fragments thereofthat are connected to each other by a peptide linker, wherein thevariable heavy chain of the Fab domain not capable of specificbinding to a target cell antigen is fused at its C-terminus to theN-terminus of the second subunit of the Fc domain and wherein thefirst polypeptide comprising two ectodomains of a TNF ligand familymember or fragments thereof is fused at its N-terminus to theC-terminus of the second subunit of the Fc domain, and (iv) asecond light chain comprising the VL domain of the Fab domain thatis not capable of specific binding to a target cell antigen.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

(i) a first light chain comprising the VL domain of the Fab domaincapable of specific binding to TnC,

(ii) a first heavy chain comprising the VH domain of the Fab domaincapable of specific binding to TnC, a first subunit of a Fc domainand a first polypeptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof that are connected to each otherby a peptide linker, wherein the variable heavy chain of the Fabdomain capable of specific binding to TnC is fused at itsC-terminus to the N-terminus of the second subunit of the Fc domainand wherein the first polypeptide comprising ectodomains of a TNFligand family member or fragments thereof is fused at itsN-terminus to the C-terminus of the first subunit of the Fc domain,(iii) a second heavy chain comprising the VH domain of a Fab domainthat is not capable of specific binding to a target cell antigen, asecond subunit of the Fc domain and a second polypeptide comprisingone ectodomain of said TNF ligand family member or a fragmentthereof, wherein the variable heavy chain of the Fab domain notcapable of specific binding to a target cell antigen is fused atit* C-terminus to the N-terminus of the second subunit of the Fcdomain and wherein the second polypeptide comprising one ectodomainof a TNF ligand family member or fragments thereof is fused at itsN-terminus to the C-terminus of the second subunit of the Fcdomain, and (iv) a second light chain comprising the VL domain ofthe Fab domain that is not capable of specific binding to a targetcell antigen.

In further particular aspects, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the antigenbinding molecule comprises

a) a first heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 104, a first light chain comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 77, asecond heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 102 and a second light chain comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%,99% or 100% identical to the amino acid sequence of SEQ ID NO: 103;or b) a first heavy chain comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 104, a first light chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 77, a second heavy chain comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 108 and a second light chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 109; or (c) a first heavy chain comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 112, a secondheavy chain comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 113 and two light chains comprising an aminoacid sequence that is at least about 95%, 96%, 97%, 98%, 99% or100% identical to the amino acid sequence of SEQ ID NO: 77; or d) afirst heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 104, a first light chain comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 77, asecond heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 116 and a second light chain comprisingan amino acid sequence that is at least about 95%, 96%, 97%, 98%,99% or 100% identical to the amino acid sequence of SEQ ID NO: 117;or e) a first heavy chain comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 104, a first light chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 77, a second heavy chain comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 120 and a second light chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 121; or (f) a first heavy chain comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 124, a secondheavy chain comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 125, and two light chains comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 77; or(g) a first heavy chain comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 127, a second heavy chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 128, and one light chains comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 77; or (h) a first heavychain comprising an amino acid sequence that is at least about 95%,96%, 97%, 98%, 99% or 100% identical to the amino acid sequence ofSEQ ID NO: 130, a second heavy chain comprising an amino acidsequence that is at least about 95%, 96%, 97%, 98%, 99% or 100%identical to the amino acid sequence of SEQ ID NO: 131, and onelight chains comprising an amino acid sequence that is at leastabout 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acidsequence of SEQ ID NO: 77; or (i) a first heavy chain comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 124, asecond heavy chain comprising an amino acid sequence that is atleast about 95%, 96%, 97%, 98%, 99% or 100% identical to the aminoacid sequence of SEQ ID NO: 133, and one light chains comprising anamino acid sequence that is at least about 95%, 96%, 97%, 98%, 99%or 100% identical to the amino acid sequence of SEQ ID NO: 77; or(j) a first heavy chain comprising an amino acid sequence that isat least about 95%, 96%, 97%, 98%, 99% or 100% identical to theamino acid sequence of SEQ ID NO: 136, a second heavy chaincomprising an amino acid sequence that is at least about 95%, 96%,97%, 98%, 99% or 100% identical to the amino acid sequence of SEQID NO: 137, and one light chains comprising an amino acid sequencethat is at least about 95%, 96%, 97%, 98%, 99% or 100% identical tothe amino acid sequence of SEQ ID NO: 77.

In further particular aspects, the invention relates to a TNFfamily ligand trimer-containing antigen binding molecule, selectedfrom the group consisting of:

a) a molecule comprising a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 104, a first light chain comprising theamino acid sequence of SEQ ID NO: 77, a second heavy chaincomprising the amino acid sequence of SEQ ID NO: 102 and a secondlight chain comprising the amino acid sequence of SEQ ID NO: 103;b) a molecule comprising a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 104, a first light chain comprising theamino acid sequence of SEQ ID NO: 77, a second heavy chaincomprising the amino acid sequence of SEQ ID NO: 108 and a secondlight chain comprising the amino acid sequence of SEQ ID NO: 109;(c) a molecule comprising a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 112, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO: 113 and two light chainscomprising the amino acid sequence of SEQ ID NO: 77; d) a moleculecomprising a first heavy chain comprising the amino acid sequenceof SEQ ID NO: 104, a first light chain comprising the amino acidsequence of SEQ ID NO: 77, a second heavy chain comprising theamino acid sequence of SEQ ID NO: 116 and a second light chaincomprising the amino acid sequence of SEQ ID NO: 117; e) a moleculecomprising a first heavy chain comprising the amino acid sequenceof SEQ ID NO: 104, a first light chain comprising the amino acidsequence of SEQ ID NO: 77, a second heavy chain comprising theamino acid sequence of SEQ ID NO: 120 and a second light chaincomprising the amino acid sequence of SEQ ID NO: 121; (f) amolecule comprising a first heavy chain comprising the amino acidsequence of SEQ ID NO: 124, a second heavy chain comprising theamino acid sequence of SEQ ID NO: 125, and two light chainscomprising the amino acid sequence of SEQ ID NO: 77; (g) a moleculecomprising a first heavy chain comprising the amino acid sequenceof SEQ ID NO: 127, a second heavy chain comprising the amino acidsequence of SEQ ID NO: 128, and one light chains comprising theamino acid sequence of SEQ ID NO: 77. (h) a molecule comprising afirst heavy chain comprising the amino acid sequence of SEQ ID NO:130, a second heavy chain comprising the amino acid sequence of SEQID NO: 131, and one light chains comprising the amino acid sequenceof SEQ ID NO: 77. (i) a molecule comprising a first heavy chaincomprising the amino acid sequence of SEQ ID NO: 124, a secondheavy chain comprising the amino acid sequence of SEQ ID NO: 133,and one light chains comprising the amino acid sequence of SEQ IDNO: 77. (j) a molecule comprising a first heavy chain comprisingthe amino acid sequence of SEQ ID NO: 136, a second heavy chaincomprising the amino acid sequence of SEQ ID NO: 137, and one lightchains comprising the amino acid sequence of SEQ ID NO: 77.

In another aspect, provided is a TNF family ligandtrimer-containing antigen binding molecule, wherein the TNF ligandfamily member is OX40L and wherein the target cell antigen isTenascin-C (TnC) and the moiety capable of specific binding to TnCcomprises a VH domain comprising

(i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 67 orSEQ ID NO: 70,

(ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68 orSEQ ID NO: 71, and

(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 orSEQ ID NO: 72, and a VL domain comprising

(iv) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55 orSEQ ID NO: 58,

(v) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 orSEQ ID NO: 59, and

(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57 orSEQ ID NO: 60.

In a particular aspect, the TNF family ligand trimer-containingantigen binding molecule of comprises

(i) a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 46 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 45 or a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 48 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 47, (ii) a second heavy chain comprising theamino acid sequence selected from the group consisting of SEQ IDNO: 195, and (iii) a second light chain comprising the amino acidsequence of SEQ ID NO: 196.

Polynucleotides

The invention further provides isolated polynucleotides encoding anantigen binding molecule as described herein or a fragmentthereof.

The isolated polynucleotides antigen binding molecules of theinvention may be expressed as a single polynucleotide that encodesthe entire antigen binding molecule or as multiple (e.g., two ormore) polynucleotides that are co-expressed. Polypeptides encodedby polynucleotides that are co-expressed may associate through,e.g., disulfide bonds or other means to form a functional antigenbinding molecule. For example, the light chain portion of animmunoglobulin may be encoded by a separate polynucleotide from theheavy chain portion of the immunoglobulin. When co-expressed, theheavy chain polypeptides will associate with the light chainpolypeptides to form the immunoglobulin.

In some aspects, the isolated polynucleotide encodes the entireantigen binding molecule according to the invention as describedherein. In particular, the isolated polynucleotide encodes apolypeptide comprised in the TNF family ligand trimer-containingantigen binding molecule according to the invention as describedherein.

In one aspect, the present invention is directed to an isolatedpolynucleotide encoding a TNF family ligand trimer-containingantigen binding molecule, wherein the polynucleotide comprises (a)a sequence that encodes a moiety capable of specific binding toTnC, (b) a sequence that encodes a polypeptide comprising twoectodomains of a TNF ligand family member or two fragments thereofthat are connected to each other by a peptide linker and (c) asequence that encodes a polypeptide comprising one ectodomain ofsaid TNF ligand family member or a fragment thereof.

In another aspect, provided is an isolated polynucleotide encodinga 4-1BB ligand trimer-containing antigen binding molecule, whereinthe polynucleotide comprises (a) a sequence that encodes a moietycapable of specific binding to TnC, (b) a sequence that encodes apolypeptide comprising two ectodomains of 4-1BBL or two fragmentsthereof that are connected to each other by a peptide linker and(c) a sequence that encodes a polypeptide comprising one ectodomainof 4-1BBL or a fragment thereof.

In one embodiment, the invention is directed to an isolatedpolynucleotide comprising a sequence encoding one or more (e.g.,one, two, three, four, five, or six) of the heavy or light chaincomplementarity determining regions (CDRs) set forth in SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 71 and SEQ ID NO: 72, or a variant or truncated formthereof containing at least the specificity-determining residues(SDRs) for said CDR.

In another embodiment, the polynucleotide comprises a sequence thatencodes three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) orthree light chain CDRs (e.g., LCDR1, LCDR2, and LCDR3) selectedfrom SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58,SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71 and SEQ ID NO: 72, or variantsor truncated forms thereof containing at least the SDRs for each ofsaid three complementarity determining regions. In yet anotherembodiment, the polynucleotide comprises a sequence encoding threeheavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) and three lightchain CDRs (e.g., LCDR1, LCDR2, and LCDR3) selected from SEQ ID NO:55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQID NO: 60, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO: 71 and SEQ ID NO: 72.

In a particular embodiment the polynucleotide encoding one or moreCDRs comprises a sequence that is at least about 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to one or more of the CDRnucleotide sequences of SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 61, SEQID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65 and SEQ IDNO: 66.

In a further embodiment, the polynucleotide comprises a sequenceencoding a heavy chain variable region selected from the group ofSEQ ID NO: 46 and SEQ ID NO: 48, and/or a sequence encoding a lightchain variable region selected from the group of SEQ ID NO: 45 andSEQ ID NO: 47. In a particular embodiment, the polynucleotideencoding a heavy chain and/or light chain variable region comprisesa sequence selected from the group of variable region nucleotidesequences consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43and SEQ ID NO: 44, or a combination thereof.

In a specific embodiment, the polynucleotide comprises a sequenceencoding a heavy chain variable region selected from the group ofSEQ ID NO: 46 and SEQ ID NO: 48, and a sequence encoding a heavychain constant region, particularly a human heavy chain constantregion. In a particular embodiment, said heavy chain constantregion is a human IgG heavy chain constant region, specifically ahuman IgG1 heavy chain constant region, comprising an Fc region. Inanother specific embodiment, the polynucleotide comprises asequence encoding a light chain variable region selected from thegroup of SEQ ID NO: 45 and SEQ ID NO: 47, and a sequence encoding alight chain constant region, particularly a human light chainconstant region.

In one embodiment, the invention is directed to a composition thatcomprises a first isolated polynucleotide encoding a polypeptidecomprising an amino acid sequence that is at least about 90%, 95%,96%, 97%, 98%, 99%, or 100% identical to a sequence selected fromthe group consisting of SEQ ID NO: 77 and SEQ ID NO: 79, and asecond isolated polynucleotide encoding a polypeptide comprising anamino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 78, and SEQ ID NO: 80.

In one embodiment, the invention is directed to a composition thatcomprises a first isolated polynucleotide comprising a sequencethat is at least about 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to a sequence selected from the group consisting of SEQID NO: 77 and SEQ ID NO: 79, and a second isolated polynucleotidecomprising a sequence that is at least about 90%, 95%, 96%, 97%,98%, 99%, or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 83, and SEQ ID NO: 84.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence that encodes a polypeptidecomprising two 4-1BBL fragments comprising an amino acid sequencethat is at least about 90%, 95%, 98% or 100% identical to an aminoacid sequence shown in SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO:174, SEQ ID NO: 175 or SEQ ID NO: 183, and to a polynucleotidecomprising a sequence that encodes a polypeptide comprising one4-1BBL fragment comprising an amino acid sequence that is at leastabout 90%, 95%, 98% or 100% identical to an amino acid sequenceshown in SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO:175 or SEQ ID NO: 183.

Furthermore, provided is an isolated polynucleotide encoding a OX40ligand trimer-containing antigen binding molecule, wherein thepolynucleotide comprises (a) a sequence that encodes a moietycapable of specific binding to TnC, (b) a sequence that encodes apolypeptide comprising two ectodomains of OX40L or two fragmentsthereof that are connected to each other by a peptide linker and(c) a sequence that encodes a polypeptide comprising one ectodomainof OX40L or a fragment thereof.

In another aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence that encodes a polypeptidecomprising two 4-1BBL fragments comprising an amino acid sequencethat is at least about 90%, 95%, 98% or 100% identical to an aminoacid sequence shown in SEQ ID NO: 181 or SEQ ID NO: 182, and to apolynucleotide comprising a sequence that encodes a polypeptidecomprising one 4-1BBL fragment comprising an amino acid sequencethat is at least about 90%, 95%, 98% or 100% identical to an aminoacid sequence shown in SEQ ID NO: 181 or SEQ ID NO: 182.

In further aspects, the invention relates to the polynucleotidescomprising a sequence that is at least about 90%, 95%, 98% or 100%identical to the specific cDNA sequences disclosed herein. In aparticular aspect, the invention relates to a polynucleotidecomprising a sequence that is identical to one of the specific cDNAsequences disclosed herein.

In other aspects, the nucleic acid molecule comprises or consistsof a nucleotide sequence that encodes an amino acid sequence as setforth in any one of SEQ ID NOs: 176, 177, 184, 185, 186, 187, 188or 189. In a further aspect, the nucleic acid molecule comprises orconsists of a nucleotide sequence that encodes an amino acidsequence as set forth in any one of SEQ ID NOs: 102, 103, 104, 108,109, 112, 113, 116, 117, 120, 121, 124, 125, 127, 130, 131, 133,136, 137, 178 and 179.

In still other aspects, the nucleic acid molecule comprises orconsists of a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 41, 42, 43, 44, 49, 50, 51, 52, 53, 54,61, 62, 63, 64, 65, 66, 73, 74, 75, 76, 81, 82, 88, 89, 90, 91, 96,98, 99, 100, 106, 107, 110, 111, 114, 115, 118, 119, 122, 123, 126,128, 129, 132, 134 or 135.

In certain aspects, the polynucleotide or nucleic acid is DNA. Inother embodiments, a polynucleotide of the present invention isRNA, for example, in the form of messenger RNA (mRNA). RNA of thepresent invention may be single stranded or double stranded.

Recombinant Methods

Antigen binding molecules of the invention may be obtained, forexample, by solid-state peptide synthesis (e.g. Merrifield solidphase synthesis) or recombinant production. For recombinantproduction one or more polynucleotide encoding the antigen bindingmolecule or polypeptide fragments thereof, e.g., as describedabove, is isolated and inserted into one or more vectors forfurther cloning and/or expression in a host cell. Suchpolynucleotide may be readily isolated and sequenced usingconventional procedures. In one aspect of the invention, a vector,preferably an expression vector, comprising one or more of thepolynucleotides of the invention is provided. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing the coding sequence of the antigenbinding molecule (fragment) along with appropriatetranscriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. See, for example,the techniques described in Maniatis et al., MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989); andAusubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing Associates and Wiley Interscience, N.Y. (1989). Theexpression vector can be part of a plasmid, virus, or may be anucleic acid fragment. The expression vector includes an expressioncassette into which the polynucleotide encoding the antigen bindingmolecule or polypeptide fragments thereof (i.e. the coding region)is cloned in operable association with a promoter and/or othertranscription or translation control elements. As used herein, a"coding region" is a portion of nucleic acid which consists ofcodons translated into amino acids. Although a "stop codon" (TAG,TGA, or TAA) is not translated into an amino acid, it may beconsidered to be part of a coding region, if present, but anyflanking sequences, for example promoters, ribosome binding sites,transcriptional terminators, introns, 5' and 3' untranslatedregions, and the like, are not part of a coding region. Two or morecoding regions can be present in a single polynucleotide construct,e.g. on a single vector, or in separate polynucleotide constructs,e.g. on separate (different) vectors. Furthermore, any vector maycontain a single coding region, or may comprise two or more codingregions, e.g. a vector of the present invention may encode one ormore polypeptides, which are post- or co-translationally separatedinto the final proteins via proteolytic cleavage. In addition, avector, polynucleotide, or nucleic acid of the invention may encodeheterologous coding regions, either fused or unfused to apolynucleotide encoding the antigen binding molecule of theinvention or polypeptide fragments thereof, or variants orderivatives thereof. Heterologous coding regions include withoutlimitation specialized elements or motifs, such as a secretorysignal peptide or a heterologous functional domain. An operableassociation is when a coding region for a gene product, e.g. apolypeptide, is associated with one or more regulatory sequences insuch a way as to place expression of the gene product under theinfluence or control of the regulatory sequence(s). Two DNAfragments (such as a polypeptide coding region and a promoterassociated therewith) are "operably associated" if induction ofpromoter function results in the transcription of mRNA encoding thedesired gene product and if the nature of the linkage between thetwo DNA fragments does not interfere with the ability of theexpression regulatory sequences to direct the expression of thegene product or interfere with the ability of the DNA template tobe transcribed. Thus, a promoter region would be operablyassociated with a nucleic acid encoding a polypeptide if thepromoter was capable of effecting transcription of that nucleicacid. The promoter may be a cell-specific promoter that directssubstantial transcription of the DNA only in predetermined cells.Other transcription control elements, besides a promoter, forexample enhancers, operators, repressors, and transcriptiontermination signals, can be operably associated with thepolynucleotide to direct cell-specific transcription.

Suitable promoters and other transcription control regions aredisclosed herein. A variety of transcription control regions areknown to those skilled in the art. These include, withoutlimitation, transcription control regions, which function invertebrate cells, such as, but not limited to, promoter andenhancer segments from cytomegaloviruses (e.g. the immediate earlypromoter, in conjunction with intron-A), simian virus 40 (e.g. theearly promoter), and retroviruses (such as, e.g. Rous sarcomavirus). Other transcription control regions include those derivedfrom vertebrate genes such as actin, heat shock protein, bovinegrowth hormone and rabbit a-globin, as well as other sequencescapable of controlling gene expression in eukaryotic cells.Additional suitable transcription control regions includetissue-specific promoters and enhancers as well as induciblepromoters (e.g. promoters inducible tetracyclins). Similarly, avariety of translation control elements are known to those ofordinary skill in the art. These include, but are not limited toribosome binding sites, translation initiation and terminationcodons, and elements derived from viral systems (particularly aninternal ribosome entry site, or IRES, also referred to as a CITEsequence). The expression cassette may also include other featuressuch as an origin of replication, and/or chromosome integrationelements such as retroviral long terminal repeats (LTRs), oradeno-associated viral (AAV) inverted terminal repeats (ITRs).

Polynucleotide and nucleic acid coding regions of the presentinvention may be associated with additional coding regions whichencode secretory or signal peptides, which direct the secretion ofa polypeptide encoded by a polynucleotide of the present invention.For example, if secretion of the antigen binding molecule orpolypeptide fragments thereof is desired, DNA encoding a signalsequence may be placed upstream of the nucleic acid encoding anantigen binding molecule of the invention or polypeptide fragmentsthereof. According to the signal hypothesis, proteins secreted bymammalian cells have a signal peptide or secretory leader sequencewhich is cleaved from the mature protein once export of the growingprotein chain across the rough endoplasmic reticulum has beeninitiated. Those of ordinary skill in the art are aware thatpolypeptides secreted by vertebrate cells generally have a signalpeptide fused to the N-terminus of the polypeptide, which iscleaved from the translated polypeptide to produce a secreted or"mature" form of the polypeptide. In certain embodiments, thenative signal peptide, e.g. an immunoglobulin heavy chain or lightchain signal peptide is used, or a functional derivative of thatsequence that retains the ability to direct the secretion of thepolypeptide that is operably associated with it. Alternatively, aheterologous mammalian signal peptide, or a functional derivativethereof, may be used. For example, the wild-type leader sequencemay be substituted with the leader sequence of human tissueplasminogen activator (TPA) or mouse .beta.-glucuronidase.

DNA encoding a short protein sequence that could be used tofacilitate later purification (e.g. a histidine tag) or assist inlabeling the fusion protein may be included within or at the endsof the polynucleotide encoding an antigen binding molecule of theinvention or polypeptide fragments thereof.

In a further aspect of the invention, a host cell comprising one ormore polynucleotides of the invention is provided. In certainembodiments a host cell comprising one or more vectors of theinvention is provided. The polynucleotides and vectors mayincorporate any of the features, singly or in combination,described herein in relation to polynucleotides and vectors,respectively. In one aspect, a host cell comprises (e.g. has beentransformed or transfected with) a vector comprising apolynucleotide that encodes (part of) an antigen binding moleculeof the invention of the invention. As used herein, the term "hostcell" refers to any kind of cellular system which can be engineeredto generate the fusion proteins of the invention or fragmentsthereof. Host cells suitable for replicating and for supportingexpression of antigen binding molecules are well known in the art.Such cells may be transfected or transduced as appropriate with theparticular expression vector and large quantities of vectorcontaining cells can be grown for seeding large scale fermenters toobtain sufficient quantities of the antigen binding molecule forclinical applications. Suitable host cells include prokaryoticmicroorganisms, such as E. coli, or various eukaryotic cells, suchas Chinese hamster ovary cells (CHO), insect cells, or the like.For example, polypeptides may be produced in bacteria in particularwhen glycosylation is not needed. After expression, the polypeptidemay be isolated from the bacterial cell paste in a soluble fractionand can be further purified. In addition to prokaryotes, eukaryoticmicrobes such as filamentous fungi or yeast are suitable cloning orexpression hosts for polypeptide-encoding vectors, including fungiand yeast strains whose glycosylation pathways have been"humanized", resulting in the production of a polypeptide with apartially or fully human glycosylation pattern. See Gerngross, NatBiotech 22, 1409-1414 (2004), and Li et al., Nat Biotech 24,210-215 (2006).

Suitable host cells for the expression of (glycosylated)polypeptides are also derived from multicellular organisms(invertebrates and vertebrates). Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains havebeen identified which may be used in conjunction with insect cells,particularly for transfection of Spodoptera frugiperda cells. Plantcell cultures can also be utilized as hosts. See e.g. U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES.TM. technology for producing antibodies intransgenic plants). Vertebrate cells may also be used as hosts. Forexample, mammalian cell lines that are adapted to grow insuspension may be useful. Other examples of useful mammalian hostcell lines are monkey kidney CV1 line transformed by SV40 (COS-7);human embryonic kidney line (293 or 293T cells as described, e.g.,in Graham et al., J Gen Virol 36, 59 (1977)), baby hamster kidneycells (BHK), mouse sertoli cells (TM4 cells as described, e.g., inMather, Biol Reprod 23, 243-251 (1980)), monkey kidney cells (CV1),African green monkey kidney cells (VERO-76), human cervicalcarcinoma cells (HELA), canine kidney cells (MDCK), buffalo ratliver cells (BRL 3A), human lung cells (W138), human liver cells(Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells (asdescribed, e.g., in Mather et al., Annals N.Y. Acad Sci 383, 44-68(1982)), MRC 5 cells, and FS4 cells. Other useful mammalian hostcell lines include Chinese hamster ovary (CHO) cells, includingdhfr-CHO cells (Urlaub et al., Proc Natl Acad Sci USA 77, 4216(1980)); and myeloma cell lines such as YO, NS0, P3X63 and Sp2/0.For a review of certain mammalian host cell lines suitable forprotein production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.),pp. 255-268 (2003). Host cells include cultured cells, e g,mammalian cultured cells, yeast cells, insect cells, bacterialcells and plant cells, to name only a few, but also cells comprisedwithin a transgenic animal, transgenic plant or cultured plant oranimal tissue. In one embodiment, the host cell is a eukaryoticcell, preferably a mammalian cell, such as a Chinese Hamster Ovary(CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell(e.g., Y0, NS0, Sp20 cell). Standard technologies are known in theart to express foreign genes in these systems. Cells expressing apolypeptide comprising either the heavy or the light chain of animmunoglobulin, may be engineered so as to also express the otherof the immunoglobulin chains such that the expressed product is animmunoglobulin that has both a heavy and a light chain.

In one aspect, a method of producing an antigen binding molecule ofthe invention or polypeptide fragments thereof is provided, whereinthe method comprises culturing a host cell comprisingpolynucleotides encoding the antigen binding molecule of theinvention or polypeptide fragments thereof, as provided herein,under conditions suitable for expression of the antigen bindingmolecule of the invention or polypeptide fragments thereof, andrecovering the antigen binding molecule of the invention orpolypeptide fragments thereof from the host cell (or host cellculture medium).

In the TNF family ligand trimer-containing antigen binding moleculeof the invention, the components (at least one moiety capable ofspecific binding to TnC, one polypeptide comprising two ectodomainsof a TNF ligand family member or fragments thereof and apolypeptide comprising one ectodomain of said TNF family ligandfamily member or a fragment thereof) are not genetically fused toeach other. The polypeptides are designed such that its components(two ectodomains of a TNF ligand family member or fragments thereofand other components such as CH or CL) are fused to each otherdirectly or through a linker sequence. The composition and lengthof the linker may be determined in accordance with methods wellknown in the art and may be tested for efficacy. Examples of linkersequences between different components of the antigen bindingmolecules of the invention are found in the sequences providedherein. Additional sequences may also be included to incorporate acleavage site to separate the individual components of the fusionprotein if desired, for example an endopeptidase recognitionsequence.

In certain embodiments the moieties capable of specific binding toTnC (e.g. Fab fragments) forming part of the antigen bindingmolecule comprise at least an immunoglobulin variable regioncapable of binding to an antigen. Variable regions can form part ofand be derived from naturally or non-naturally occurring antibodiesand fragments thereof. Methods to produce polyclonal antibodies andmonoclonal antibodies are well known in the art (see e.g. Harlowand Lane, "Antibodies, a laboratory manual", Cold Spring HarborLaboratory, 1988). Non-naturally occurring antibodies can beconstructed using solid phase-peptide synthesis, can be producedrecombinantly (e.g. as described in U.S. Pat. No. 4,186,567) or canbe obtained, for example, by screening combinatorial librariescomprising variable heavy chains and variable light chains (seee.g., U.S. Pat. No. 5,969,108 to McCafferty).

Any animal species of immunoglobulin can be used in the invention.Non-limiting immunoglobulins useful in the present invention can beof murine, primate, or human origin. If the fusion protein isintended for human use, a chimeric form of immunoglobulin may beused wherein the constant regions of the immunoglobulin are from ahuman. A humanized or fully human form of the immunoglobulin canalso be prepared in accordance with methods well known in the art(see e.g., U.S. Pat. No. 5,565,332 to Winter). Humanization may beachieved by various methods including, but not limited to (a)grafting the non-human (e.g., donor antibody) CDRs onto human(e.g., recipient antibody) framework and constant regions with orwithout retention of critical framework residues (e.g., those thatare important for retaining good antigen binding affinity orantibody functions), (b) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residuescritical for the antibody-antigen interaction) onto human frameworkand constant regions, or (c) transplanting the entire non-humanvariable domains, but "cloaking" them with a human-like section byreplacement of surface residues. Humanized antibodies and methodsof making them are reviewed, e.g., in Almagro and Fransson, FrontBiosci 13, 1619-1633 (2008), and are further described, e.g., inRiechmann et al., Nature 332, 323-329 (1988); Queen et al., ProcNatl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337,7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321,522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855(1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen etal., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3),169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol Immunol 28, 489-498(1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36,43-60 (2005) (describing "FR shuffling"); and Osbourn et al.,Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260(2000) (describing the "guided selection" approach to FRshuffling). Particular immunoglobulins according to the inventionare human immunoglobulins. Human antibodies and human variableregions can be produced using various techniques known in the art.Human antibodies are described generally in van Dijk and van deWinkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr OpinImmunol 20, 450-459 (2008). Human variable regions can form part ofand be derived from human monoclonal antibodies made by thehybridoma method (see e.g. Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., NewYork, 1987)). Human antibodies and human variable regions may alsobe prepared by administering an immunogen to a transgenic animalthat has been modified to produce intact human antibodies or intactantibodies with human variable regions in response to antigenicchallenge (see e.g. Lonberg, Nat Biotech 23, 1117-1125 (2005).Human antibodies and human variable regions may also be generatedby isolating Fv clone variable region sequences selected fromhuman-derived phage display libraries (see e.g., Hoogenboom et al.in Methods in Molecular Biology 178, 1-37 (O'Brien et al., ed.,Human Press, Totowa, N.J., 2001); and McCafferty et al., Nature348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phagetypically display antibody fragments, either as single-chain Fv(scFv) fragments or as Fab fragments.

In certain aspects, the moieties capable of specific binding to atarget cell antigen (e.g., Fab fragments) comprised in the antigenbinding molecules of the present invention are engineered to haveenhanced binding affinity according to, for example, the methodsdisclosed in PCT publication WO 2012/020006 or U.S. Pat. Appl.Publ. No. 2004/0132066. The ability of the antigen bindingmolecules of the invention to bind to a specific antigenicdeterminant can be measured either through an enzyme-linkedimmunosorbent assay (ELISA) or other techniques familiar to one ofskill in the art, e.g., surface plasmon resonance technique(Liljeblad, et al., Glyco J 17, 323-329 (2000)), and traditionalbinding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competitionassays may be used to identify an antigen binding molecule thatcompetes with a reference antibody for binding to a particularantigen. In certain embodiments, such a competing antigen bindingmolecule binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by the reference antigenbinding molecule. Detailed exemplary methods for mapping an epitopeto which an antigen binding molecule binds are provided in Morris(1996) "Epitope Mapping Protocols", in Methods in Molecular Biologyvol. 66 (Humana Press, Totowa, N.J.). In an exemplary competitionassay, immobilized antigen is incubated in a solution comprising afirst labeled antigen binding molecule that binds to the antigenand a second unlabeled antigen binding molecule that is beingtested for its ability to compete with the first antigen bindingmolecule for binding to the antigen. The second antigen bindingmolecule may be present in a hybridoma supernatant. As a control,immobilized antigen is incubated in a solution comprising the firstlabeled antigen binding molecule but not the second unlabeledantigen binding molecule. After incubation under conditionspermissive for binding of the first antibody to the antigen, excessunbound antibody is removed, and the amount of label associatedwith immobilized antigen is measured. If the amount of labelassociated with immobilized antigen is substantially reduced in thetest sample relative to the control sample, then that indicatesthat the second antigen binding molecule is competing with thefirst antigen binding molecule for binding to the antigen. SeeHarlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

Antigen binding molecules of the invention prepared as describedherein may be purified by art-known techniques such as highperformance liquid chromatography, ion exchange chromatography, gelelectrophoresis, affinity chromatography, size exclusionchromatography, and the like. The actual conditions used to purifya particular protein will depend, in part, on factors such as netcharge, hydrophobicity, hydrophilicity etc., and will be apparentto those having skill in the art. For affinity chromatographypurification an antibody, ligand, receptor or antigen can be usedto which the antigen binding molecule binds. For example, foraffinity chromatography purification of fusion proteins of theinvention, a matrix with protein A or protein G may be used.Sequential Protein A or G affinity chromatography and sizeexclusion chromatography can be used to isolate an antigen bindingmolecule essentially as described in the Examples. The purity ofthe antigen binding molecule or fragments thereof can be determinedby any of a variety of well-known analytical methods including gelelectrophoresis, high pressure liquid chromatography, and the like.For example, the antigen binding molecules expressed as describedin the Examples were shown to be intact and properly assembled asdemonstrated by reducing and non-reducing SDS-PAGE.

Assays

The antigen binding molecules provided herein may be identified,screened for, or characterized for their physical/chemicalproperties and/or biological activities by various assays known inthe art.

1. Affinity Assays

The affinity of the antigen binding molecule provided herein forTnC and/or for the corresponding TNF receptor can be determined inaccordance with the methods set forth in the Examples by surfaceplasmon resonance (SPR), using standard instrumentation such as aBIAcore instrument (GE Healthcare), and receptors or targetproteins such as may be obtained by recombinant expression. Theaffinity of the TNF family ligand trimer-containing antigen bindingmolecule for the target cell antigen can also be determined bysurface plasmon resonance (SPR), using standard instrumentationsuch as a BIAcore instrument (GE Healthcare), and receptors ortarget proteins such as may be obtained by recombinant expression.A specific illustrative and exemplary embodiment for measuringbinding affinity is described in Example 4. According to oneaspect, K.sub.D is measured by surface plasmon resonance using aBIACORE.RTM. T100 machine (GE Healthcare) at 25.degree. C.

2. Binding Assays and Other Assays

Binding of the TNF family ligand trimer-containing antigen bindingmolecule provided herein to the corresponding receptor expressingcells may be evaluated using cell lines expressing the particularreceptor or target cell antigen, for example by flow cytometry(FACS). In one aspect, fresh peripheral blood mononuclear cells(PBMCs) expressing the TNF receptor are used in the binding assay.These cells are used directly after isolation (naive PMBCs) orafter stimulation (activated PMBCs). In another aspect, activatedmouse splenocytes (expressing the TNF receptor molecule) were usedto demonstrate the binding of the TNF family ligandtrimer-containing antigen binding molecule of the invention to thecorresponding TNF receptor expressing cells.

In a further aspect, cancer cell lines expressing TnC, were used todemonstrate the binding of the antigen binding molecules to thetarget cell antigen.

In another aspect, competition assays may be used to identify anantigen binding molecule that competes with a specific antibody orantigen binding molecule for binding to the target or TNF receptor,respectively. In certain embodiments, such a competing antigenbinding molecule binds to the same epitope (e.g., a linear or aconformational epitope) that is bound by a specific anti-targetantibody or a specific anti-TNF receptor antibody. Detailedexemplary methods for mapping an epitope to which an antibody bindsare provided in Morris (1996) "Epitope Mapping Protocols," inMethods in Molecular Biology vol. 66 (Humana Press, Totowa,N.J.).

3. Activity Assays

In one aspect, assays are provided for identifying TNF familyligand trimer-containing antigen binding molecules that bind to aspecific target cell antigen and to a specific TNF receptor havingbiological activity. Biological activity may include, e.g.,agonistic signalling through the TNF receptor on cells expressingthe target cell antigen. TNF family ligand trimer-containingantigen binding molecules identified by the assays as having suchbiological activity in vitro are also provided.

In certain aspects, a TNF family ligand trimer-containing antigenbinding molecule of the invention is tested for such biologicalactivity. Assays for detecting the biological activity of themolecules of the invention are those described in Example 12.Furthermore, assays for detecting cell lysis (e.g. by measurementof LDH release), induced apoptosis kinetics (e.g. by measurement ofCaspase 3/7 activity) or apoptosis (e.g. using the TUNEL assay) arewell known in the art. In addition the biological activity of suchcomplexes can be assessed by evaluating their effects on survival,proliferation and lymphokine secretion of various lymphocytesubsets such as NK cells, NKT-cells or 145 T-cells or assessingtheir capacity to modulate phenotype and function of antigenpresenting cells such as dendritic cells, monocytes/macrophages orB-cells.

Pharmaceutical Compositions, Formulations and Routes ofAdministration

In a further aspect, the invention provides pharmaceuticalcompositions comprising any of the antigen binding moleculesprovided herein, e.g., for use in any of the below therapeuticmethods. In one embodiment, a pharmaceutical composition comprisesany of the antigen binding molecules provided herein and at leastone pharmaceutically acceptable excipient. In another embodiment, apharmaceutical composition comprises any of the antigen bindingmolecules provided herein and at least one additional therapeuticagent, e.g., as described below.

Pharmaceutical compositions of the present invention comprise atherapeutically effective amount of one or more antigen bindingmolecules dissolved or dispersed in a pharmaceutically acceptableexcipient. The phrases "pharmaceutical or pharmacologicallyacceptable" refers to molecular entities and compositions that aregenerally non-toxic to recipients at the dosages and concentrationsemployed, i.e., do not produce an adverse, allergic or otheruntoward reaction when administered to an animal, such as, forexample, a human, as appropriate. The preparation of apharmaceutical composition that contains at least one antigenbinding molecule and optionally an additional active ingredientwill be known to those of skill in the art in light of the presentdisclosure, as exemplified by Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, incorporated herein byreference. In particular, the compositions are lyophilizedformulations or aqueous solutions. As used herein,"pharmaceutically acceptable excipient" includes any and allsolvents, buffers, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, salts, stabilizers and combinationsthereof, as would be known to one of ordinary skill in the art.

Parenteral compositions include those designed for administrationby injection, e.g., subcutaneous, intradermal, intralesional,intravenous, intraarterial intramuscular, intrathecal orintraperitoneal injection. For injection, the antigen bindingmolecules of the invention may be formulated in aqueous solutions,preferably in physiologically compatible buffers such as Hanks'solution, Ringer's solution, or physiological saline buffer. Thesolution may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the fusionproteins may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use. Sterileinjectable solutions are prepared by incorporating the fusionproteins of the invention in the required amount in the appropriatesolvent with various of the other ingredients enumerated below, asrequired. Sterility may be readily accomplished, e.g., byfiltration through sterile filtration membranes. Generally,dispersions are prepared by incorporating the various sterilizedactive ingredients into a sterile vehicle which contains the basicdispersion medium and/or the other ingredients. In the case ofsterile powders for the preparation of sterile injectablesolutions, suspensions or emulsion, the preferred methods ofpreparation are vacuum-drying or freeze-drying techniques whichyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered liquid mediumthereof. The liquid medium should be suitably buffered if necessaryand the liquid diluent first rendered isotonic prior to injectionwith sufficient saline or glucose. The composition must be stableunder the conditions of manufacture and storage, and preservedagainst the contaminating action of microorganisms, such asbacteria and fungi. It will be appreciated that endotoxincontamination should be kept minimally at a safe level, forexample, less that 0.5 ng/mg protein. Suitable pharmaceuticallyacceptable excipients include, but are not limited to: buffers suchas phosphate, citrate, and other organic acids; antioxidantsincluding ascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben;catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); lowmolecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids suchas glycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol;salt-forming counter-ions such as sodium; metal complexes (e.g.Zn-protein complexes); and/or non-ionic surfactants such aspolyethylene glycol (PEG). Aqueous injection suspensions maycontain compounds which increase the viscosity of the suspension,such as sodium carboxymethyl cellulose, sorbitol, dextran, or thelike. Optionally, the suspension may also contain suitablestabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentratedsolutions. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesameoil, or synthetic fatty acid esters, such as ethyl cleats ortriglycerides, or liposomes.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosedin Remington's Pharmaceutical Sciences (18th Ed. Mack PrintingCompany, 1990). Sustained-release preparations may be prepared.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing thepolypeptide, which matrices are in the form of shaped articles,e.g. films, or microcapsules. In particular embodiments, prolongedabsorption of an injectable composition can be brought about by theuse in the compositions of agents delaying absorption, such as, forexample, aluminum monostearate, gelatin or combinationsthereof.

Exemplary pharmaceutically acceptable excipients herein furtherinclude interstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example,human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20(HYLENEX.RTM., Baxter International, Inc.). Certain exemplarysHASEGPs and methods of use, including rHuPH20, are described in USPatent Publication Nos. 2005/0260186 and 2006/0104968. In oneaspect, a sHASEGP is combined with one or more additionalglycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S.Pat. No. 6,267,958. Aqueous antibody formulations include thosedescribed in U.S. Pat. No. 6,171,586 and WO2006/044908, the latterformulations including a histidine-acetate buffer.

In addition to the compositions described previously, the fusionproteins may also be formulated as a depot preparation. Such longacting formulations may be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection. Thus, for example, the fusion proteins may be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly solublesalt.

Pharmaceutical compositions comprising the fusion proteins of theinvention may be manufactured by means of conventional mixing,dissolving, emulsifying, encapsulating, entrapping or lyophilizingprocesses. Pharmaceutical compositions may be formulated inconventional manner using one or more physiologically acceptablecarriers, diluents, excipients or auxiliaries which facilitateprocessing of the proteins into preparations that can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen.

The antigen binding molecules may be formulated into a compositionin a free acid or base, neutral or salt form. Pharmaceuticallyacceptable salts are salts that substantially retain the biologicalactivity of the free acid or base. These include the acid additionsalts, e.g. those formed with the free amino groups of aproteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or suchorganic acids as acetic, oxalic, tartaric or mandelic acid. Saltsformed with the free carboxyl groups can also be derived frominorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases asisopropylamine, trimethylamine, histidine or procaine.Pharmaceutical salts tend to be more soluble in aqueous and otherprotic solvents than are the corresponding free base forms.

The composition herein may also contain more than one activeingredients as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such active ingredients are suitablypresent in combination in amounts that are effective for thepurpose intended.

The formulations to be used for in vivo administration aregenerally sterile. Sterility may be readily accomplished, e.g., byfiltration through sterile filtration membranes.

Therapeutic Methods and Compositions

Any of the antigen binding molecules provided herein may be used intherapeutic methods. For use in therapeutic methods, antigenbinding molecules of the invention can be formulated, dosed, andadministered in a fashion consistent with good medical practice.Factors for consideration in this context include the particulardisorder being treated, the particular mammal being treated, theclinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners.

In one aspect, antigen binding molecules of the invention for useas a medicament are provided. In further aspects, antigen bindingmolecules of the invention for use in treating a disease, inparticular for use in the treatment of cancer, are provided. Incertain aspects, antigen binding molecules of the invention for usein a method of treatment are provided. In one aspect, the inventionprovides an antigen binding molecule as described herein for use inthe treatment of a disease in an individual in need thereof. Incertain aspects, the invention provides an antigen binding moleculefor use in a method of treating an individual having a diseasecomprising administering to the individual a therapeuticallyeffective amount of the fusion protein. In certain aspects, thedisease to be treated is cancer. Examples of cancers include solidtumors, bladder cancer, renal cell carcinoma, brain cancer, headand neck cancer, pancreatic cancer, lung cancer, breast cancer,ovarian cancer, uterine cancer, cervical cancer, endometrialcancer, esophageal cancer, colon cancer, colorectal cancer, rectalcancer, gastric cancer, prostate cancer, blood cancer, skin cancer,squamous cell carcinoma, bone cancer, and kidney cancer, melanoma,B-cell lymphoma, B-cell leukemia, non-Hodgkin lymphoma and acutelymphoblastic leukemia. Thus, an antigen binding molecule asdescribed herein for use in the treatment of cancer is provided.The subject, patient, or "individual" in need of treatment istypically a mammal, more specifically a human.

In another aspect, provided is an antigen binding molecule asdescribed herein for use in the treatment of infectious diseases,in particular for the treatment of viral infections. In a furtheraspect, provided is an antigen binding molecule as described hereinfor use in the treatment of autoimmune diseases such as for exampleLupus disease.

In one aspect, provided is an antigen binding molecule according tothe invention for use in treating head and neck squamous cellcarcinoma (HNSCC), breast cancer, colorectal cancer (CRC),pancreatic cancer (PAC), gastric cancer, non-small-cell lungcarcinoma (NSCLC) and Mesothelioma, wherein the target cell antigenis TnC.

In a further aspect, the invention relates to the use of an antigenbinding molecule in the manufacture or preparation of a medicamentfor the treatment of a disease in an individual in need thereof. Inone aspect, the medicament is for use in a method of treating adisease comprising administering to an individual having thedisease a therapeutically effective amount of the medicament. Incertain embodiments the disease to be treated is a proliferativedisorder, particularly cancer. Thus, in one aspect, the inventionrelates to the use of an antigen binding molecule of the inventionin the manufacture or preparation of a medicament for the treatmentof cancer. Examples of cancers include solid tumors, bladdercancer, renal cell carcinoma, brain cancer, head and neck cancer,pancreatic cancer, lung cancer, breast cancer, ovarian cancer,uterine cancer, cervical cancer, endometrial cancer, esophagealcancer, colon cancer, colorectal cancer, rectal cancer, gastriccancer, prostate cancer, blood cancer, skin cancer, squamous cellcarcinoma, bone cancer, and kidney cancer, melanoma, B-celllymphoma, B-cell leukemia, non-Hodgkin lymphoma and acutelymphoblastic leukemia. Other cell proliferation disorders that canbe treated using an antigen binding molecule of the presentinvention include, but are not limited to neoplasms located in the:abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicl*s, ovary, thymus, thyroid), eye, head and neck, nervoussystem (central and peripheral), lymphatic system, pelvic, skin,soft tissue, spleen, thoracic region, and urogenital system. Alsoincluded are pre-cancerous conditions or lesions and cancermetastases. In certain embodiments the cancer is chosen from thegroup consisting of renal cell cancer, skin cancer, lung cancer,colorectal cancer, breast cancer, brain cancer, head and neckcancer. A skilled artisan may recognize that in some cases theantigen binding molecule may not provide a cure but may onlyprovide partial benefit. In some aspects, a physiological changehaving some benefit is also considered therapeutically beneficial.Thus, in some aspects, an amount of antigen binding molecule thatprovides a physiological change is considered an "effective amount"or a "therapeutically effective amount".

In a further aspect, the invention relates to the use of an antigenbinding molecule as described herein in the manufacture orpreparation of a medicament for the treatment of infectiousdiseases, in particular for the treatment of viral infections orfor the treatment of autoimmune diseases, for example Lupusdisease.

In a further aspect, the invention provides a method for treating adisease in an individual, comprising administering to saidindividual a therapeutically effective amount of an antigen bindingmolecule of the invention. In one aspect a composition isadministered to said individual, comprising a fusion protein of theinvention in a pharmaceutically acceptable form. In certainaspects, the disease to be treated is a proliferative disorder. Ina particular aspect, the disease is cancer. In another aspect, thedisease is an infectious disease or an autoimmune disease. Incertain aspects, the method further comprises administering to theindividual a therapeutically effective amount of at least oneadditional therapeutic agent, e.g. an anti-cancer agent if thedisease to be treated is cancer. An "individual" according to anyof the above embodiments may be a mammal, preferably a human.

For the prevention or treatment of disease, the appropriate dosageof an antigen binding molecule of the invention (when used alone orin combination with one or more other additional therapeuticagents) will depend on the type of disease to be treated, the routeof administration, the body weight of the patient, the type offusion protein, the severity and course of the disease, whether thefusion protein is administered for preventive or therapeuticpurposes, previous or concurrent therapeutic interventions, thepatient's clinical history and response to the fusion protein, andthe discretion of the attending physician. The practitionerresponsible for administration will, in any event, determine theconcentration of active ingredient(s) in a composition andappropriate dose(s) for the individual subject. Various dosingschedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

The antigen binding molecule is suitably administered to thepatient at one time or over a series of treatments. Depending onthe type and severity of the disease, about 1 .mu.g/kg to 15 mg/kg(e.g., 0.1 mg/kg-10 mg/kg) of antigen binding molecule can be aninitial candidate dosage for administration to the patient,whether, for example, by one or more separate administrations, orby continuous infusion. One typical daily dosage might range fromabout 1 .mu.g/kg to 100 mg/kg or more, depending on the factorsmentioned above. For repeated administrations over several days orlonger, depending on the condition, the treatment would generallybe sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the antigen binding molecule wouldbe in the range from about 0.005 mg/kg to about 10 mg/kg. In otherexamples, a dose may also comprise from about 1 .mu.g/kg bodyweight, about 5 .mu.g/kg body weight, about 10 .mu.g/kg bodyweight, about 50 .mu.g/kg body weight, about 100 .mu.g/kg bodyweight, about 200 .mu.g/kg body weight, about 350 .mu.g/kg bodyweight, about 500 .mu.g/kg body weight, about 1 mg/kg body weight,about 5 mg/kg body weight, about 10 mg/kg body weight, about 50mg/kg body weight, about 100 mg/kg body weight, about 200 mg/kgbody weight, about 350 mg/kg body weight, about 500 mg/kg bodyweight, to about 1000 mg/kg body weight or more per administration,and any range derivable therein. In examples of a derivable rangefrom the numbers listed herein, a range of about 5 mg/kg bodyweight to about 100 mg/kg body weight, about 5 .mu.g/kg body weightto about 500 mg/kg body weight etc., can be administered, based onthe numbers described above. Thus, one or more doses of about 0.5mg/kg, 2.0 mg/kg, 5.0 mg/kg or 10 mg/kg (or any combinationthereof) may be administered to the patient. Such doses may beadministered intermittently, e.g., every week or every three weeks(e.g., such that the patient receives from about two to abouttwenty, or e.g., about six doses of the fusion protein). An initialhigher loading dose, followed by one or more lower doses may beadministered. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventionaltechniques and assays.

The antigen binding molecules of the invention will generally beused in an amount effective to achieve the intended purpose. Foruse to treat or prevent a disease condition, the antigen bindingmolecules of the invention, or pharmaceutical compositions thereof,are administered or applied in a therapeutically effective amount.Determination of a therapeutically effective amount is well withinthe capabilities of those skilled in the art, especially in lightof the detailed disclosure provided herein.

For systemic administration, a therapeutically effective dose canbe estimated initially from in vitro assays, such as cell cultureassays. A dose can then be formulated in animal models to achieve acirculating concentration range that includes the IC.sub.50 asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g.,animal models, using techniques that are well known in the art. Onehaving ordinary skill in the art could readily optimizeadministration to humans based on animal data.

Dosage amount and interval may be adjusted individually to provideplasma levels of the antigen binding molecules which are sufficientto maintain therapeutic effect. Usual patient dosages foradministration by injection range from about 0.1 to 50 mg/kg/day,typically from about 0.5 to 1 mg/kg/day. Therapeutically effectiveplasma levels may be achieved by administering multiple doses eachday. Levels in plasma may be measured, for example, by HPLC.

In cases of local administration or selective uptake, the effectivelocal concentration of the antigen binding molecule may not berelated to plasma concentration. One skilled in the art will beable to optimize therapeutically effective local dosages withoutundue experimentation.

A therapeutically effective dose of the antigen binding moleculesdescribed herein will generally provide therapeutic benefit withoutcausing substantial toxicity. Toxicity and therapeutic efficacy ofa fusion protein can be determined by standard pharmaceuticalprocedures in cell culture or experimental animals. Cell cultureassays and animal studies can be used to determine the LD.sub.50(the dose lethal to 50% of a population) and the ED.sub.50 (thedose therapeutically effective in 50% of a population). The doseratio between toxic and therapeutic effects is the therapeuticindex, which can be expressed as the ratio LD.sub.50/ED.sub.50.Antigen binding molecules that exhibit large therapeutic indicesare preferred. In one embodiment, the antigen binding moleculeaccording to the present invention exhibits a high therapeuticindex. The data obtained from cell culture assays and animalstudies can be used in formulating a range of dosages suitable foruse in humans. The dosage lies preferably within a range ofcirculating concentrations that include the ED.sub.50 with littleor no toxicity. The dosage may vary within this range dependingupon a variety of factors, e.g., the dosage form employed, theroute of administration utilized, the condition of the subject, andthe like. The exact formulation, route of administration and dosagecan be chosen by the individual physician in view of the patient'scondition (see, e.g., Fingl et al., 1975, in: The PharmacologicalBasis of Therapeutics, Ch. 1, p. 1, incorporated herein byreference in its entirety).

The attending physician for patients treated with fusion proteinsof the invention would know how and when to terminate, interrupt,or adjust administration due to toxicity, organ dysfunction, andthe like. Conversely, the attending physician would also know toadjust treatment to higher levels if the clinical response were notadequate (precluding toxicity). The magnitude of an administereddose in the management of the disorder of interest will vary withthe severity of the condition to be treated, with the route ofadministration, and the like. The severity of the condition may,for example, be evaluated, in part, by standard prognosticevaluation methods. Further, the dose and perhaps dose frequencywill also vary according to the age, body weight, and response ofthe individual patient.

Other Agents and Treatments

The antigen binding molecules of the invention may be administeredin combination with one or more other agents in therapy. Forinstance, a fusion protein of the invention may be co-administeredwith at least one additional therapeutic agent. The term"therapeutic agent" encompasses any agent that can be administeredfor treating a symptom or disease in an individual in need of suchtreatment. Such additional therapeutic agent may comprise anyactive ingredients suitable for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. In certain embodiments, an additionaltherapeutic agent is another anti-cancer agent.

Such other agents are suitably present in combination in amountsthat are effective for the purpose intended. The effective amountof such other agents depends on the amount of fusion protein used,the type of disorder or treatment, and other factors discussedabove. The antigen binding molecules of the invention are generallyused in the same dosages and with administration routes asdescribed herein, or about from 1 to 99% of the dosages describedherein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

Such combination therapies noted above encompass combinedadministration (where two or more therapeutic agents are includedin the same or separate compositions), and separate administration,in which case, administration of the antigen binding molecule ofthe invention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/oradjuvant.

Articles of Manufacture

In another aspect of the invention, an article of manufacturecontaining materials useful for the treatment, prevention and/ordiagnosis of the disorders described above is provided. The articleof manufacture comprises a container and a label or package inserton or associated with the container. Suitable containers include,for example, bottles, vials, syringes, IV solution bags, etc. Thecontainers may be formed from a variety of materials such as glassor plastic. The container holds a composition which is by itself orcombined with another composition effective for treating,preventing and/or diagnosing the condition and may have a sterileaccess port (for example the container may be an intravenoussolution bag or a vial having a stopper that is pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an antigen binding molecule of the invention.

The label or package insert indicates that the composition is usedfor treating the condition of choice. Moreover, the article ofmanufacture may comprise (a) a first container with a compositioncontained therein, wherein the composition comprises an antigenbinding molecule of the invention; and (b) a second container witha composition contained therein, wherein the composition comprisesa further cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can beused to treat a particular condition.

Alternatively, or additionally, the article of manufacture mayfurther comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic waterfor injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, and syringes.

TABLE-US-00003 TABLE C (Sequences): SEQ ID NO: Name Sequence 1Nucleotide sequence huTNC Table 3 2 Nucleotide sequence Table 3muTNC 3 Nucleotide sequence Table 3 cynoTNC 4 huTNC Table 3 5 muTNCTable 3 6 cynoTNC Table 3 7 GST huTNC fn5 A1234 BC Table 4 fn6 B 8GST huTNCfn5 mu A124 Table 4 BC hu fn6 B 9 GST TNC hu fn5 B-C fn6 BTable 4 10 GST huTNC fn5 A1234 fn6 B Table 4 11 huTNC A4 B Table 412 huTNC A1 B Table 4 13 Nucleotide sequence Table 6 pRJH33 librarytemplate DP88-4 library 14 Nucleotide sequence Fab Table 7 lightchain V1_5 15 Nucleotide sequence Fab Table 7 heavy chain VH1_69 16Fab light chain Vk1_5 Table 8 17 Fab heavy chain VH1_69 Table 8(DP88) 18 Nucleotide sequence LMB3 Table 9 19 Nucleotide sequenceTable 9 Vk1_5_L3r_S 20 Nucleotide sequence Table 9 Vk1_5_L3r_SY 21Nucleotide sequence Table 9 Vk1_5_L3r_SPY 22 Nucleotide sequenceRJH31 Table 9 23 Nucleotide sequence RJH32 Table 9 24 Nucleotidesequence DP88- Table 9 v4-4 25 Nucleotide sequence DP88- Table 9v4-6 26 Nucleotide sequence DP88- Table 9 v4-8 27 Nucleotidesequence Table 9 fdseqlong 28 Nucleotide sequence Table 10 pRJH53library template of lambda-DP47 library Vl3_19/VH3_23 29 Nucleotidesequence Fab Table 11 light chain Vl3_19 30 Nucleotide sequence FabTable 11 heavy chain VH3_23 31 Fab light chain Vl3_19 Table 12 32Fab heavy chain VH3_23 Table 12 (DP47) 33 Nucleotide sequence Table13 Vl_3_19_L3r_V 34 Nucleotide sequence Table 13 Vl_3_19_L3r_HV 35Nucleotide sequence Table 13 Vl_3_19_L3r_HLV 36 Nucleotide sequenceRJH80 Table 13 37 Nucleotide sequence Table 13 DP47CDR3_ba (mod.)38 Nucleotide sequence DP47- Table 13 v4-4 39 Nucleotide sequenceDP47- Table 13 v4-6 40 Nucleotide sequence DP47- Table 13 v4-8 41Nucleotide sequence Table 14 18D4 VL 42 Nucleotide sequence Table14 18D4 VH 43 Nucleotide sequence Table 14 11C7 VL 44 Nucleotidesequence Table 14 11C7 VH 45 18D4 VL Table 15 46 18D4 VH Table 1547 11C7 VL Table 15 48 11C7 VH Table 15 49 Nucleotide sequenceTable 16 18D4 LCDR1 50 Nucleotide sequence Table 16 18D4 LCDR2 51Nucleotide sequence Table 16 18D4 LCDR3 52 Nucleotide sequenceTable 16 11C7 LCDR1 53 Nucleotide sequence Table 16 11C7 LCDR2 54Nucleotide sequence Table 16 11C7 LCDR3 55 18D4 LCDR1 Table 17 5618D4 LCDR2 Table 17 57 18D4 LCDR3 Table 17 58 11C7 LCDR1 Table 1759 11C7 LCDR2 Table 17 60 11C7 LCDR3 Table 17 61 Nucleotidesequence Table 18 18D4 HCDR1 62 Nucleotide sequence Table 18 18D4HCDR2 63 Nucleotide sequence Table 18 18D4 HCDR3 64 Nucleotidesequence Table 18 11C7 HCDR1 65 Nucleotide sequence Table 18 11C7HCDR2 66 Nucleotide sequence Table 18 11C7 HCDR3 67 18D4 HCDR1Table 19 68 18D4 HCDR2 Table 19 69 18D4 HCDR3 Table 19 70 11C7HCDR1 Table 19 71 11C7 HCDR2 Table 19 72 11C7 HCDR3 Table 19 73Nucleotide sequence Table 20 18D4 Light chain 74 Nucleotidesequence Table 20 18D4 Heavy chain 75 Nucleotide sequence Table 2011C7 Light chain 76 Nucleotide sequence Table 20 11C7 Heavy chain77 18D4 Light chain Table 21 78 18D4 Heavy chain Table 21 79 11C7Light chain Table 21 80 11C7 Heavy chain Table 21 81 Nucleotidesequence Table 22 18D4 Heavy chain PGLALA 82 Nucleotide sequence11C7 Table 22 Heavy chain PGLALA 83 18D4 Heavy chain Table 23PGLALA 84 11C7 Heavy chain PGLALA Table 23 85 Human 4-1BB Fc(kih)Table 27 86 Cynomolgus 4-1BB Fc(kih) Table 27 87 Murine 4-1BBFc(kih) Table 27 88 nucleotide sequence Table 28 Fc hole chain 89nucleotide sequence Table 28 Human 4-1BB Fc(kih) 90 nucleotidesequence Table 28 Cynomolgus 4-1BB Fc(kih) 91 nucleotide sequenceTable 28 Murine 4-1BB Fc(kih) 92 Fc hole chain Table 28 93 human4-1BB antigen Fc Table 28 knob chain

94 Cynomolgus 4-1BB Fc(kih) Table 28 95 Murine 4-1BB Fc(kih) Table28 96 nucleotide sequence Table 29 Human 4-1BB His 97 Human 4-1BBHis Table 29 98 Nucleotide sequence dimeric Table 30 ligand(71-254)-CL* Fc knob chain 99 Nucleotide sequence Table 30monomeric ligand (71-254)- CH1* 100 Nucleotide sequence anti- Table30 TnC(18D4) Fc hole chain 101 Randomized sequence 1 NNNNNNNNNN 102Dimeric ligand (71-254)- Table 31 CL* Fc knob chain 103 Monomericligand (71-254)- Table 31 CH1* 104 anti-TnC(18D4) Fc hole Table 31chain 105 Randomized sequence 2 NNNNNNNNNNN 106 Nucleotide sequencedimeric Table 32 ligand (71-254)-CL Fc knob chain 107 Nucleotidesequence Table 32 monomeric ligand (71-254)- CH1 108 Dimeric ligand(71-254)- Table 33 CL Fc knob chain 109 Monomeric ligand (71-254)-Table 33 CH1 110 Nucleotide sequence anti- Table 34 TnC(18D4) Fchole dimeric ligand (71-254) chain 111 Nucleotide sequence anti-Table 34 TnC(18D4) Fc knob monomeric ligand (71-254) chain 112anti-TnC(18D4) Fc hole Table 35 dimeric ligand (71-254) chain 113anti-TnC(18D4) Fc knob Table 35 monomeric ligand (71-254) chain 114Nucleotide sequence dimeric Table 36 ligand (71-248)-CL* Fc knobchain 115 Nuceotide sequence Table 36 monomeric ligand (71-248)-CH1* 116 Dimeric ligand (71-248)- Table 37 CL* Fc knob chain 117Monomeric ligand (71-248)- Table 37 CH1* 118 Nucleotide sequencedimeric Table 38 ligand (71-248)-CL Fc knob chain 119 Nucleotidesequence Table 38 monomeric ligand (71-248)- CH1 120 Dimeric ligand(71-248)- Table 39 CL Fc knob chain 121 Monomeric ligand (71-248)-Table 39 CH1 122 Nucleotide sequence anti- Table 40 TnC(18D4) Fchole dimeric ligand (71-248) chain 123 Nucleotide sequence anti-Table 40 TnC(18D4) Fc knob monomeric (71-248) ligand 124anti-TnC(18D4) Fc hole Table 41 dimeric ligand (71-248) chain 125anti-TnC(18D4) Fc knob Table 41 monomeric (71-248) ligand 126Nucleotide sequence Fc hole Table 42 dimeric ligand (71-248) chain127 Fc hole dimeric ligand (71- Table 43 248) chain 128 Nucleotidesequence Fc hole Table 44 monomeric ligand (71-248) chain 129Nucleotide sequence anti- Table 44 TnC(18D4) Fc knob dimeric ligand(71-248) chain 130 Fc hole monomeric ligand Table 45 (71-248) chain131 anti-TnC(18D4) Fc knob Table 45 dimeric ligand (71-248) chain132 Nucleotide sequence Fc Table 46 knob monomeric ligand (71- 248)chain 133 Fc knob monomeric ligand Table 47 (71-248) chain 134Nucleotide sequence anti- Table 48 TnC(18D4) Fc hole monomericligand (71-248) chain 135 Nucleotide sequence Fc Table 48 knobdimeric ligand (71- 248) chain 136 anti-TnC(18D4) Fc hole Table 49monomeric ligand (71-248) chain 137 Fc knob dimeric ligand (71-Table 49 248) chain 138 nucleotide sequence DP47 Table 50 Fc-holechain 139 nucleotide sequence DP47 Table 50 light chain 140 DP47Fc-hole chain Table 50 141 DP47 light chain Table 50 142 nucleotidesequence DP47 Table 51 Fc hole chain fused to dimeric hu 4-1BBL(71-254) 143 nucleotide sequence DP47 Table 51 Fc knob chain fusedto monomeric hu 4-1BBL (71- 254) 144 DP47 Fc hole chain fused toTable 51 dimeric hu 4-1BBL (71-254) 145 DP47 Fc knob chain fusedTable 51 to monomeric hu 4-1BBL (71-254) 146 nucleotide sequenceDP47 Table 54 heavy chain (hu IgG1 PGLALA) 147 DP47 heavy chain (huIgG1 Table 54 PGLALA) 148 nucleotide sequence anti- Table 55TnC(18D4) heavy chain (huIgG1 PGLALA) 149 anti-TnC(18D4) heavychain Table 55 (huIgG1 PGLALA) 150 Peptide linker (G4S).sub.2GGGGSGGGGS 151 Peptide linker (SG4).sub.2 SGGGGSGGGG 152 Peptidelinker G4(SG4).sub.2 GGGGSGGGGSGGGG 153 Peptide linker 1 GSPGSSSSGS154 Peptide linker (G4S).sub.4 GGGGSGGGGSGGGGSGGGGS 155 Peptidelinker 2 GSGSGNGS 156 Peptide linker 3 GGSGSGSG 157 Peptide linker4 GGSGSG 158 Peptide linker 5 GGSG 159 Peptide linker 6 GGSGNGSG160 Peptide linker 7 GGNGSGSG 161 Peptide linker 8 GGNGSG 162Peptide linker 9 GGGGS 163 Leader Sequence 1 MDWTWRILFLVAAATGAHS164 Leader Sequence 1 (DNA1) ATGGACTGGACCTGGAGAATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC 165 Leader Sequence 1 (DNA2)ATGGACTGGACCTGGAGGATCCTCTTCTTGGT GGCAGCAGCCACAGGAGCCCACTCC 166Leader Sequence 2 MDMRVPAQLLGLLLLWFPGA 167 Leader Sequence 2 (DNA)ATGGACATGAGGGTCCCCGCTCAGCTCCTGGG CCTCCTGCTGCTCTGGTTCCCAGGTGCCAGGTGT 168 Leader Sequence 3 MGWSCIILFLVATATGVHS 169 Leader Sequence 3(DNA 1) ATGGGATGGAGCTGTATCATCCTCTTCTTGGT AGCAACAGCTACCGGTGTGCATTCG170 Leader Sequence 3 (DNA 2) ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTGGCTACCGCCACTGGAGTGCATTCC 171 Leader Sequence 3 (DNA 3)ATGGGCTGGTCCTGCATCATCCTGTTTCTGGTC GCCACAGCCACCGGCGTGCACTCT 172Human (hu) 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254)DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 173 hu 4-1BBL (85-254)LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA

GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLEL RRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVH LHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 174 hu 4-1BBL (80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAG QRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 175 hu 4-1BBL (52-254)PWAVSGARASPGSAASPRLREGPELSPDDPAGLL DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 176 dimeric hu 4-1BBL (71-254)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI connected by (G4S).sub.2 (SEQDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA ID NO: 150) linkerKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGL FRVTPEIPAGLPSPRSE 177 monomeric hu4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254) plus (G4S).sub.2(SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 150) linkerKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGS 178dimeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254)-CH1Fc knob chain DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRS AAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGL FRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDEL TKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK 179monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254)-CLDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 180 hu 4-1BBL (50-254) ACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGL AGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALA LTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPA GLPSPRSE 181 hu OX40L (51-183)QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 182 hu OX40L (52-183)VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 183 Human (hu) 4-1BBL (71-REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248)DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 184 dimeric hu 4-1BBL (71-248)REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI connected by (G4S).sub.2 (SEQDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA ID NO: 150) linkerKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDP GLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGL 185 dimeric hu 4-1BBL(80-254) DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS connected by (G4S).sub.2(SEQ DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV ID NO: 150) linkerFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGLPSPRSEGGGGSGGGGSDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGG LSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPA SSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 186 dimeric hu 4-1BBL (52-254)PWAVSGARASPGSAASPRLREGPELSPDDPAGLL connected by (G4S).sub.2 (SEQDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAG ID NO: 150) linkerVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSPWAVSGARASPGSAASPR LREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVV AKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQG RLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE 187 monomeric hu 4-1BBL (71-REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 254) plus (G4S).sub.1 (SEQ IDDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 162) linkerKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLPSPRSEGGGGS 188monomeric hu 4-1BBL (71- REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248)plus (G4S).sub.2 (SEQ ID DGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 150)linker KAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGR LLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGS 189 monomeric hu 4-1BBL (71-REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLI 248) plus (G4S).sub.1 (SEQ IDDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVA NO: 162) linkerKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQP LRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATV LGLFRVTPEIPAGLGGGGS 190 dimerichuOX40L (51-183) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI connected by(G4S).sub.2 (SEQ MKVQNNSVIINCDGFYLISLKGYFSQEVNISLHY ID NO: 150)linker QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 191 dimeric huOX40L (52-183)VSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIM connected by (G4S)2 (SEQKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ ID NO: 150) linkerKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLN VTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEF CVL 192 hu 4-1BBL (85-248)LDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLA GVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALT VDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL 193 hu 4-1BBL (80-248)DPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYS DPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGA AALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVT PEIPAGL 194 hu 4-1BBL (52-248)PWAVSGARASPGSAASPRLREGPELSPDDPAGLL DLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELR RVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHL HTEARARHAWQLTQGATVLGLFRVTPEIPAGL195 Dimeric hu OX40L (51-183)- QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEICL* Fc knob chain MKVQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQDNSVIINCDGFYLISLKGYFSQEVDISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSRTVAAPSVFIFPPSDRK LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 196 Monomeric hu OX40L (51-QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEI 183)-CH1*MKVQDNSVIINCDGFYLISLKGYFSQEVDISLHY QKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDEKVEPKSC 197 human TnC UniProtno. P24821 198 human MCSP UniProt no. Q6UVK1 199 human EGFR UniProtno. P00533 200 human CD19 UniProt no. P15391 201 human CD20 Uniprotno. P11836 202 human CD33 UniProt no. P20138 203 human Lymphotoxin.alpha. UniProt no. P01374 204 human TNF UniProt no. P01375 205human Lymphotoxin .beta. UniProt no. Q06643 206 human OX40L UniProtno. P23510 207 human CD40L UniProt no. P29965

208 human FasL UniProt no. P48023 209 human CD27L UniProt no.P32970 210 human CD30L UniProt no. P32971 211 human 4-1BBL UniProtno. P41273 212 human TRAIL UniProt no. P50591 213 human RANKLUniProt no. O14788 214 human TWEAK UniProt no. O43508 215 humanAPRIL UniProt no. O75888 216 human BAFF UniProt no. Q9Y275 217human LIGHT UniProt no. O43557 218 human TL1A UniProt no. O95150219 human GITRL UniProt no. Q9UNG2 220 human ectodysplasin AUniProt no. Q92838

General information regarding the nucleotide sequences of humanimmunoglobulins light and heavy chains is given in: Kabat, E. A.,et al., Sequences of Proteins of Immunological Interest, 5th ed.,Public Health Service, National Institutes of Health, Bethesda, Md.(1991). Amino acids of antibody chains are numbered and referred toaccording to the EU numbering systems according to Kabat (Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5thed., Public Health Service, National Institutes of Health,Bethesda, Md. (1991)) as defined above.

Particular Embodiments

1. A TNF family ligand trimer-containing antigen binding moleculecomprising (a) at least one moiety capable of specific binding toTenascin-C (TnC) and (b) a first and a second polypeptide that arelinked to each other by a disulfide bond, wherein the antigenbinding molecule is characterized in that the first polypeptidecomprises two ectodomains of a TNF ligand family member or afragment thereof that are connected to each other by a peptidelinker and in that the second polypeptide comprises only oneectodomain of said TNF ligand family member or a fragment thereof.2. A TNF family ligand trimer-containing antigen binding moleculecomprising (a) at least one antigen binding moiety capable ofspecific binding to Tenascin-C (TnC) and (b) a first and a secondpolypeptide that are linked to each other by a disulfide bond,wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises two ectodomains of a TNF ligand familymember or a fragment thereof that are connected to each other by apeptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof. 3. The TNF family ligand trimer-containing antigen bindingmolecule of embodiment 1 or 2, further comprising (c) an Fc domaincomposed of a first and a second subunit capable of stableassociation. 4. The TNF family ligand trimer-containing antigenbinding molecule of any one of embodiments 1 to 3, comprising (a)at least one moiety capable of specific binding to TnC and (b) afirst and a second polypeptide that are linked to each other by adisulfide bond, wherein the antigen binding molecule ischaracterized in that (i) the first polypeptide contains a CH1 orCL domain and the second polypeptide contains a CL or CH1 domain,respectively, wherein the second polypeptide is linked to the firstpolypeptide by a disulfide bond between the CH1 and CL domain, andwherein the first polypeptide comprises two ectodomains of a TNFligand family member or fragments thereof that are connected toeach other and to the CH1 or CL domain by a peptide linker andwherein the second polypeptide comprises one ectodomain of said TNFligand family member or a fragment thereof connected via a peptidelinker to the CL or CH1 domain of said polypeptide, or (ii) thefirst polypeptide contains a CH3 domain and the second polypeptidecontains a CH3 domain, respectively, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to theC-terminus of the CH3 domain by a peptide linker and wherein thesecond polypeptide comprises only one ectodomain of said TNF ligandfamily member or a fragment thereof connected via a peptide linkerto C-terminus of the CH3 domain of said polypeptide, or (iii) thefirst polypeptide contains a VH-CL or a VL-CH1 domain and thesecond polypeptide contains a VL-CH1 domain or a VH-CL domain,respectively, wherein the second polypeptide is linked to the firstpolypeptide by a disulfide bond between the CH1 and CL domain, andwherein the first polypeptide comprises two ectodomains of a TNFligand family member or fragments thereof that are connected toeach other and to to VH or VL by a peptide linker and wherein thesecond polypeptide comprises one ectodomain of said TNF ligandfamily member or a fragment thereof connected via a peptide linkerto VL or VH of said polypeptide. 5. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 4, wherein the TNF ligand family membercostimulates human T-cell activation. 6. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 5, wherein the TNF ligand family member isselected from 4-1BBL and OX40L. 7. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 6, wherein the TNF ligand family member is 4-1BBL.8. The TNF family ligand trimer-containing antigen binding moleculeof any one of embodiments 1 to 7, wherein the ectodomain of a TNFligand family member comprises the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 172, SEQ ID NO: 173, SEQ IDNO: 174, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 192, SEQ ID NO:193 and SEQ ID NO: 194, particularly the amino acid sequence of SEQID NO: 172 or SEQ ID NO: 183. 9. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 8, wherein the ectodomain of a TNF ligand familymember comprises the amino acid sequence of SEQ ID NO: 183. 10. TheTNF family ligand trimer-containing antigen binding molecule of anyone of embodiments 1 to 9, comprising (a) at least one moietycapable of specific binding to TnC and (b) a first and a secondpolypeptide that are linked to each other by a disulfide bond,wherein the antigen binding molecule is characterized in that thefirst polypeptide comprises the amino acid sequence selected fromthe group consisting of SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO:185 and SEQ ID NO: 186 and in that the second polypeptide comprisesthe amino acid sequence selected from the group consisting of SEQID NO: 172, SEQ ID NO: 174, SEQ ID NO: 175 and SEQ ID NO: 183. 11.The TNF family ligand trimer-containing antigen binding molecule ofany one of embodiments 1 to 10, comprising (a) at least one moietycapable of specific binding to TnC, and (b) a first polypeptidecontaining a CH1 or CL domain and a second polypeptide containing aCL or CH1 domain, respectively, wherein the second polypeptide islinked to the first polypeptide by a disulfide bond between the CH1and CL domain, and wherein the antigen binding molecule ischaracterized in that the first polypeptide comprises twoectodomains of a TNF ligand family member or fragments thereof thatare connected to each other and to the CH1 or CL domain by apeptide linker and in that the second polypeptide comprises onlyone ectodomain of said TNF ligand family member or a fragmentthereof connected via a peptide linker to the CL or CH1 domain ofsaid polypeptide. 12. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 11, whereinthe moiety capable of specific binding to TnC is selected from thegroup consisting of an antibody fragment, a Fab molecule, acrossover Fab molecule, a single chain Fab molecule, a Fv molecule,a scFv molecule, a single domain antibody, an aVH and a scaffoldantigen binding protein. 13. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 12, wherein the molecule comprises one or twomoieties capable of specific binding to TnC. 14. The TNF familyligand trimer-containing antigen binding molecule of any one ofembodiments 1 to 13, wherein the moiety capable of specific bindingto TnC is a Fab molecule capable of specific binding to TnC. 15.The TNF family ligand trimer-containing antigen binding molecule ofany one of embodiments 1 to 14, wherein the moiety capable ofspecific binding to TnC comprises a VH domain comprising (i) CDR-H1comprising the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO:70, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 68or SEQ ID NO: 71, and (iii) CDR-H3 comprising the amino acidsequence of SEQ ID NO: 69 or SEQ ID NO: 72, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO: 55 or SEQ ID NO: 58, (v) CDR-L2 comprising the amino acidsequence of SEQ ID NO: 56 or SEQ ID NO: 59, and (vi) CDR-L3comprising the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO:60. 16. The TNF family ligand trimer-containing antigen bindingmolecule of any one of embodiments 1 to 15, wherein the moietycapable of specific binding to TnC comprises a variable heavy chaincomprising an amino acid sequence of SEQ ID NO: 46 and a variablelight chain comprising an amino acid sequence of SEQ ID NO: 45 orwherein the moiety capable of specific binding to TNC comprises avariable heavy chain comprising an amino acid sequence of SEQ IDNO: 48 and a variable light chain comprising an amino acid sequenceof SEQ ID NO: 47. 17. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 3 to 16, whereinthe Fc domain is an IgG, particularly an IgG1 Fc domain or an IgG4Fc domain. 18. The TNF family ligand trimer-containing antigenbinding molecule of any one of embodiments 3 to 17, wherein the Fcdomain is an IgG1 Fc domain comprising the amino acid substitutionsat positions 234 and 235 (EU numbering) and/or 329 (EU numbering).19. The TNF family ligand trimer-containing antigen bindingmolecule of any one of embodiments 1 to 18, wherein the antigenbinding molecule comprises a first heavy chain and a first lightchain, both comprising a Fab molecule capable of specific bindingto TnC, a first peptide comprising two ectodomains of a TNF ligandfamily member or fragments thereof connected to each other by afirst peptide linker fused at its C-terminus by a second peptidelinker to a second heavy or light chain, and a second peptidecomprising one ectodomain of said TNF ligand family member fused atit* C-terminus by a third peptide linker to a second light or heavychain, respectively. 20. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 19, whereinthe first peptide comprising two ectodomains of a TNF ligand familymember or fragments thereof connected to each other by a firstpeptide linker is fused at its C-terminus by a second peptidelinker to a CH1 domain that is part of a heavy chain, and thesecond peptide comprising one ectodomain of said TNF ligand familymember or a fragment thereof is fused at its C-terminus by a thirdpeptide linker to a CL domain that is part of a light chain. 21.The TNF family ligand trimer-containing antigen binding molecule ofany one of embodiments 1 to 20, wherein the first peptidecomprising two ectodomains of a TNF ligand family member orfragments thereof connected to each other by a first peptide linkeris fused at its C-terminus by a second peptide linker to a CLdomain that is part of a heavy chain, and the second peptidecomprising one ectodomain of said TNF ligand family member or afragment thereof is fused at its C-terminus by a third peptidelinker to a CH1 domain that is part of a light chain. 22. The TNFfamily ligand trimer-containing antigen binding molecule of any oneof embodiments 1 to 21, wherein the first peptide comprising twoectodomains of a TNF ligand family member or fragments thereofconnected to each other by a first peptide linker is fused at itsC-terminus by a second peptide linker to a VH domain that is partof a heavy chain, and the second peptide comprising one ectodomainof said TNF ligand family member or a fragment thereof is fused atit* C-terminus by a third peptide linker to a VL domain that ispart of a light chain. 23. The TNF family ligand trimer-containingantigen binding molecule of embodiments 20 or 21, wherein in the CLdomain adjacent to the TNF ligand family member the amino acid atposition 123 (EU numbering) has been replaced by arginine (R) andthe amino acid at position 124 (EU numbering) has been substitutedby lysine (K), and wherein in the CH1 domain adjacent to the TNFligand family member the amino acids at position 147 (EU numbering)and at position 213 (EU numbering) have been substituted byglutamic acid (E). 24. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 23, whereinthe antigen binding molecule comprises (a) a first heavy chain anda first light chain, both comprising a Fab molecule capable ofspecific binding to TnC, (b) a second heavy chain comprising anamino acid sequence selected from the group consisting of SEQ IDNO: 176, SEQ ID NO: 184, SEQ ID NO: 185 and SEQ ID NO: 186, and asecond light chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO:175 and SEQ ID NO: 183. 25. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 20, whereinthe antigen binding molecule comprises (i) a first heavy chaincomprising the VH domain comprising the amino acid sequence of SEQID NO:46 and a first light chain comprising the VL domaincomprising the amino acid sequence of SEQ ID NO: 45 or a firstheavy chain comprising the VH domain comprising the amino acidsequence of SEQ ID NO: 48 and a first light chain comprising the VLdomain comprising the amino acid sequence of SEQ ID NO: 47, (ii) asecond heavy chain comprising the amino acid sequence of SEQ ID NO:178, and (iii) a second light chain comprising the amino acidsequence of SEQ ID NO: 179. 26. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 19 and 21, wherein the antigen binding moleculecomprises (i) a first heavy chain comprising the VH domaincomprising the amino acid sequence of SEQ ID NO: 46 and a firstlight chain comprising the VL domain comprising the amino acidsequence of SEQ ID NO: 45 or a first heavy chain comprising the VHdomain comprising the amino acid sequence of SEQ ID NO: 48 and afirst light chain comprising the VL domain comprising the aminoacid sequence of SEQ ID NO: 47, (ii) a second heavy chaincomprising the amino acid sequence selected from the groupconsisting of SEQ ID NO: 102, SEQ ID NO: 108, SEQ ID NO: 116 andSEQ ID NO: 120, and (iii) a second light chain comprising the aminoacid sequence selected from the group consisting of SEQ ID NO: 103,SEQ ID NO: 109, SEQ ID NO: 117 and SEQ ID NO: 121. 27. The TNFfamily ligand trimer-containing antigen binding molecule of any oneof embodiments 1 to 18, comprising (a) at least one moiety capableof specific binding TnC, and (b) a first and a second polypeptidethat are linked to each other by a disulfide bond, wherein theantigen binding molecule is characterized in that the firstpolypeptide contains a CH3 domain and the second polypeptidecontains a CH3 domain, respectively, and wherein the firstpolypeptide comprises two ectodomains of a TNF ligand family memberor fragments thereof that are connected to each other and to theC-terminus of the CH3 domain by a peptide linker and wherein thesecond polypeptide comprises one ectodomain of said TNF ligandfamily member or a fragment thereof connected via a peptide linkerto C-terminus of the CH3 domain of said polypeptide. 28. The TNFfamily ligand trimer-containing antigen binding molecule ofembodiment 27, wherein the antigen binding molecule comprises (i) afirst heavy chain comprising the amino acid sequence of SEQ ID NO:127, a second heavy chain comprising the amino acid sequence of SEQID NO: 125, and one light chain comprising the amino acid sequenceof SEQ ID NO: 77, or (ii) a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 130, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO: 131, and one light chaincomprising the amino acid sequence of SEQ ID NO: 77, or (ii) afirst heavy chain comprising the amino acid sequence of SEQ ID NO:124, a second heavy chain comprising the amino acid sequence of SEQID NO: 133, and one light chain comprising the amino acid sequenceof SEQ ID NO: 77, or (ii) a first heavy chain comprising the aminoacid sequence of SEQ ID NO: 136, a second heavy chain comprisingthe amino acid sequence of SEQ ID NO: 137, and one light chaincomprising the amino acid sequence of SEQ ID NO: 77. 29. The TNFfamily ligand trimer-containing antigen binding molecule ofembodiment 27, comprising two moieties capable of specific bindingto a target cell antigen. 30. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 27 or 29, wherein the antigen binding moleculecomprises (i) a first heavy chain comprising the amino acidsequence of SEQ ID NO: 112, a second heavy chain comprising theamino acid sequence of SEQ ID NO: 113, and two light chainscomprising the amino acid sequence of SEQ ID NO: 77, or (ii) afirst heavy chain comprising the amino acid sequence of SEQ ID NO:124, a second heavy chain comprising the amino acid sequence of SEQID NO: 125, and two light chains comprising the amino acid sequenceof SEQ ID NO: 77. 31. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 6, 11 to 23and 27 to 29, wherein the TNF ligand family member is OX40L. 32.The TNF family ligand trimer-containing antigen binding molecule ofany one of embodiments 1 to 6, 11 to 23 and 27 to 29, wherein theectodomain of a TNF ligand family member comprises the amino acidsequence of SEQ ID NO: 181 or SEQ ID NO: 182, particularly theamino acid sequence of SEQ ID NO: 181. 33. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 6, 11 to 23, 27 to 29, 31 and 32, comprising (a)at least one moiety capable of specific binding to a target cellantigen and (b) a first and a second polypeptide that are linked toeach other by a disulfide bond, wherein the antigen bindingmolecule is characterized in that the first polypeptide comprisesthe amino acid sequence of SEQ ID NO: 190 or SEQ ID: 191 and inthat the second polypeptide comprises the amino acid sequence ofSEQ ID NO: 181 or SEQ ID NO: 182. 34. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33, wherein thetarget cell antigen is Tenascin-C (TnC) and the moiety capable ofspecific binding to TnC comprises a VH domain comprising (i) CDR-H1comprising the amino acid

sequence of SEQ ID NO: 67 or SEQ ID NO: 70, (ii) CDR-H2 comprisingthe amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 71, and(iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 69 orSEQ ID NO: 72, and a VL domain comprising (iv) CDR-L1 comprisingthe amino acid sequence of SEQ ID NO: 55 or SEQ ID NO: 58, (v)CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 or SEQID NO: 59, and (vi) CDR-L3 comprising the amino acid sequence ofSEQ ID NO: 57 or SEQ ID NO: 60. 35. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33, wherein theantigen binding moiety capable of specific binding to TnC comprisesa VH domain comprising (i) CDR-H1 comprising the amino acidsequence of SEQ ID NO: 67, (ii) CDR-H2 comprising the amino acidsequence of SEQ ID NO: 68, and (iii) CDR-H3 comprising the aminoacid sequence of SEQ ID NO: 69, and a VL domain comprising (iv)CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, (v)CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and(vi) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57.36. TNF family ligand trimer-containing antigen binding molecule ofany one of embodiments 1 to 6, 11 to 23, 27 to 29 and 31 to 33,wherein the antigen binding moiety capable of specific binding toTnC comprises a VH domain comprising (i) CDR-H1 comprising theamino acid sequence of SEQ ID NO: 70, (ii) CDR-H2 comprising theamino acid sequence of SEQ ID NO: 71, and (iii) CDR-H3 comprisingthe amino acid sequence of SEQ ID NO: 72, and a VL domaincomprising (iv) CDR-L1 comprising the amino acid sequence of SEQ IDNO: 58, (v) CDR-L2 comprising the amino acid sequence of SEQ ID NO:59, and (vi) CDR-L3 comprising the amino acid sequence of SEQ IDNO: 60. 37. The TNF family ligand trimer-containing antigen bindingmolecule of any one of embodiments 1 to 6, 11 to 23, 27 to 29 and31 to 36, wherein the antigen binding molecule comprises (i) afirst heavy chain comprising the VH domain comprising the aminoacid sequence of SEQ ID NO: 46 and a first light chain comprisingthe VL domain comprising the amino acid sequence of SEQ ID NO: 45or a first heavy chain comprising the VH domain comprising theamino acid sequence of SEQ ID NO: 48 and a first light chaincomprising the VL domain comprising the amino acid sequence of SEQID NO: 47, (ii) a second heavy chain comprising the amino acidsequence selected from the group consisting of SEQ ID NO: 195, and(iii) a second light chain comprising the amino acid sequence ofSEQ ID NO: 196. 38. The TNF family ligand trimer-containing antigenbinding molecule of any one of embodiments 1 to 37, wherein theantigen binding moiety has an improved affinity. 39. The TNF familyligand trimer-containing antigen binding molecule of any one ofembodiments 1 to 38, wherein the antigen binding moiety binds tohuman TnC with a K.sub.D value lower than about 1 nM. 40. The TNFfamily ligand trimer-containing antigen binding molecule of any oneof embodiments 1 to 39, wherein the antigen binding moiety hascross-species reactivity. 41. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 40, wherein the antigen binding moiety binds to atleast one of human, mouse and cynomolgus TnC with a K.sub.D valuelower than about 2 nM. 42. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 41, whereinthe antigen binding moiety binds to human, mouse and cynomolgusTnC. 43. The TNF family ligand trimer-containing antigen bindingmolecule of any one of embodiments 40 to 42, wherein the antigenbinding moiety binds to the target antigen from all indicatedspecies with similar affinity. 44. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 40 to 43, wherein the antigen binding moiety binds tothe target antigen from all indicated species with similaraffinity, in particular within a K.sub.D range of a factor of 100,within a K.sub.D range of a factor of 50, within a K.sub.D range ofa factor of 20, within a K.sub.D range of a factor of 10, within aK.sub.D range of a factor of 5. 45. The TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 40 to 44, wherein the antigen binding moiety binds tothe target antigen from all indicated species with similar affinitywithin a K.sub.D range of a factor of 10. 46. An isolatedpolynucleotide encoding the TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 45. 47. Avector, particularly an expression vector, comprising the isolatedpolynucleotide of embodiment 46. 48. A host cell comprising theisolated polynucleotide of embodiment 46 or the vector ofembodiment 47. 49. A method for producing the TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 45, comprising the steps of (i) culturing the hostcell of embodiment 48 under conditions suitable for expression ofthe antigen binding molecule, and (ii) recovering the antigenbinding molecule. 50. A pharmaceutical composition comprising theTNF family ligand trimer-containing antigen binding molecule of anyone of embodiments 1 to 45 and at least one pharmaceuticallyacceptable excipient. 51. The TNF family ligand trimer-containingantigen binding molecule of any one of embodiments 1 to 45, or thepharmaceutical composition of embodiment 50, for use as amedicament. 52. The TNF family ligand trimer-containing antigenbinding molecule of any one of embodiments 1 to 45, or thepharmaceutical composition of embodiment 50, for use in thetreatment of cancer. 53. Use of the TNF family ligandtrimer-containing antigen binding molecule of any one ofembodiments 1 to 45 for the manufacture of a medicament for thetreatment of cancer. 54. A method of treating a disease in anindividual, comprising administering to said individual atherapeutically effective amount of a composition comprising a TNFfamily ligand trimer-containing antigen binding molecule of any oneof embodiments 1 to 45 in a pharmaceutically acceptable form. 55.The method of embodiment 54, wherein said disease is cancer.

EXAMPLES

The following are examples of methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described inSambrook, J. et al., Molecular cloning: A laboratory manual; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Themolecular biological reagents were used according to themanufacturer's instructions. DNA sequences were determined bydouble strand sequencing. In some cases desired gene segments wereprepared by Geneart AG (Regensburg, Germany) from syntheticoligonucleotides and PCR products by automated gene synthesis. Thegene segments which are flanked by singular restrictionendonuclease cleavage sites were cloned into indicated plasmids.The plasmid DNA was purified from transformed bacteria andconcentration determined by UV spectroscopy. The DNA sequence ofthe subcloned gene fragments was confirmed by DNA sequencing. GeneSegments were designed with suitable restriction sites to allowsub-cloning into the respective expression vectors.

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A. etal., (1991) Sequences of Proteins of Immunological Interest, FifthEd., NIH Publication No 91-3242. For expression, all constructscontained a 5'-end DNA sequence coding for a leader peptide whichtargets proteins for secretion in eukaryotic cells. Exemplaryleader peptides and polynucleotide sequences encoding them aregiven in SEQ ID NO 163 to SEQ ID NO 171.

Cell Culture Techniques

Standard cell culture techniques were used as described in CurrentProtocols in Cell Biology (2000), Bonifacino, J. S., Dasso, M.,Harford, J. B., Lippincott-Schwartz, J. and Yamada, K. M. (eds.),John Wiley & Sons, Inc.

Protein Purification

Proteins were purified from filtered cell culture supernatantsreferring to standard protocols. In brief, antibodies were appliedto a Protein A Sepharose column (GE healthcare) and washed withPBS. Elution of antibodies was achieved at pH 2.8 followed byimmediate neutralization of the sample. Aggregated protein wasseparated from monomeric antibodies by size exclusionchromatography (Superdex 200, GE Healthcare) in PBS or in 20 mMHistidine, 150 mM NaCl pH 6.0. Monomeric antibody fractions werepooled, concentrated (if required) using e.g., a MILLIPORE AmiconUltra (30 MWCO) centrifugal concentrator, frozen and stored at-20.degree. C. or -80.degree. C. Part of the samples were providedfor subsequent protein analytics and analytical characterizatione.g. by SDS-PAGE, size exclusion chromatography (SEC) or massspectrometry.

SDS-PAGE

The NuPAGE.RTM. Pre-Cast gel system (Invitrogen) was used accordingto the manufacturer's instruction. In particular, 10% or 4-12%NuPAGE.RTM. Novex.RTM. Bis-TRIS Pre-Cast gels (pH 6.4) and aNuPAGE.RTM. MES (reduced gels, with NuPAGE.RTM. Antioxidant runningbuffer additive) or MOPS (non-reduced gels) running buffer wasused.

Analytical Size Exclusion Chromatography

Size exclusion chromatography (SEC) for the determination of theaggregation and oligomeric state of antibodies was performed byHPLC chromatography. Briefly, Protein A purified antibodies wereapplied to a Tosoh TSKgel G3000SW column in 300 mM NaCl, 50 mMKH.sub.2PO.sub.4/K.sub.2HPO.sub.4, pH 7.5 on an Agilent HPLC 1100system or to a Superdex 200 column (GE Healthcare) in 2.times.PBSon a Dionex HPLC-System. The eluted protein was quantified by UVabsorbance and integration of peak areas. BioRad Gel FiltrationStandard 151-1901 served as a standard.

Mass Spectrometry

This section describes the characterization of the multispecificantibodies with VH/VL exchange (VH/VL CrossMabs) with emphasis ontheir correct assembly. The expected primary structures wereanalyzed by electrospray ionization mass spectrometry (ESI-MS) ofthe deglycosylated intact CrossMabs and deglycosylated/plasmindigested or alternatively deglycosylated/limited LysC digestedCrossMabs.

The VH/VL CrossMabs were deglycosylated with N-Glycosidase F in aphosphate or Tris buffer at 37.degree. C. for up to 17 h at aprotein concentration of 1 mg/ml. The plasmin or limited LysC(Roche) digestions were performed with 100 .mu.g deglycosylatedVH/VL CrossMabs in a Tris buffer pH 8 at room temperature for 120hours and at 37.degree. C. for 40 min, respectively. Prior to massspectrometry the samples were desalted via HPLC on a Sephadex G25column (GE Healthcare). The total mass was determined via ESI-MS ona maXis 4G UHR-QTOF MS system (Bruker Daltonik) equipped with aTriVersa NanoMate source (Advion).

Determination of Binding and Binding Affinity of MultispecificAntibodies to the Respective Antigens Using Surface PlasmonResonance (SPR) (BIACORE)

Binding of the generated antibodies to the respective antigens isinvestigated by surface plasmon resonance using a BIACOREinstrument (GE Healthcare Biosciences AB, Uppsala, Sweden).Briefly, for affinity measurements Goat-Anti-Human IgG, JIR109-005-098 antibodies are immobilized on a CMS chip via aminecoupling for presentation of the antibodies against the respectiveantigen. Binding is measured in HBS buffer (HBS-P (10 mM HEPES, 150mM NaCl, 0.005% Tween 20, ph 7.4), 25.degree. C. (or alternativelyat 37.degree. C.). Antigen (R&D Systems or in house purified)was added in various concentrations in solution. Association wasmeasured by an antigen injection of 80 seconds to 3 minutes;dissociation was measured by washing the chip surface with HBSbuffer for 3-10 minutes and a KD value was estimated using a 1:1Langmuir binding model. Negative control data (e.g. buffer curves)are subtracted from sample curves for correction of systemintrinsic baseline drift and for noise signal reduction. Therespective Biacore Evaluation Software is used for analysis ofsensorgrams and for calculation of affinity data.

Example 1

TnC Antigen Sequences and Production of Antigens

All constructs of Table 3 and Table 4 are fused to the C-term ofGST and are expressed in E. coli BL21(DE3). For site specificbiotinylation, the Avi-tag was added to the C-term of the Tenascinsequence, and the BirA biotin ligase was coexpressed on a separateplasmid (Avidity, Colo., USA). Growth medium was 2YT with 100.mu.g/ml ampicillin and 20 .mu.g/ml chloramphenicol. Biotin wasadded to a final concentration of 50 .mu.M. Protein Expression wasinduced with 1 mM IPTG at 22.degree. C. overnight. Cells wereharvested by centrifugation, and cell-lysis was performed bysonication in the presence of B-PER reagent (pierce 78260), and 1mg/ml lysozyme (Sigma L6876). Lysate was centrifuged and clearedlysate was loaded on Glutathione Sepharose columns (GE Healthcare;Product No 17-0756-01). After washing, the TnC molecules were cleftfrom the GST via Thrombin (Sigma Aldrich; Product No 10602400001)over night at 4.degree. C. Elution was performed in 50 mM Trisbuffer pH 8.0; 200 mM NaCl, 5 mM MgCl2, 1 mM DTT and 10% glycerol.The final purification step was on a gelfiltration column (Superdex75 16/60; GE Healthcare). Samples were flash frozen in liquidnitrogen until processing.

TABLE-US-00004 TABLE 3 Sequences of TnC antigens used forcross-species affinity determination (reference Table 26) AntigenSequence SEQ ID NO huTNC ATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCT SEQID NO: 1 TGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGTCCATGGGATATCGGGGATCCGAGCTGGACACCCCCAAGGACCTGCAGGTGTCCGAGACAGCCGAGACAAGCCTGACCCTGCTGTGGAAAACCCCCCTGGCCAAGTTCGACCGGTACAGACTGAACTACAGCCTGCCCACTGGACAGTGGGTCGGCGTGCAGCTGCCCCGGAACACCACCTCCTACGTGCTGCGGGGCCTGGAACCCGGCCAGGAATACAACGTCCTGCTGACGGCCGAGAAGGGCCGGCACAAGAGCAAGCCCGCCAGAGTGAAGGCCAGCACCGAGCAGGCCCCCGAGCTGGAAAACCTGACCGTGACCGAAGTGGGCTGGGACGGCCTGCGGCTGAACTGGACCGCGGCTGACCAGGCCTATGAGCACTTTATCATTCAGGTGCAGGAGGCCAACAAGGTGGAGGCAGCTCGGAACCTCACCGTGCCTGGCAGCCTTCGGGCTGTGGACATACCGGGCCTCAAGGCTGCTACGCCTTATACAGTCTCCATCTATGGGGTGATCCAGGGCTATAGAACACCAGTGCTCTCTGCTGAGGCCTCCACAGGCGAAACACCGAACCTGGGCGAAGTGGTGGTGGCGGAAGTGGGTTGGGATGCGCTGAAACTGAACTGGACCGCGCCGGAAGGCGCGTATGAATATTTTTTCATCCAGGTGCAGGAAGCGGATACCGTTGAAGCGGCGCAGAACCTGACCGTTCCGGGCGGTCTGCGTAGCACCGATCTGCCGGGCCTGAAAGCGGCGACCCATTATACCATTACCATCCGTGGGGTGACCCAGGACTTCTCTACCACCCCTCTGAGCGTGGAGGTGCTGACCGAGGAGGTACCCGACATGGGCAACCTGACCGTGACCGAGGTGTCCTGGGACGCCCTGCGGCTGAACTGGACCACCCCCGACGGCACCTACGACCAGTTCACAATCCAGGTGCAGGAAGCCGACCAGGTGGAAGAAGCACATAATCTGACCGTTCCGGGTAGCCTGCGTAGCATGGAAATTCCGGGTCTGCGTGCAGGCACCCCGTATACCGTTACCCTGCATGGTGAAGTTCGTGGTCATAGCACCCGTCCGCTGGCAGTTGAAGTTGTTACCGAAGATCTGCCGCAGCTGGGTGATCTGGCAGTTAGCGAAGTTGGTTGGGATGGTCTGCGTCTGAATTGGACCGCAGCAGATAATGCATATGAACATTTTGTGATCCAGGTGCAAGAGGTGAATAAAGTTGAAGCAGCCCAGAATCTGACCCTGCCTGGTTCACTGCGTGCAGTTGATATTCCGGGACTCGAGGCAGCAACCCCGTATCGTGTTAGCATTTATGGTGTTATTCGCGGTTATCGTACACCGGTTCTGAGCGCAGAAGCAAGCACCGCAAAAGAACCGGAAATTGGTAATCTGAACGTGAGCGATATTACACCGGAATCATTTAATCTGAGCTGGATGGCAACCGATGGTATTTTTGAAACCTTTACCATCGAGATCATCGATAGCAATCGTCTGCTGGAAACCGTGGAATATAATATTAGCGGTGCAGAACGTACCGCACATATTAGCGGTCTGCCTCCGAGCACCGATTTTATTGTTTATCTGAGCGGTCTGGCACCGAGCATTCGTACCAAAACCATTAGCGCAACCGCAACCACCGAAGCACTGCCGCTGCTGGAAAATCTGACCATTAGCGATATTAACCCGTATGGTTTTACCGTTTCATGGATGGCAAGCGAAAATGCATTTGATAGCTTTCTGGTTACAGTTGTGGATAGCGGTAAACTGCTGGACCCGCAAGAATTTACCCTGAGCGGCACCCAGCGCAAACTGGAACTGCGTGGTCTGATTACCGGTATTGGTTATGAAGTTATGGTGAGCGGTTTTACCCAGGGTCATCAGACCAAACCGCTGCGTGCAGAAATTGTTACCGAAGCAATGGGTAGCCCGAAAGAAGTTATTTTTTCCGATATCACCGAGAATTCGGCAACCGTTAGCTGGCGTGCACCGACCGCACAGGTTGAAAGCTTTCGTATTACCTATGTTCCGATTACCGGTGGCACCCCGAGCATGGTTACAGTTGATGGCACCAAAACCCAGACCCGTCTGGTTAAACTGATTCCGGGTGTTGAATATCTGGTTAGCATTATTGCCATGAAAGGCTTTGAAGAAAGCGAACCGGTTAGCGGTAGCTTTACCACAGCTAGCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAGGTACCCATCACCATCACC ACCACTAA muTNCTATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCC SEQ ID NO: 2TTGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGTCCATGGGATATCGGGGATCCGAGCTGGACACCCCCAAGGACCTGCAGGTGTCCGAGACAGCCGAGACAAGCCTGACCCTGCTGTGGAAAACCCCCCTGGCCAAGTTCGACCGGTACAGACTGAACTACAGCCTGCCCACTGGACAGTGGGTCGGCGTGCAGCTGCCCCGGAACACCACCTCCTACGTGCTGCGGGGCCTGGAACCCGGCCAGGAATACAACGTCCTGCTGACGGCCGAGAAGGGCCGGCACAAGAGCAAGCCCGCCAGAGTGAAGGCCAGCACCGAGGAAGTGCCCAGCCTGGAAAACCTGACCGTGACCGAGGCCGGCTGGGACGGCCTGCGGCTGAACTGGACCGCCGACGACCTGGCCTACGAGTACTTCGTGATCCAGGTGCAGGAAGCCAACAACGTCGAGACAGCCCACAACTTCACCGTGCCCGGCAACCTGAGAGCCGCCGACATCCCCGGCCTGAAGGTGGCCACATCCTACCGGGTGTCCATCTACGGCGTGGCCAGGGGCTACCGGACCCCCGTGCTGTCCGCCGAGACAAGCACCGGCACCACGCCGAACCTGGGCGAAGTGACCGTGGCGGAAGTGGGTTGGGATGCGCTGACCCTGAATTGGACCGCACCGGAAGGCGCGTATAAAAACTTTTTCATCCAGGTGCTGGAAGCGGATACCACCCAGACCGTGCAGAACCTGACCGTGCCGGGTGGTCTGCGTAGCGTAGATCTGCCTGGTCTGAAAGCAGCAACCCGCTATTACATTACCCTGCGTGGTGTTACCCAGGATTTTGGCACCGCACCGCTGAGCGTTGAAGTTCTGACCGAGGATCTGCCGCAGCTGGGTGGTCTGAGCGTTACCGAAGTTAGTTGGGATGGTCTGACCCTGAATTGGACCACCGATGATCTGGCATATAAACATTTTGTGGTGCAGGTTCAAGAGGCCAATAATGTTGAAGCAGCACAGAATCTGACCGTTCCGGGTAGCCTGCGTGCAGTTGATATTCCGGGACTGAAAGCCGATACCCCGTATCGTGTTAGCATTTATGGTGTTATTCAGGGTTATCGTACCCCGATGCTGAGCACCGATGTTAGCACAGCACGTGAACCGGAAATTGGTAATCTGAATGTTAGTGATGTGACCCCGAAATCATTTAATCTGAGCTGGACCGCAACCGATGGTATTTTTGATATGTTTACCATTGAAATTATTGATAGCAATCGCCTGCTGCAGACCGCAGAACATAACATTAGCGGTGCAGAACGTACCGCACATATTAGCGGTCTGCCTCCGAGCACCGATTTTATTGTTTATCTGAGCGGTATTGCACCGAGCATTCGTACCAAAACCATTAGCACCACCGCAACCACCGAAGCACTGACCGCAATGGGTAGCCCGAAAGAAGTGATTTTTAGCGATATTACCGAAAATAGCGCCACCGTTTCATGGCGTGCACCGACCGCACAGGTTGAAAGCTTTCGTATTACCTATGTTCCGATTACCGGTGGCACCCCGAGCATGGTTACCGTTGATGGCACCAAAACCCAGACCCGTCTGGTTAAACTGATTCCGGGTGTTGAATATCTGGTTAGCATTATTGCCATGAAAGGCTTTGAAGAAAGCGAACCGGTTAGCGGTAGCTTTACCACAGCTAGCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAGGTACCCATCACCATCACCACC ACTAA cynoTNCATGTCCCCTATACTAGGTTATTGGAAAATTAAGGGCCT SEQ ID NO: 3TGTGCAACCCACTCGACTTCTTTTGGAATATCTTGAAGAAAAATATGAAGAGCATTTGTATGAGCGCGATGAAGGTGATAAATGGCGAAACAAAAAGTTTGAATTGGGTTTGGAGTTTCCCAATCTTCCTTATTATATTGATGGTGATGTTAAATTAACACAGTCTATGGCCATCATACGTTATATAGCTGACAAGCACAACATGTTGGGTGGTTGTCCAAAAGAGCGTGCAGAGATTTCAATGCTTGAAGGAGCGGTTTTGGATATTAGATACGGTGTTTCGAGAATTGCATATAGTAAAGACTTTGAAACTCTCAAAGTTGATTTTCTTAGCAAGCTACCTGAAATGCTGAAAATGTTCGAAGATCGTTTATGTCATAAAACATATTTAAATGGTGATCATGTAACCCATCCTGACTTCATGTTGTATGACGCTCTTGATGTTGTTTTATACATGGACCCAATGTGCCTGGATGCGTTCCCAAAATTAGTTTGTTTTAAAAAACGTATTGAAGCTATCCCACAAATTGATAAGTACTTGAAATCCAGCAAGTATATAGCATGGCCTTTGCAGGGCTGGCAAGCCACGTTTGGTGGTGGCGACCATCCTCCAAAATCGGATGGTTCAACTAGTGGTTCTGGTCATCACCATCACCATCACTCCGCGGGTCTGGTGCCACGCGGTAGTACTGCAATTGGTATGAAAGAAACCGCTGCTGCTAAATTCGAACGCCAGCACATGGACAGCCCAGATCTGGGTACCGGTGGTGGCTCCGGTATTGAGGGACGCGGGTCCATGGGATATCGGGGATCCGAACTGGATACCCCGAAAGATCTGCGTGTTAGCGAAACCGCAGAAACCAGCCTGACCCTGTTTTGGAAAACACCGCTGGCAAAATTTGATCGTTATCGTCTGAATTATAGCCTGCCGACCGGTCAGTGGGTTGGTGTTCAGCTGCCTCGTAATACCACCAGTTATGTTCTGCGTGGTCTGGAACCGGGTCAAGAATATAACGTTCTGCTGACCGCAGAAAAAGGTCGTCATAAAAGCAAACCGGCACGTGTTAAAGCAAGCACCGAACAGGCACCGGAACTGGAAAATCTGACCGTTACCGAAGTTGGCTGGGATGGCCTGCGCCTGAACTGGACGGCTGCGGACCAGGCCTACGAACACTTCGTTATCCAGGTGCAAGAAGCCAACAAAGTAGAAGCCGCTCAGAATCTGACGGTTCCGGGAAATCTGCGTGCAGTTGATATTCCGGGTCTGAAAGCAGCAACCCCGTATACCGTTAGCATTTATGGTGTTATTCAGGGTTATCGTACACCGGTTCTGAGTGCCGAAGCCAGCACCGGTGAAACCCCGAATCTGGGTGAAGTTATGGTTAGCGAAGTGGGCTGGGATGCACTGAAACTGAATTGGACAGTTCCGGAAGGTGCCTATGAATACTTTTTCATTCAGGTTCAAGAAGCGGATACCGTTGAAGCCGCTCAGAATCATACCGTTCCGGGTGGTCTGCGTAGCACCGATCTGCCTGGCCTGAAAGCCGCTACCCATTACACCATTACCATTCGTGGTGTTACCCAGGATTTTAGCACCACACCGCTGAGCGTTGAAGTTCTGACAGAAGAACTGCCGCAGCTGGGTGATCTGGCAGTTAGCGAAGTTGGTTGGGATGGTCTGCGTCTGAATTGGACCGCAGCAGATCAGGCATATGAACATTTTGTTATCCAGGTGCAAGAAGTGAACAAAGTTGAAGCAGCACAGAATCTGACCGTTCCGGGTAGCCTGCGTGCAGTTGATATTCCGGGTCTGAAAGCAGCAACCCCGTATACCGTTAGCATTTATGGTGTTATTCGCGGTTATCGTACACCGGTTCTGAGCGCAGAAGCAAGCACCGCAAAAGAACCGGAAATTGGTAATCTGAACGTGAGCGATATTACACCGGAAAGTTTTAGCCTGAGCTGGACCGCAACCGATGGTATTTTTGAAACCTTTACCATCGAGATCATCGATAGCAATCGTCTGCTGGAAATCGTGGAATATAACATTAGCGGTGCAGAACGTACCGCACATATTAGCGGTCTGCCTCCGAGCACCGATTTTATTGTTTATCTGAGCGGTCTGGCACCGAGCTTTCGTACCAAAACCATTAGCGCAACCGCAACCACCGAAGCACTGACCGCAATGGGTAGCCCGAAAGAAGTGATTTTTAGCGATATTACCGAAAATAGCGCCACCGTTTCATGGCGTGCACCGACCGCACAGGTTGAAAGCTTTCGTATTACCTATGTTCCGATTACCGGTGGCACCCCGAGCATGGTTACCGTGGATGGCACCAAAACCCAGACCCGTCTGGTTAAACTGGTTCCGGGTGTTGAATATCTGGTGAATATCATTGCCATGAAAGGCTTTGAAGAAAGCGAACCGGTTAGCGGTAGCTTTACCACCGCTAGCGGCCTGAACGACATCTTCGAGGCTCAGAAAATCGAATGGCACGAAGGTACCCATCACCATCACCACCACTAA huTNCMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 4KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATA

TTEALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTEAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEG THHHHHH muTNCMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 5KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEEVPSLENLTVTEAGWDGLRLNWTADDLAYEYFVIQVQEANNVETAHNFTVPGNLRAADIPGLKVATSYRVSIYGVARGYRTPVLSAETSTGTTPNLGEVTVAEVGWDALTLNWTAPEGAYKNFFIQVLEADTTQTVQNLTVPGGLRSVDLPGLKAATRYYITLRGVTQDFGTAPLSVEVLTEDLPQLGGLSVTEVSWDGLTLNWTTDDLAYKHFVVQVQEANNVEAAQNLTVPGSLRAVDIPGLKADTPYRVSIYGVIQGYRTPMLSTDVSTAREPEIGNLNVSDVTPKSFNLSWTATDGIFDMFTIEIIDSNRLLQTAEHNISGAERTAHISGLPPSTDFIVYLSGIAPSIRTKTISTTATTEALTAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEGTHHHHHH cynoTNCMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGD SEQ ID NO: 6KWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLRVSETAETSLTLFWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRLNWTAADQAYEHFVIQVQEANKVEAAQNLTVPGNLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEVMVSEVGWDALKLNWTVPEGAYEYFFIQVQEADTVEAAQNHTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEELPQLGDLAVSEVGWDGLRLNWTAADQAYEHFVIQVQEVNKVEAAQNLTVPGSLRAVDIPGLKAATPYTVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVSDITPESFSLSWTATDGIFETFTIEIIDSNRLLEIVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSFRTKTISATATTEALTAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLVPGVEYLVNIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEGTHHHHHH

TABLE-US-00005 TABLE 4 Sequences of TnC antigens used for affinitydetermination (reference Table 5) Antigen pETR # batch ID ProteinSequence SEQ ID NO GST huTNCMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 7 fn5 A1234 BCWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN fn6 BMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTEALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTEAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEGTHHHHHH GSTMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 8 huTNCfn5 muWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHN A124 BC huMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKV fn6 BDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEEVPSLENLTVTEAGWDGLRLNWTADDLAYEYFVIQVQEANNVETAHNFTVPGNLRAADIPGLKVATSYRVSIYGVARGYRTPVLSAETSTGTTPNLGEVTVAEVGWDALTLNWTAPEGAYKNFFIQVLEADTTQTVQNLTVPGGLRSVDLPGLKAATRYYITLRGVTQDFGTAPLSVEVLTEDLPQLGGLSVTEVSWDGLTLNWTTDDLAYKHFVVQVQEANNVEAAQNLTVPGSLRAVDIPGLKADTPYRVSIYGVIQGYRTPMLSTDVSTAREPEIGNLNVSDVTPKSFNLSWTATDGIFDMFTIEIIDSNRLLQTAEHNISGAERTAHISGLPPSTDFIVYLSGIAPSIRTKTISTTATTEALTAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDI FEAQKIEWHEGTHHHHHH GSTTNC hu MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 9 fn5B-C fn6 B WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTAKEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSNRLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTEALPLLENLTISDINPYGFTVSWMASENAFDSFLVTVVDSGKLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTASGLNDIFEAQKIEWHEGTHHHHHH GST huTNCMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 10 fn5 A1234fn6 B WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSELDTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPTGQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPARVKASTEQAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTEEVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEADQVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEVVTEDLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEAMGSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITGGTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAS GLNDIFEAQKIEWHEGTHHHHHHhuTNC A4 B MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 11WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSEDLPQLGDLAVSEVGWDGLRLNWTAADNAYEHFVIQVQEVNKVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTASGLNDIFEAQKIEWHEGT HHHHHH huTNC A1 BMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDK SEQ ID NO: 12WRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGSTSGSGHHHHHHSAGLVPRGSTAIGMKETAAAKFERQHMDSPDLGTGGGSGIEGRGSMGYRGSEQAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEANKVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTASGLNDIFEAQKIEWHEGTH HHHHH

Example 2

Selection of Anti-TnC Antibodies from Generic Fab Libraries

Anti-TnC antibodies were selected from two different generic phagedisplay libraries: DP88-4 (clone 18D4) and lambda-DP47 (clone11C7).

Library Construction

The DP88-4 library was constructed on the basis of human germlinegenes using the V-domain pairing Vk1_5 (kappa light chain) andVH1_69 (heavy chain) comprising randomized sequence space in CDR3of the light chain (L3, 3 different lengths) and CDR3 of the heavychain (H3, 3 different lengths). Library generation was performedby assembly of 3 PCR-amplified fragments applying splicing byoverlapping extension (SOE) PCR. Fragment 1 comprises the 5' end ofthe antibody gene including randomized L3, fragment 2 is a centralconstant fragment spanning from L3 to H3 whereas fragment 3comprises randomized H3 and the 3' portion of the antibody gene.The following primer combinations were used to generate theselibrary fragments for DP88-4 library: fragment 1 (forward primerLMB3 combined with reverse primers Vk1_5_L3r_S or Vk1_5_L3r_SY orVk1_5_L3r_SPY), fragment 2 (forward primer RJH31 combined withreverse primer RJH32) and fragment 3 (forward primers DP88-v4-4 orDP88-v4-6 or DP88-v4-8 combined with reverse primer fdseqlong),respectively. PCR parameters for production of library fragmentswere 5 min initial denaturation at 94.degree. C., 25 cycles of 1min 94.degree. C., 1 min 58.degree. C., 1 min 72.degree. C. andterminal elongation for 10 min at 72.degree. C. For assembly PCR,using equimolar ratios of the gel-purified single fragments astemplate, parameters were 3 min initial denaturation at 94.degree.C. and 5 cycles of 30 s 94.degree. C., 1 min 58.degree. C., 2 min72.degree. C. At this stage, outer primers (LMB3 and fdseqlong)were added and additional 20 cycles were performed prior to aterminal elongation for 10 min at 72.degree. C. After assembly ofsufficient amounts of full length randomized Fab constructs, theywere digested NcoI/NheI and ligated into similarly treated acceptorphagemid vector. Purified ligations were used for .about.60transformations into electrocompetent E. coli TG1. Phagemidparticles displaying the Fab library were rescued and purified byPEG/NaCl purification to be used for selections. These libraryconstruction steps were repeated three times to obtain a finallibrary size of 4.4.times.10.sup.9. Percentages of functionalclones, as determined by C-terminal tag detection in dot blot, were92.6% for the light chain and 93.7% for the heavy chain,respectively.

The lambda-DP47 library was constructed on the basis of humangermline genes using the following V-domain pairings: V13_19 lambdalight chain with VH3_23 heavy chain. The library was randomized inCDR3 of the light chain (L3) and CDR3 of the heavy chain (H3) andwas assembled from 3 fragments by "splicing by overlappingextension" (SOE) PCR. Fragment 1 comprises the 5' end of theantibody gene including randomized L3, fragment 2 is a centralconstant fragment spanning from the end of L3 to the beginning ofH3 whereas fragment 3 comprises randomized H3 and the 3' portion ofthe Fab fragment. The following primer combinations were used togenerate library fragments for library: fragment 1(LMB3-V1_3_19_L3r_V/V1_3_19_L3r_HV/V1_3_19_L3r_HLV), fragment 2(RJH80-DP47CDR3_ba (mod)) and fragment 3(DP47-v4-4/DP47-v4-6/DP47-v4-8-fdseqlong). PCR parameters forproduction of library fragments were 5 min initial denaturation at94.degree. C., 25 cycles of 60 sec 94.degree. C., 60 sec 55.degree.C., 60 sec 72.degree. C. and terminal elongation for 10 min at72.degree. C. For assembly PCR, using equimolar ratios of the 3fragments as template, parameters were 3 min initial denaturationat 94.degree. C. and 5 cycles of 60 s 94.degree. C., 60 sec55.degree. C., 120 sec 72.degree. C. At this stage, outer primerswere added and additional 20 cycles were performed prior to aterminal elongation for 10 min at 72.degree. C. After assembly ofsufficient amounts of full length randomized Fab fragments, theywere digested with NcoI/NheI alongside with similarly treatedacceptor phagemid vector. 15 .mu.g of Fab library insert wereligated with 13.3 .mu.g of phagemid vector. Purified ligations wereused for 60 transformations resulting in 1.5.times.10.sup.9transformants Phagemid particles displaying the Fab library wererescued and purified by PEG/NaCl purification to be used forselections.

Phage Display Selections & ELISA Screening

Human GST-fused TnC fn5 A1234 BC fn6 as antigen for the phagedisplay selections was expressed in E. coli and in vivosite-specifically biotinylated via co-expression of BirA biotinligase at the avi-tag recognition sequence located at theC-terminus of the fusion protein (production of antigens accordingto Example 1, sequences derived from Table 4). This antigencomprises the human TnC extra splice domains A1, A2, A3, A4, B, andC, located between the two fibronectin type III domains 5 and 6.The phage display selections aimed at selecting binders to any ofthese extra splice domains and determine the domain specificity ina subsequent step by surface plasmon resonance using additionalantigen constructs comprising fewer extra splice domains.

Selection rounds (biopanning) were performed in solution accordingto the following pattern: 1. Pre-clearing of .about.10.sup.12phagemid particles with an unrelated GST-fusion protein that alsocarried an avi-tag and His6-tag similar to the TnC target cellantigen to deplete the libraries of antibodies recognizing thethree different tags, 2. incubation of the pre-cleared phagemidparticles in the supernatant with 100 nM biotinylated humanGST-fused TnC fn5 A1234 BC fn6 for 0.5 hours in the presence of anunrelated non-biotinylated GST-fusion protein for further depletionof tag-binders in a total volume of 1 ml, 3. capture ofbiotinylated human GST-fused TnC fn5 A1234 BC fn6 and attachedspecifically binding phage by transfer to 4 wells of a neutravidinpre-coated microtiter plate for 10 minutes (in rounds 1 & 3),4. washing of respective wells using 5.times.PBS/Tween20 and5.times.PBS, 5. elution of phage particles by addition of 250 .mu.l100 mM TEA (triethylamine) per well for 10 minutes andneutralization by addition of 500 .mu.l 1 M Tris/HCl pH 7.4 to thepooled eluates from 4 wells, 6. re-infection of log-phase E. coliTG1 cells with the supernatant of eluted phage particles, infectionwith helperphage VCSM13, incubation on a shaker at 30.degree. C.over night and subsequent PEG/NaCl precipitation of phagemidparticles to be used in the next selection round. Selections werecarried out over 3 rounds using constant antigen concentrations of100 nM. In round 2, in order to avoid enrichment of binders toneutravidin, capture of antigen: phage complexes was performed byaddition of 5.4.times.10.sup.7 streptavidin-coated magnetic beads.Specific binders were identified by ELISA after rounds 2 and 3 asfollows: 100 .mu.l of 100 nM biotinylated human GST-fused TnC fn5A1234 BC fn6 were coated on neutravidin plates. Fab-containingbacterial supernatants were added and binding Fabs were detectedvia their Flag-tags using an anti-Flag/HRP secondary antibody.Clones exhibiting signals on human GST-fused TnC fn5 A1234 BC fn6and being negative on an unrelated GST-fusion protein carrying thesame tags as the target, were short-listed for further analyses.They were bacterially expressed in a 0.5 liter culture volume,affinity purified and further characterized by SPR-analysis usingBioRad's ProteOn XPR36 biosensor to test cross-reactivity to murineTnC and to determine which extra splice domains the antibodiesrecognize.

SPR-Analysis Using BioRad's ProteOn XPR36 Biosensor

Affinities (K.sub.D) of selected clones were measured by surfaceplasmon resonance (SPR) using a ProteOn XPR36 instrument (Biorad)at 25.degree. C. with biotinylated human and murine TnC antigensimmobilized on NLC chips by neutravidin capture. Immobilization ofantigens (ligand): Recombinant antigens were diluted with PBST (10mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20) to 10.mu.g/ml, then injected at 30 .mu.l/minute in vertical orientation.Injection of analytes: For `one-shot kinetics` measurements,injection direction was changed to horizontal orientation, two-folddilution series of purified Fab were injected simultaneously alongseparate channels 1-5, with association times of 200 s, anddissociation times of 240 s, respectively. Buffer (PBST) wasinjected along the sixth channel to provide an "in-line" blank forreferencing. Association rate constants (k.sub.on) and dissociationrate constants (k.sub.off) were calculated using a simpleone-to-one Langmuir binding model in ProteOn Manager v3.1 softwareby simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K.sub.D) wascalculated as the ratio k.sub.off/k.sub.on. Table 5 lists theequilibrium dissociation constants (K.sub.D) of the two selectedclones 18D4 and 11C7 for several TnC antigens differing in speciesand composition of the extra splice domains.

TABLE-US-00006 TABLE 5 Equilibrium dissociation constants (K.sub.D)for clones 18D4 and 11C7 clone 18D4 clone 11C7 antigen K.sub.D [nM]K.sub.D [nM] GST huTNC fn5 A1234 BC fn6 B 4.0 2.3 GST huTNCfn5 muA124 BC hu fn6 B 11.2 1.9 GST TNC hu fn5 B-C fn6 B n.a. 1.0 GSThuTNC fn5 A1234 fn6 B 5.0 n.a. huTNC A4 B 2.0 n.a. huTNC A1 B 5.6n.a.

TABLE-US-00007 TABLE 6 DNA sequence of generic phage-displayedDP88-4 library (Vk1_5/VH1_69) template used for PCRs Construct Basepair sequence SEQ ID NO pRJH33 libraryATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTAT SEQ ID NO: 13 templateDP88-4 TACTCGCGGCCCAGCCGGCCATGGCCGACATCCAGATGAC library; completeCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACCGTG Fab codingTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTG regionGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA comprising PelBGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTC leader sequence +CCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCA Vk1_5 kappaCTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACT V-domain + CLTATTACTGCCAACAGTATAATAGTTATTCTACGTTTGGCCA constant domainGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACC for light chainATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT and PelB +CTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTAT VH1_69 V-CCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCC domain + CH1CTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG constant domainGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTG for heavy chain)ACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTGCCGCATAATAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCAC CATCACCATCACGCCGCGGCA

TABLE-US-00008 TABLE 7 Base pair sequence of DP88-4 library(Vk1_5/VH1_69) germline template Construct Base pair sequence SEQID NO Fab light chain GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATC SEQID NO: 14 V1_5 TGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCTACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTG CCGCA Fab heavy chainCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 15 VH1_69CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAGACTATCCCCAGGCGGTTACTATGTTATGGATGCCTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCGCG GCA

TABLE-US-00009 TABLE 8 Amino acid sequence of DP88-4 library(Vk1_5/VH1_69) germline template Construct Base pair sequence SEQID NO Fab light chain DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKSEQ ID NO: 16 Vk1_5 APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGAAEQKLISEEDLNGAADYKDDDDKGAA Fab heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 17 VH1_69(DP88) GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSPGGYYVMDAWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDAAASTSAHHHHHHAAA

TABLE-US-00010 TABLE 9 Primer sequences used for generation ofDP88-4 library (Vk1_5/VH1_69) Primer name Primer sequence 5'-3' SEQID NO LMB3 CAGGAAACAGCTATGACCATGATTAC SEQ ID NO: 18 Vk1_5_L3r_SCTCGACTTTGGTGCCCTGGCCAAACGTSBAATA SEQ ID NO: 19CGAATTATACTGTTGGCAGTAATAAGTTGCAAAATCAT Vk1_5_L3r_SYCTCGACTTTGGTGCCCTGGCCAAACGTMHRSGRATACGA SEQ ID NO: 20ATTATACTGTTGGCAGTAATAAGTTGCAAAATCAT Vk1_5_L3r_SPYCTCGACTTTGGTGCCCTGGCCAAACGTMHHMSSSGRATAC SEQ ID NO: 21GAATTATACTGTTGGCAGTAATAAGTTGCAAAATCAT RJH31ACGTTTGGCCAGGGCACCAAAGTCGAG SEQ ID NO: 22 RJH32TCTCGCACAGTAATACACGGCGGTGTCC SEQ ID NO: 23 DP88-v4-4GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-3-4-GAC- SEQ ID NO: 24TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC DP88-v4-6GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-3-4- SEQ ID NO: 25GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC DP88-v4-8GGACACCGCCGTGTATTACTGTGCGAGA-1-2-2-2-2-2-2-3-4- SEQ ID NO: 26GAC-TAC-TGGGGCCAAGGGACCACCGTGACCGTCTCC fdseqlongGACGTTAGTAAATGAATTTTCTGTATGAGG SEQ ID NO: 27 Underlined bases: 60%given sequence and 40% N; Bases in italics: 60% given sequence and40% M. 1: G/D = 20%, E/V/S = 10%, A/P/R/L/T/Y = 5%; 2: G/Y/S = 15%,A/D/T/R/P/L/V/N/W/F/I/E = 4.6%; 3: G/A/Y = 20%, P/W/S/D/T = 8%; 4:F = 46%, L/M = 15%, G/I/Y = 8%.

TABLE-US-00011 TABLE 10 DNA sequence of generic phage-displayedlambda-DP47 library (Vl3_19/VH3_23) template used for PCRsConstruct Base pair sequence SEQ ID NO pRJH53 libraryATGAAATACCTATTGCCTACGGCAGCCGCTGGATTGTTAT SEQ ID NO: 28 template ofTACTCGCGGCCCAGCCGGCCATGGCCTCGTCTGAGCTGAC lambda-DP47TCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTC libraryAGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATG Vl3_19/VH3_23;CAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTAC complete FabTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCC coding regionAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCC comprising PelBTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACT leader sequence +ATTACTGTAACTCCCGTGATAGTAGCGGTAATCATGTGGT Vl3_19 lambdaATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAACC V-domain + CLCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGC constant domainGAGGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGA for light chainTCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAA and PelB +GGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCAC VH3_23 V-CACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAG domain + CH1CAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCA constant domainCAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCAC for heavy chainCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGCGGAGC including tagsCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACGACAAGGGTGCCGCATAATAAGGCGCGCCAATTCTATTTCAAGGAGACAGTCATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCGAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCA CCATCACGCCGCGGCA

TABLE-US-00012 TABLE 11 Base pair sequence of lambda-DP47 library(Vl3_19/VH3_23) germline template Construct Base pair sequence SEQID NO Fab light chain TCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTT SEQID NO: 29 Vl3_19 GGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGTGATAGTAGCGGTAATCATGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGACAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAATTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGCGGAGCCGCAGAACAAAAACTCATCTCAGAAGAGGATCTGAATGGAGCCGCAGACTACAAGGACGACGACG ACAAGGGTGCCGCA Fab heavychain GAGGTGCAATTGCTGGAGTCTGGGGGAGGCTTGGTACAGC SEQ ID NO: 30 VH3_23CTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCGTTTCCGTATTTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAAGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAAGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACGCGGCCGCAAGCACTAGTGCCCATCACCATCACCATCACGCCGCGGCA

TABLE-US-00013 TABLE 12 Amino acid sequence of lambda-DP47 library(Vl3_19/VH3_23) germline template Construct Base pair sequence SEQID NO Fab light chain SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQ SEQID NO: 31 Vl3_19 APVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRDSSGNHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSGAAEQKLISEEDLNGAADYKDDDDKGAA Fab heavy chainEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 32 VH3_23(DP47) GKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDAAASTSAHHHHHHAAA

TABLE-US-00014 TABLE 13 Primer sequences used for generation oflambda-DP47 library (Vl3_19/VH3_23) Primer name Primer sequence5'-3' SEQ ID NO LMB3 See Table 9 SEQ ID NO: 18 Vl_3_19_L3r_VGGACGGTCAGCTTGGTCCCTCCGCCGAATAC V A SEQ ID NO: 33 A G A A AGGAGTTACAGTAATAGTCAGCCTCATCTTCCGC underlined: 60% original base and40% randomization as M bold and italic: 60% original base and 40%randomization as N Vl_3_19_L3r_H V GGACGGTCAGCTTGGTCCCTCCGCCGAATACC A SEQ ID NO: 34 A A G A A A GGAGTTACAGTAATAGTCAGCCTCATCTTCCGCunderlined: 60% original base and 40% randomization as M bolded anditalic: 60% original base and 40% randomization as N Vl_3_19_L3r_HGGACGGTCAGCTTGGTCCCTCCGCCGAATAC R V SEQ ID NO: 35 LV A A A G A A AGGAGTTACAGTAATAGTCAGCCTCATCTTC CGC underlined: 60% original baseand 40% randomization as M bolded and italic: 60% original base and40% randomization as N RJH80 TTCGGCGGAGGGACCAAGCTGACCGTCC SEQ IDNO: 36 DP47CDR3_ba CGCACAGTAATATACGGCCGTGTCC SEQ ID NO: 37 (mod.)DP47-v4-4 CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-3-4-GAC- SEQ ID NO:38 TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG DP47-v4-6CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-3-4- SEQ ID NO: 39GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG DP47-v4-8CGAGGACACGGCCGTATATTACTGTGCG-5-1-2-2-2-2-2-2-3- SEQ ID NO: 404-GAC-TAC-TGGGGCCAAGGAACCCTGGTCACCGTCTCG fdseqlong See Table 9 SEQID NO: 27 Trinucleotide mixtures in randomized primers: 1 (G/D =20%, E/V/S = 10%, A/P/R/L/T/Y = 5%); 2 (G/Y/S = 15%,A/D/T/R/P/L/V/N/W/F/I/E = 4.6%); 3 (G/A/Y = 20, P/W/S/D/T = 8%); 4(F = 46%, L/M = 15%, G/I/Y = 8%); 5 (K = 70%, R = 30%)

Example 3

Cloning of Variable Antibody Domains into Expression Vectors

The variable regions of heavy and light chain DNA sequences of theselected anti-TnC binders (Table 14 to Table 19) were subcloned inframe with either the constant heavy chain or the constant lightchain of human IgG1. The antibodies have been prepared either aswild type human IgG1 backbone or as variant containing Pro329Gly,Leu234Ala and Leu235Ala mutations, which have been introduced toabrogate binding to Fc gamma receptors according to the methoddescribed in International Patent Appl. Publ. No. WO 2012/130831A1.

The CDR sequences of the anti-TnC binder are shown in Table 16 toTable 19. The base pair and amino acid sequences of the anti-TnCIgGs are shown in Table 20 and Table 21. The base pair and aminoacid sequences of the anti-TnC P319GLALA IgGs are shown in Table 22and Table 23. All antibody-encoding sequences were cloned into anexpression vector, which drives transcription of the insert with achimeric MPSV promoter and contains a synthetic polyA signalsequence located at the 3' end of the CDS. In addition, the vectorcontains an EBV OriP sequence for episomal maintenance of theplasmid.

LCDR3 of clone 11C7 (NSINSTRNEV (SEQ ID NO: 60)), as selected byphage display, contains a potential N-glycosylation site, i.e. NST,which can potentially be removed by amino acid substitutions tofacilitate production of a hom*ogeneous product. At the same time,binding to the target should be retained. N (position 1) couldpreferentially be substituted by Q, S or T. Alternatively, S(position 2) could be replaced by P. Alternatively, T (position 3)could be substituted preferentially by G or N or by any otherproteinogenic amino acid except for S or C. Whicheversubstitution(s) would be the best, can be determined empirically bythose skilled in the art.

TABLE-US-00015 TABLE 14 Variable region base pair sequences forphage-derived anti-TnC antibodies Clone Chain Base pair sequenceSEQ ID NO 18D4 VL GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCAT SEQ IDNO: 41 CTGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGAATAAGAAGTTTCCTTCGGGGACGTTTGGCCAGGGCACCAAAGTCGAGA TCAAG VHCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 42CCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCA 11C7 VLTCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCT SEQ ID NO: 43TGGGACAGACAGTCAGGGTCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCATTAATAGTACTCGTAATGAGGTATTCGGCGGAGGGACCAAGCTGACCG TCCTA VHGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG SEQ ID NO: 44CCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAAAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTCCGCGTGTTCCGCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGT

TABLE-US-00016 TABLE 15 Variable region polypeptide sequences forphage-derived anti-TnC antibodies Clone Chain Polypeptide sequence18D4 VL DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK SEQ ID NO: 45APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATY YCQQNKKFPSGTFGQGTKVEIKVH QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 46GQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSS 11C7 VLSSELTQDPAVSVALGQTVRVTCQGDSLRSYYASWYQQKPG SEQ ID NO: 47QAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSINSTRNEVFGGGTKLTVL VHEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 48GKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSS

TABLE-US-00017 TABLE 16 CDR base pairs sequences of the anti-TnCantibody light chains SEQ ID SEQ ID SEQ ID clone NO LCDR1 NO LCDR2NO LCDR3 18D4 SEQ ID CGTGCCAGTCA SEQ ID GATGCCTCCA SEQ IDCAACAGAATAAG NO: 49 GAGTATTAGTA NO: 50 GTTTGGAAAGT NO: 51AAGTTTCCTTCGG GCTGGTTGGCC GGACG 11C7 SEQ ID CAAGGAGACA SEQ IDGGTAAAAACA SEQ ID AACTCCATTAATA NO: 52 GCCTCAGAAGT NO: 53ACCGGCCCTCA NO: 54 GTACTCGTAATGA TATTATGCAAGC GGTA

TABLE-US-00018 TABLE 17 CDR polypeptides sequences of the anti-TnCantibody light chains SEQ ID SEQ ID SEQ ID clone NO LCDR1 NO LCDR2NO LCDR3 18D4 SEQ ID RASQSISSWLA SEQ ID DASSLES SEQ ID QQNKKFPSGTNO: 55 NO: 56 NO: 57 11C7 SEQ ID QGDSLRSYYAS SEQ ID GKNNRPS SEQ IDNSINSTRNEV NO: 58 NO: 59 NO: 60

TABLE-US-00019 TABLE 18 CDR base pairs sequences of the anti-TnCantibody heavy chains SEQ ID SEQ ID SEQ ID clone NO HCDR1 NO HCDR2NO HCDR3 18D4 SEQ ID AGCTACGCTAT SEQ ID GGGATCATCC SEQ IDGGTAACTTCTACGG NO: 61 AAGC NO: 62 CTATCTTTGG NO: 63 TGGTCTGGACTACTACAGCAAAC TACGCACAGA AGTTCCAGGGC 11C7 SEQ ID GGATTCACCTT SEQ IDGCTATTAGCG SEQ ID ACTTCTCCGCGTGT NO: 64 TAGCAGTTATG NO: 65GTAGTGGTGG NO: 66 TCCGCTGGACTAC CCATGAGC TAGCACATAC TACGCAGACTCCGTGAAGGGC

TABLE-US-00020 TABLE 19 CDR polypeptide sequences of the anti-TnCantibody heavy chains SEQ ID SEQ ID SEQ ID clone NO HCDR1 NO HCDR2NO HCDR3 18D4 SEQ ID SYAIS SEQ ID GIIPIFGTANYA SEQ ID GNFYGGLDY NO:67 NO: 68 QKFQG NO: 69 11C7 SEQ ID GFTFSSYAMS SEQ ID AISGSGGSTYYSEQ ID TSPRVPLDY NO: 70 NO: 71 ADSVKG NO: 72

TABLE-US-00021 TABLE 20 Base pair sequences of anti-TnC clones inwild type human IgG1 format Clone Chain Base pair sequence SEQ IDNO 18D4 Light GACATCCAGATGACCCAGTCTCCATCCACCCTGTCTGCATC SEQ ID NO:73 chain TGTAGGAGACCGTGTCACCATCACTTGCCGTGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCACGTTTCAGCGGCAGTGGATCCGGGACAGAATTCACTCTCACCATCAGCAGCTTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGAATAAGAAGTTTCCTTCGGGGACGTTTGGCCAGGGCACCAAAGTCGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT HeavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAGC SEQ ID NO: 74 chainCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA 11C7 LightTCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTT SEQ ID NO: 75 chainGGGACAGACAGTCAGGGTCACATGCCAAGGAGACAGCCTCAGAAGTTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGACTATTACTGTAACTCCATTAATAGTACTCGTAATGAGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAACCCAAGGCTGCCCCCAGCGTGACCCTGTTCCCCCCCAGCAGCGAGGAACTGCAGGCCAACAAGGCCACCCTGGTCTGCCTGATCAGCGACTTCTACCCAGGCGCCGTGACCGTGGCCTGGAAGGCCGACAGCAGCCCCGTGAAGGCCGGCGTGGAGACCACCACCCCCAGCAAGCAGAGCAACAACAAGTACGCCGCCAGCAGCTACCTGAGCCTGACCCCCGAGCAGTGGAAGAGCCACAGGTCCTACAGCTGCCAGGTGACCCACGAGGGCAGCACCGTGGAGAAAACCGTGGCCCCCACCGAGTGCAGC HeavyGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC SEQ ID NO: 76 chainTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAAAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTCCGCGTGTTCCGCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

TABLE-US-00022 TABLE 21 Polypeptide sequences of anti-TnC clones inwild type human IgG1 format Clone Chain Polypeptide sequence SEQ IDNO 18D4 Light DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGK SEQ ID NO:77 chain APKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQNKKFPSGTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC HeavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 78 chainGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11C7 LightSSELTQDPAVSVALGQTVRVTCQGDSLRSYYASWYQQKPGQ SEQ ID NO: 79 chainAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSINSTRNEVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS HeavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ ID NO: 80 chainGKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

TABLE-US-00023 TABLE 22 Base pair sequences of anti-TnC clones inP329GLALA human IgG1 format Clone Chain Base pair sequence 18D4Light See Table 20 SEQ ID NO: 73 chain HeavyCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAGAAG SEQ ID NO: 81 chainCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCTCCGGAG PGLALAGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAA 11C7 Light SeeTable 20 SEQ ID NO: 75 chain HeavyGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTACAG SEQ ID NO: 82 chainCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGAT PGLALATCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGCGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAAAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTCCGCGTGTTCCGCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA

TABLE-US-00024 TABLE 23 Polypeptide sequences of anti-TnC clones inP329GLALA human IgG1 format Clone Chain Polypeptide sequence 18D4Light See Table 21 SEQ ID NO: 77 chain HeavyQVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAP SEQ ID NO: 83 chainGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11C7 Light See Table 21 SEQ IDNO: 79 chain Heavy EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAP SEQ IDNO: 84 chain GKGLEWVSAISGSGGSTYYADSVKGRFTISKDNSKNTLYLQMNSLRAEDTAVYYCAKTSPRVPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Example 4

Purification of Anti-TnC IgGs

All genes were transiently expressed under control of a chimericMPSV promoter consisting of the MPSV core promoter combined withthe CMV promoter enhancer fragment. The expression vector alsocontains the oriP region for episomal replication in EBNA (EpsteinBarr Virus Nuclear Antigen) containing host cells.

The anti-TnC IgGs were produced by co-transfecting HEK293-EBNAcells with the mammalian expression vectors using polyethylenimine.The cells were transfected with the corresponding expressionvectors in a 1:1 ratio ("vector HC":"vector HLC").

For a 200 mL production in 500 mL shake flasks, 250 million HEK293EBNA cells were seeded 24 hours before transfection in Excell mediawith supplements. For transfection, the cells were centrifuged for5 minutes at 210.times.g, and supernatant was replaced bypre-warmed CD-CHO medium. Expression vectors were mixed in 20 mLCD-CHO medium to a final amount of 200 .mu.g DNA. After addition of540 .mu.L PEI (1 mg/mL), the solution was vortexed for 15 secondsand incubated for 10 minutes at room temperature. Afterwards, cellswere mixed with the DNA/PEI solution, transferred to a 500 mL shakeflask and incubated for 3 hours at 37.degree. C. in an incubatorwith a 5% CO.sub.2 atmosphere and shaking at 165 rpm. After theincubation, 160 mL Excell medium with supplements was added andcells were cultured for 24 hours. At this point the valproic acidconcentration is 1 mM (the media comprises additionally g/L PepSoyand 6 mM L-Glutamine) 24 hours after transfection the cells aresupplement with an amino acid and glucose feed at 12% final volume(24 mL) and 3 g/L glucose (1.2 mL from 500 g/L stock). Afterculturing for 7 days, the cell supernatant was collected bycentrifugation for 45 minutes at 2000-3000.times.g. The solutionwas sterile filtered (0.22 .mu.m filter), supplemented with sodiumazide to a final concentration of 0.01% (w/v), and kept at4.degree. C.

Purification of anti-TnC IgGs from cell culture supernatants wascarried out by affinity chromatography using MabSelectSure. Theprotein was concentrated and filtered prior to loading on a HiLoadSuperdex 200 column (GE Healthcare) equilibrated with 20 mMHistidine, 140 mM NaCl, 0.01% Tween-20 solution of pH 6.0.

For affinity chromatography, the supernatant was loaded on a ProtAMabSelect Sure column (CV=6 mL, GE Healthcare) equilibrated with 36mL 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5. Unboundprotein was removed by washing with 6-10 column volumes of a buffercontaining 20 mM Sodium Citrate, 20 mM Sodium Phosphate, pH 7.5.The bound protein was eluted using a linear pH-gradient of 15 CVsof sodium chloride (from 20 to 100%) of 20 mM Sodium Citrate, 100mM Sodium Chloride, 100 mM Glycine, 0.01% (v/v) Tween-20, pH 3.0.The column was then washed with 4 column volumes of a solutioncontaining 20 mM Sodium Citrate, 100 mM Sodium Chloride, 100 mMGlycine, 0.01% (v/v) Tween-20, pH 3.0 followed by are-equilibration step.

The pH of the collected fractions was adjusted by adding 1/10 (v/v)of 0.5 M Na.sub.2HPO.sub.4, pH 8.0. The protein was concentratedand filtered prior to loading on a HiLoad Superdex 200 column (GEHealthcare) equilibrated with 20 mM Histidine, 140 mM NaCl, pH 6.0,0.01% Tween20.

The protein concentration of purified IgGs was determined bymeasuring the OD at 280 nm, using the molar extinction coefficientcalculated on the basis of the amino acid sequence.

Purity and molecular weight of the IgGs were analyzed by CE-SDS inthe presence and absence of a reducing agent (Invitrogen, USA)using a LabChipGXII (Caliper). The aggregate content of thepurified protein was analyzed using a TSKgel G3000 SW XL analyticalsize-exclusion column (Tosoh) equilibrated in a 25 mMK.sub.2HPO.sub.4, 125 mM NaCl, 200 mM L-Arginine Monohydrocloride,0.02% (w/v) NaN.sub.3, pH 6.7 running buffer at 25.degree. C.(Table 24).

TABLE-US-00025 TABLE 24 Biochemical analysis of anti-TnC IgG1 YieldMonomer Clone [mg/l] [%] a-TnC(18D4) 6.8 98.3 huIgG1 a-TnC(18D4)26.7 100 P329GLALA huIgG1

Example 5

Surface Plasmon Resonance (TnC Binding)

Binding of the anti-TnC IgGs 18D4, 11C7 and the Fab fragment ofanti-TnC 18D4 to human, murine and cynomolgus TnC (antigensaccording to Table 4) was assessed by surface plasmon resonance(SPR). All SPR experiments were performed on a Biacore T200 at25.degree. C. with HBS-EP as running buffer (0.01 M HEPES pH 7.4,0.15 M NaCl, 3 mM EDTA, 0.005% Surfactant P20, Biacore,Freiburg/Germany).

A 20 .mu.g/ml stock solution of biotinylated human, murine orcynomolgus TnC was injected on a SA chip, with the aim toimmobilize up to 100 RU using the immobilization wizard function.Final immobilization levels were between 79-400 RU.

The anti-TnC IgGs 18D4 (PGLALA and wtFc), 11C7 (rb IgG) and 18D4Fab fragment were then immediately passed over the chip surface ata concentration ranging from 0.02-12.5 nM (IgGs) and 0.02-50 nM(Fab fragment) with a flow rate of 30 .mu.l/min for 180 s followedby a dissociation step of 180s. An additional dissociation step of1800s was performed for the highest concentration of IgG. Aftereach step the surface was regenerated following two sequentialinjections of 10 mM glycine pH2.1 for 60 s. Bulk refractive indexdifferences were corrected for by subtracting the response obtainedin a reference flow cell without TNC. Affinity and avidity werecalculated using the Langmuir 1:1 kinetic model. However, as theK.sub.D for the 1:1 fitting of bivalent interactions is an apparentvalue only, it should only be used for comparisons.

TABLE-US-00026 TABLE 26 KD values and species cross-reactivity huTNC cyno TNC mu TNC Construct KD (nM) KD (nM) KD (nM) 18D4 IgG wtFc 0.093 0.036 0.061 18D4 IgG PG/LALA 0.032 0.011 0.030 llC7 rb IgG0.029 nd nd 18D4 Fab fragment 1.44 6.69 6.5

The anti-TnC binder 18D4 cross-reacts with mouse and cynomolgus TnCwith similar avidity in the pM range. Monovalent binding of the18D4 Fab fragment to TnC results in a K.sub.D in the low nM range.Anti-TNC binder 11C7 also binds human TnC in the pM range. Speciescross-reactivity could not be assessed for binder 11C7 due toabsence of the C domain in murine and cynomolgus TNC constructsused in this Biacore experiment (Table 26).

Thermal Stability

The thermal stability was monitored by Static Light Scattering(SLS) and by measuring the intrinsic protein fluorescence inresponse to applied temperature stress. 30 .mu.g of filteredprotein sample with a protein concentration of 1 mg/ml was appliedin duplicate to an Optim 2 instrument (Avacta Analytical Ltd). Thetemperature was ramped from 25 to 85.degree. C. at 0.1.degree.C./min, with the radius and total scattering intensity beingcollected. For determination of intrinsic protein fluorescence thesample was excited at 266 nm and emission was collected between 275nm and 460 nm. Both IgGs have an aggregation temperature of62.degree. C. (Table 26).

TABLE-US-00027 TABLE 26 Thermal stability Construct Tagg (.degree.C.) 18D4 IgG PGLALA 62 18D4 IgG wtFc 62

Example 6

Results of TnC Staining in LS174T Xenograft Derived Tumor Tissueand Human Tissue Array

Anti-TnC clones 18D4 and 11C7 as rabbit IgG antibodies were testedin LS174T xenograft derived tumor tissue and human tissue array fordetection of TnC with an immunohistochemistry technique.

The LS174T colorectal carcinoma frozen tumor samples were sectionedin a Cryostat at 12 .mu.m thickness, mounted on superfrost slides(Thermo Scientific, Germany). Frozen samples of human tissue arrayincluding paired normal and tumor samples were purchased fromBiochain (San Francisco, USA). Tissue sections were allowed to thawfor 30 minutes. Slides were washed in PBS, fixed in cold acetonefor 10 minutes and an incubation step with 0.03% hydrogenperoxidase in water was performed to block the endogeneousperoxidase. The sections were then incubated for 1 h with 5% goats*erum in PBS followed by overnight incubation with 0.5 .mu.g/mlanti-TnC clone 18D4 or 11C7 or an isotype control antibody(Serotec, Germany) at 4.degree. C. Afterwards, the sections werewashed with PBS three times for 5 minutes each and developed usingthe peroxidase Rabbit Vectastain ABC kit following themanufacturers' instructions (PK-6101, Vector laboratories,California, USA). Slides were then dehydrated in increasingconcentration of ethanol and incubated 2 minutes in Xylene. Onedrop of Permount mounting medium (Fischer Chemical, Germany) wasadded to the sections and coverslip. Images were obtained withOlympus scanner VS120 (Olympus, Germany) and analyzed.

The pattern of histological staining in LS174T xenografts tumorscorresponds to specific TnC stroma fibers (FIGS. 1A-1C). The TnCstaining, for both clones 18D4 and 11C7, is overall expressed withmoderate intensity. Negative isotype control signal validates thespecificity of the technique. Specificity of the staining isvalidated by negative isotype control signals in the correspondinghistological staining in with a rabbit isotype control (FIG. 2).Histological staining in human tumor array with anti-TnC clone 18D4corresponds to specific TnC stroma fibers. The TnC staining isexpressed at higher levels in most tumor tissues compared tocontrol normal pair tissue (FIG. 3). Histological staining in humantumor array with anti-TnC clone 11C7 corresponds to specific TnCstroma fibers. The TnC staining is expressed at higher levels inmost tumor tissues compared to control normal pair tissue (FIG.4).

Example 7

Preparation, Purification and Characterization of 4-1BB

DNA sequences encoding the ectodomains of human, mouse orcynomolgus 4-1BB (Table 27) were subcloned in frame with the humanIgG1 heavy chain CH2 and CH3 domains on the knob (Merchant et al.,1998). An AcTEV protease cleavage site was introduced between anantigen ectodomain and the Fc of human IgG1. An Avi tag fordirected biotinylation was introduced at the C-terminus of theantigen-Fc knob. Combination of the antigen-Fc knob chaincontaining the S354C/T366W mutations, with a Fc hole chaincontaining the Y349C/T366S/L368A/Y407V mutations allows generationof a heterodimer which includes a single copy of 4-1BB ectodomaincontaining chain, thus creating a monomeric form of Fc-linkedantigen. Table 28 shows the cDNA and amino acid sequences of theantigen Fc-fusion constructs.

TABLE-US-00028 TABLE 27 Amino acid numbering of antigen ectodomains(ECD) and their origin SEQ ID NO: Construct Origin ECD SEQ ID NO:85 human Synthetized aa 24-186 4-1BB ECD according to Q07011 SEQ IDNO: 86 cynomolgus isolated from aa 24-186 4-1BB ECD cynomolgusblood SEQ ID NO: 87 murine Synthetized aa 24-187 4-1BB ECDaccording to P20334

TABLE-US-00029 TABLE 28 cDNA and Amino acid sequences of monomericantigen Fc(kih) fusion molecules SEQ ID NO: Antigen Sequence SEQ IDNO: 88 Nucleotide GACAAAACTCACACATGCCCACCGTGCCCAGCACC sequenceTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCC Fc hole chainCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAG CCTCTCCCTGTCTCCGGGTAAA SEQ IDNO: 89 Nucleotide CTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCAC sequenceCTTCTGCGACAACAACCGGAACCAGATCTGCAGCC human 4-1BBCCTGCCCCCCCAACAGCTTCAGCTCTGCCGGCGGA antigen Fc knobCAGCGGACCTGCGACATCTGCAGACAGTGCAAGG chainGCGTGTTCAGAACCCGGAAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGACCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGCAAGAGCGTGCTGGTCAACGGCACCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCA GAAGATTGAATGGCACGAG SEQ ID NO:90 Nucleotide TTGCAGGATCTGTGTAGTAACTGCCCAGCTGGTAC sequenceATTCTGTGATAATAACAGGAGTCAGATTTGCAGTC cynomolgus 4-CCTGTCCTCCAAATAGTTTCTCCAGCGCAGGTGGA 1BB antigenCAAAGGACCTGTGACATATGCAGGCAGTGTAAAG Fc knob chainGTGTTTTCAAGACCAGGAAGGAGTGTTCCTCCACCAGCAATGCAGAGTGTGACTGCATTTCAGGGTATCACTGCCTGGGGGCAGAGTGCAGCATGTGTGAACAGGATTGTAAACAAGGTCAAGAATTGACAAAAAAAGGTTGTAAAGACTGTTGCTTTGGGACATTTAATGACCAGAAACGTGGCATCTGTCGCCCCTGGACAAACTGTTCTTTGGATGGAAAGTCTGTGCTTGTGAATGGGACGAAGGAGAGGGACGTGGTCTGCGGACCATCTCCAGCCGACCTCTCTCCAGGAGCATCCTCTGCGACCCCGCCTGCCCCTGCGAGAGAGCCAGGACACTCTCCGCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCAGA AGATTGAATGGCACGAG SEQ ID NO: 91Nucleotide GTGCAGAACAGCTGCGACAACTGCCAGCCCGGCA sequence murineCCTTCTGCCGGAAGTACAACCCCGTGTGCAAGAGC 4-1BB antigen FcTGCCCCCCCAGCACCTTCAGCAGCATCGGCGGCCA knob chainGCCCAACTGCAACATCTGCAGAGTGTGCGCCGGCTACTTCCGGTTCAAGAAGTTCTGCAGCAGCACCCACAACGCCGAGTGCGAGTGCATCGAGGGCTTCCACTGCCTGGGCCCCCAGTGCACCAGATGCGAGAAGGACTGCAGACCCGGCCAGGAACTGACCAAGCAGGGCTGTAAGACCTGCAGCCTGGGCACCTTCAACGACCAGAACGGGACCGGCGTGTGCCGGCCTTGGACCAATTGCAGCCTGGACGGGAGAAGCGTGCTGAAAACCGGCACCACCGAGAAGGACGTCGTGTGCGGCCCTCCCGTGGTGTCCTTCAGCCCTAGCACCACCATCAGCGTGACCCCTGAAGGCGGCCCTGGCGGACACTCTCTGCAGGTCCTGGTCGACGAACAGTTATATTTTCAGGGCGGCTCACCCAAATCTGCAGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATCCGGAGGCCTGAACGACATCTTCGAGGCCCA GAAGATTGAATGGCACGAG SEQ ID NO:92 Fc hole chain DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 93 human4-1BB LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQR antigen Fc knobTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGA chainGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE SEQ IDNO: 94 cynomolgus 4- LQDLCSNCPAGTFCDNNRSQICSPCPPNSFSSAGGQR 1BBantigen TCDICRQCKGVFKTRKECSSTSNAECDCISGYHCLGA Fc knob chainECSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSATPPAPAREPGHSPQVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE SEQ IDNO: 95 murine 4-1BB VQNSCDNCQPGTFCRKYNPVCKSCPPSTFSSIGGQPN antigenFc knob CNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQ chainCTRCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLDGRSVLKTGTTEKDVVCGPPVVSFSPSTTISVTPEGGPGGHSLQVLVDEQLYFQGGSPKSADKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGKSGGLNDIFEAQKIEWHE

All 4-1BB-Fc-fusion molecule encoding sequences were cloned into aplasmid vector, which drives expression of the insert from an MPSVpromoter and contains a synthetic polyA signal sequence located atthe 3' end of the CDS. In addition, the vector contains an EBV OriPsequence for episomal maintenance of the plasmid.

For preparation of the biotinylated monomeric antigen/Fc fusionmolecules, exponentially growing suspension HEK293 EBNA cells wereco-transfected with three vectors encoding the two components offusion protein (knob and hole chains) as well as BirA, an enzymenecessary for the biotinylation reaction. The corresponding vectorswere used at a 2:1:0.05 ratio ("antigen ECD-AcTEV-Fc knob":"Fchole":"BirA").

For protein production in 500 ml shake flasks, 400 million HEK293EBNA cells were seeded 24 hours before transfection. Fortransfection cells were centrifuged for 5 minutes at 210 g, and thesupernatant was replaced by pre-warmed CD CHO medium. Expressionvectors were resuspended in 20 mL of CD CHO medium containing 200.mu.g of vector DNA. After addition of 540 .mu.L ofpolyethylenimine (PEI), the solution was vortexed for 15 secondsand incubated for 10 minutes at room temperature. Afterwards, cellswere mixed with the DNA/PEI solution, transferred to a 500 mL shakeflask and incubated for 3 hours at 37.degree. C. in an incubatorwith a 5% CO.sub.2 atmosphere. After the incubation, 160 mL of F17medium was added and cells were cultured for 24 hours. Theproduction medium was supplemented with 5 mM kifunensine. One dayafter transfection, 1 mM valproic acid and 7% Feed 1 withsupplements were added to the culture. After 7 days of culturing,the cell supernatant was collected by spinning down cells for 15min at 210 g. The solution was sterile filtered (0.22 .mu.mfilter), supplemented with sodium azide to a final concentration of0.01% (w/v), and kept at 4.degree. C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant wasloaded on a HiTrap ProteinA HP column (CV=5 mL, GE Healthcare)equilibrated with 40 mL 20 mM sodium phosphate, 20 mM sodiumcitrate pH 7.5. Unbound protein was removed by washing with atleast 10 column volumes of 20 mM sodium phosphate, 20 mM sodiumcitrate, 0.5 M sodium chloride containing buffer (pH 7.5). Thebound protein was eluted using a linear pH-gradient of sodiumchloride (from 0 to 500 mM) created over 20 column volumes of 20 mMsodium citrate, 0.01% (v/v) Tween-20, pH 3.0. The column was thenwashed with 10 column volumes of 20 mM sodium citrate, 500 mMsodium chloride, 0.01% (v/v) Tween-20, pH 3.0.

The pH of collected fractions was adjusted by adding 1/40 (v/v) of2M Tris, pH8.0. The protein was concentrated and filtered prior toloading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 2 mM MOPS, 150 mM sodium chloride, 0.02% (w/v)sodium azide solution of pH 7.4.

For affinity determination to the human receptor, the ectodomain ofhuman 4-1BB was also subcloned in frame with an avi(GLNDIFEAQKIEWHE (SEQ ID NO: 225)) and a hexahistidine tag.

Protein production was performed as described above for theFc-fusion protein. Secreted proteins were purified from cellculture supernatants by chelating chromatography, followed by sizeexclusion chromatography. The first chromatographic step wasperformed on a NiNTA Superflow Cartridge (5 ml, Qiagen)equilibrated in 20 mM sodium phosphate, 500 nM sodium chloride,pH7.4. Elution was performed by applying a gradient over 12 columnvolume from 5% to 45% of elution buffer (20 mM sodium phosphate,500 nM sodium chloride, 500 mM Imidazole, pH7.4). The protein wasconcentrated and filtered prior to loading on a HiLoad Superdex 75column (GE Healthcare) equilibrated with 2 mM MOPS, 150 mM sodiumchloride, 0.02% (w/v) sodium azide solution of pH 7.4 (Table29).

TABLE-US-00030 TABLE 29 Sequences of monomeric human 4-1BB Hismolecule SEQ ID NO: antigen Sequence SEQ ID NO: 96 nucleotideCTGCAGGACCCCTGCAGCAACTGCCCTGCCGGCA sequenceCCTTCTGCGACAACAACCGGAACCAGATCTGCAG humanCCCCTGCCCCCCCAACAGCTTCAGCTCTGCCGGC 4-1BB HisGGACAGCGGACCTGCGACATCTGCAGACAGTGC AAGGGCGTGTTCAGAACCCGGAAAGAGTGCAGCAGCACCAGCAACGCCGAGTGCGACTGCACCCCCGGCTTCCATTGTCTGGGAGCCGGCTGCAGCATGTGCGAGCAGGACTGCAAGCAGGGCCAGGAACTGA CCAAGAAGGGCTGCAAGGACTGCTGCTTCGGCACCTTCAACGACCAGAAGCGGGGCATCTGCCGGCCCTGGACCAACTGTAGCCTGGACGGCAAGAGCGT GCTGGTCAACGGCACCAAAGAACGGGACGTCGTGTGCGGCCCCAGCCCTGCTGATCTGTCTCCTGGGGCCAGCAGCGTGACCCCTCCTGCCCCTGCCAGAGAGCCTGGCCACTCTCCTCAGGTCGACGAACAGTTATATTTTCAGGGCGGCTCAGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTGGCACGAGGCT CGAGCTCACCACCATCACCATCAC SEQ IDNO: 97 human LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQ 4-1BB HisRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQVDEQLYFQGGSGLN DIFEAQKIEWHEARAHHHHHH

Example 8

Preparation of TnC Targeted Split Trimeric 4-1BB Ligand Fc FusionMolecules

The DNA sequence encoding part of the ectodomain (amino acid 71-254and 71-248) of human 4-1BB ligand was synthesized according to theP41273 sequence of Uniprot database. The TnC binder used to targetthe trimeric 4-1BB ligand was clone 18D4.

Construct 6.1: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-254) Fc (kih) Fusion with CH-CL Cross and ChargedResidues

A polypeptide containing two ectodomains of 4-1BB ligand (71-254),separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to thehuman IgG1-CL domain, was cloned as depicted in FIG. 5A: human4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254)and fused to the human IgG1-CH domain, was cloned as described inFIG. 5B: human 4-1BB ligand, (G4S)2 connector, human CH.

To improve correct pairing the following mutations have beenintroduced in the crossed CH-CL. In the dimeric 4-1BB ligand fusedto human CL, E123R and Q124K. In the monomeric 4-1BB ligand fusedto human CH1, K147E and K213E.

The variable region of heavy and light chain DNA sequences encodinga binder specific for Tenescin (TnC), clone 18D4, were subcloned inframe with either the constant heavy chain of the hole or theconstant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (as described inInternational Patent Appl. Publ. No. WO 2012/130831).

Combination of the dimeric ligand-Fc knob chain containing theS354C/T366W mutations, the monomeric CH1 fusion, the targetedanti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407Vmutations and the anti-TnC light chain (as described e.g. in U.S.Pat. Nos. 5,731,168; 7,695,936) allows generation of a heterodimer,which includes an assembled trimeric 4-1BB ligand and a TnC bindingFab (FIG. 2).

Table 30 and Table 31 show, respectively, the cDNA and amino acidsequences of the monovalent TnC (18D4) targeted split trimeric4-1BB ligand (71-254) Fc (kih) fusion with CH-CL cross and chargedresidues (construct 6.1).

TABLE-US-00031 TABLE 30 Base pair sequences of monovalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusioncontaining CH-CL cross with charged residues (construct 6.1). SEQID NO Construct Sequence SEQ ID NO: 98 Dimeric ligandAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGC (71-254)-CL*TGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTC Fc knob chainAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTGCCAAGCCCTAGATCAGAAGGGGGCGGAGGTTCCGGAGGGGGAGGATCTCGTACGGTGGCTGCACCATCTGTCTTTATCTTCCCACCCAGCGACCGGAAGCTGAAGTCTGGCACAGCCAGCGTCGTGTGCCTGCTGAATAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCC CTGTCTCCGGGTAAA SEQ ID NO: 99Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGC ligand (71-254)-TGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTC CH1*AGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCCGGCTGCTGCACCTGTCTGCCGGCCAGAGACTGGGAGTGCATCTGCACACAGAGGCCAGAGCCAGGCACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCTGCCGGCCTGCCTAGCCCTAGATCTGAAGGCGGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACG AGAAGGTGGAACCCAAGTCCTGC SEQ IDNO: 100 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) FcGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG hole chainCCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG AGCCTCTCCCTGTCTCCGGGTAAA SEQ IDNO: 73 anti- See Table 20 TnC(18D4) light chain *for chargedresidues

TABLE-US-00032 TABLE 31 Amino acid sequences of monovalentTnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih)fusion containing CH-CL cross with charged residues (construct6.1). SEQ ID NO Construct Sequence SEQ ID NO: 102 Dimeric ligandREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP (71-254)-CL*LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY Fc knob chainYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK SEQ ID NO: 103 MonomericREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGP ligand (71-254)-LSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVY CH1*YVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD EKVEPKSC SEQ ID NO: 104 anti-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW TnC(18D4) FcVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITA hole chainDKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK SEQ ID NO: 77anti- See Table 21 TnC(18D4) light chain *for charged residues

Construct 6.2: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-254) Fc (kih) Fusion Containing CH-CL Cross withoutCharged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-254),separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to thehuman IgG1-CL domain, was cloned as depicted in FIG. 7A: human4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254)and fused to the human IgG1-CH domain, was cloned as described inFIG. 7B: human 4-1BB ligand, (G4S)2 connector, human CH.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole or the constant lightchain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (patent accessionnumber).

Combination of the dimeric ligand-Fc knob chain containing theS354C/T366W mutations, the monomeric CH1 fusion, the targetedanti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407Vmutations and the anti-TnC light chain allows generation of aheterodimer, which includes an assembled trimeric 4-1BB ligand anda TnC binding Fab (FIG. 8).

Table 32 and Table 33 show, respectively, the cDNA and amino acidsequences of the monovalent TnC (18D4) targeted split trimeric4-1BB ligand (71-254) Fc (kih) fusion containing CH-CL crosswithout charged residues (construct 6.2).

TABLE-US-00033 TABLE 32 Base pair sequences of monovalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-254) Fc (kih) fusioncontaining CH-CL cross without charged residues (construct 6.2).SEQ ID NO Construct Sequence SEQ ID NO: 106 DimericAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-254)-GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC CL FcTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT knob chainGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTGCCAAGCCCTAGATCAGAAGGGGGCGGAGGTTCCGGAGGGGGAGGATCTCGTACGGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT AAA SEQ ID NO: 107 MonomericAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 254)-CH1TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCTCTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCCGGCTGCTGCACCTGTCTGCCGGCCAGAGACTGGGAGTGCATCTGCACACAGAGGCCAGAGCCAGGCACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCTGCCGGCCTGCCTAGCCCTAGATCTGAAGGCGGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAACCC AAGTCTTGT SEQ ID NO: 98 anti-See Table 30 TnC(18D4) Fc hole chain SEQ ID NO: 99 anti- See Table30 TnC(18D4) light chain

TABLE-US-00034 TABLE 33 Amino acid sequences of monovalentTnC(18D4) targeted split trimeric 4-1BB ligand (71-254) Fc (kih)fusion containing CH-CL cross without charged residues (construct6.2). SEQ ID NO Construct Sequence SEQ ID NO: 108 Dimeric ligandREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS (71-254)-CLWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF Fc knob chainQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 109 MonomericREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS ligand (71-254)-WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF CH1QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 104 anti- See Table 31TnC(18D4) Fc hole chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4)light chain

Construct 6.3: Bivalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-254) Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand (71-254),separated by (G4S)2 (SEQ ID NO: 150) linkers was fused to theC-terminus of human IgG1 Fc hole chain, as depicted in FIG. 9A:human IgG1 Fc hole, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human 4-1BB ligand.

A polypeptide containing one ectodomain of 4-1BB ligand (71-254)and fused to the C-terminus of human IgG1 Fc knob chain asdescribed in FIG. 9B: human IgG1 Fc knob, (G4S)2 connector, human4-1BB ligand.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole, the knob or theconstant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (described inInternational Patent Appl. Publ. No. WO 2012/130831).

Combination of the anti-TnC huIgG1 hole dimeric ligand chaincontaining the Y349C/T366S/L368A/Y407V mutations, the anti-TnChuIgG1 knob monomeric ligand chain containing the S354C/T366Wmutations and the anti-TnC light chain allows generation of aheterodimer, which includes an assembled trimeric 4-1BB ligand andtwo TnC binding Fabs (FIG. 10).

Table 34 and Table 35 show, respectively, the cDNA and amino acidsequences of the bivalent TnC (18D4)-targeted split trimeric 4-1BBligand (71-254) Fc (kih) fusion (construct 6.3).

TABLE-US-00035 TABLE 34 Base pair sequences of bivalent TnC(18D4)targeted split trimeric 4-1BB ligand Fc (kih) PGLALA fusion(construct 6.3) SEQ IO NO Construct Sequence SEQ ID NO: 110anti-TnC(18D4) CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA Fc hole dimericAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG ligand (71-254)GCCTCCGGAGGCACATTCAGCAGCTACGCTATAAG chainCTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCTGCCGGACTGCCAAGC CCTAGATCAGAA SEQ ID NO: 111anti-TnC(18D4) CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGA Fc knobAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAG monomericGCCTCCGGAGGCACATTCAGCAGCTACGCTATAAG ligand (71-254)CTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAG chainTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGG CCTGCCTTCTCCAAGAAGCGAA SEQ IDNO: 77 anti- See Table 21 TnC(18D4) light chain

TABLE-US-00036 TABLE 35 Amino acid sequences of bivalent TnC(18D4)targeted split trimeric 4-1BB ligand Fc (kih) PGLALA fusion(construct 6.3) SEQ ID NO Construct Sequence SEQ ID NO: 112 anti-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS TnC(18D4) FcWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI hole dimericTADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGL ligand (71-254)DYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA chainALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSE SEQID NO: 113 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAIS TnC(18D4) FcWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTI knobTADKSTSTAYMELSSLRSEDTAVYYCAKGNFYGGL monomericDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTA ligand (71-254)ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ chainSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIP AGLPSPRSE SEQ ID NO: 77 anti-See Table 21 TnC(18D4) light chain

Construct 6.4: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-248) Fc (kih) Fusion Containing CH-CL Cross with ChargedResidues

A polypeptide containing two ectodomains of 4-1BB ligand (71-248),separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to thehuman IgG1-CL domain, was cloned as depicted in FIG. 11A: human4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248)and fused to the human IgG1-CH domain, was cloned as described inFIG. 11B: human 4-1BB ligand, (G4S)2 connector, human CH.

To improve correct pairing the following mutations have beenintroduced in the crossed CH-CL. In the dimeric 4-1BB ligand fusedto human CL, E123R and Q124K. In the monomeric 4-1BB ligand fusedto human CH1, K147E and K213E.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole, the knob or theconstant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (patent accessionnumber).

Combination of the dimeric ligand-Fc knob chain containing theS354C/T366W mutations, the monomeric CH1 fusion, the targetedanti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407Vmutations and the anti-TnC light chain allows generation of aheterodimer, which includes an assembled trimeric 4-1BB ligand anda TnC binding Fab (FIG. 12).

Table 36 and Table 37 show, respectively, the cDNA and amino acidsequences of the monovalent TnC targeted split trimeric 4-1BBligand (71-248) Fc (kih) fusion containing CH-CL cross with chargedresidues (construct 6.4).

TABLE-US-00037 TABLE 36 Base pair sequences of monovalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusioncontaining CH-CL cross with charged residues (construct 6.4). SEQID NO Construct Sequence SEQ ID NO: 114 DimericAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 248)-CL* FcTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCT knob chainGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGACTGGGCGGAGGCGGTTCCGGAGGGGGAGGATCTCGTACGGTGGCTGCACCATCTGTCTTTATCTTCCCACCCAGCGACCGGAAGCTGAAGTCTGGCACAGCCAGCGTCGTGTGCCTGCTGAATAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGAGCGGCAACAGCCAGGAAAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACCCACCAGGGCCTGTCTAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGCGACAAGACCCACACCTGTCCTCCATGCCCTGCCCCTGAAGCTGCTGGCGGCCCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA SEQ ID NO: 115Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGC 248)-CH1*TGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACAAAGGGCCCCAGCGTGTTCCCTCTGGCCCCTAGCAGCAAGAGCACATCTGGCGGAACAGCCGCCCTGGGCTGCCTGGTGGAAGATTACTTCCCCGAGCCCGTGACCGTGTCCTGGAATTCTGGCGCCCTGACAAGCGGCGTGCACACCTTTCCAGCCGTGCTGCAGAGCAGCGGCCTGTACTCTCTGAGCAGCGTCGTGACAGTGCCCAGCAGCTCTCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGT GGACGAGAAGGTGGAACCCAAGTCCTGCSEQ ID NO: 100 anti- See Table 30 TnC(18D4) Fc hole chain SEQ IDNO: 73 anti- See Table 20 TnC(18D4) light chain *chargedresidues

TABLE-US-00038 TABLE 37 Amino acid sequences of monovalentTnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih)fusion containing CH-CL cross with charged residues (construct6.4). SEQ ID NO Construct Sequence SEQ ID NO: 116 DimericREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLS ligand (71-WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF 248)-CL* FcQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTV knob chainDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSRTVAAPSVFIFPPSDRKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 117 Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSligand (71- WYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFF 248)-CH1*QLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT QTYICNVNHKPSNTKVDEKVEPKSCSEQ ID NO: 104 anti- See Table 31 TnC(18D4) Fc hole chain SEQ IDNO: 77 anti- See Table 21 TnC(18D4) light chain *chargedresidues

Construct 6.5: Monovalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-248) Fc (kih) Fusion Containing CH-CL Cross withoutCharged Residues

A polypeptide containing two ectodomains of 4-1BB ligand (71-248),separated by (G4S)2 (SEQ ID NO: 150) linkers, and fused to thehuman IgG1-CL domain, was cloned as depicted in FIG. 13A: human4-1BB ligand, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human CL.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248)and fused to the human IgG1-CH domain, was cloned as described inFIG. 13B: human 4-1BB ligand, (G4S)2 connector, human CH.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole, the knob or theconstant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (patent accessionnumber).

Combination of the dimeric ligand-Fc knob chain containing theS354C/T366W mutations, the monomeric CH1 fusion, the targetedanti-TnC-Fc hole chain containing the Y349C/T366S/L368A/Y407Vmutations and the anti-TnC light chain allows generation of aheterodimer, which includes an assembled trimeric 4-1BB ligand anda TnC binding Fab (FIG. 14).

Table 38 and Table 39 show, respectively, the cDNA and amino acidsequences of the monovalent TnC (18D4) targeted split trimeric4-1BB ligand (71-248) with charged residues in the CH-CL cross Fc(kih) fusion (construct 6.5).

TABLE-US-00039 TABLE 38 Base pair sequences of monovalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusioncontaining CH-CL cross without charged residues (construct 6.5).SEQ ID NO Construct Sequence SEQ ID NO: 118 Dimeric ligandAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT (71-248)-CLGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAG Fc knob chainCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCCGCTGGACTGGGCGGAGGCGGTTCCGGAGGGGGAGGATCTCGTACGGTGGCCGCTCCCTCCGTGTTTATCTTTCCCCCATCCGATGAACAGCTGAAAAGCGGCACCGCCTCCGTCGTGTGTCTGCTGAACAATTTTTACCCTAGGGAAGCTAAAGTGCAGTGGAAAGTGGATAACGCACTGCAGTCCGGCAACTCCCAGGAATCTGTGACAGAACAGGACTCCAAGGACAGCACCTACTCCCTGTCCTCCACCCTGACACTGTCTAAGGCTGATTATGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGACAAGACCCACACCTGTCCCCCTTGTCCTGCCCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAAGTGACCTGCGTGGTGGTGGATGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAATGCCAAGACCAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCT CTCCCTGTCTCCGGGTAAA SEQ ID NO:119 Monomeric AGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCT ligand (71-248)-GGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAG CH1CTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGACTGGGAGGCGGAGGTTCCGGAGGCGGAGGATCTGCTAGCACCAAAGGCCCTTCCGTGTTTCCTCTGGCTCCTAGCTCCAAGTCCACCTCTGGAGGCACCGCTGCTCTCGGATGCCTCGTGAAGGATTATTTTCCTGAGCCTGTGACAGTGTCCTGGAATAGCGGAGCACTGACCTCTGGAGTGCATACTTTCCCCGCTGTGCTGCAGTCCTCTGGACTGTACAGCCTGAGCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGA ACCCAAGTCTTGT SEQ ID NO: 100anti- See Table 30 TnC(18D4) Fc hole chain SEQ ID NO: 73 anti- SeeTable 20 TnC(18D4) light chain

TABLE-US-00040 TABLE 39 Amino acid sequences of monovalentTnC(18D4) targeted split trimeric 4-1BB ligand (71-248) Fc (kih)fusion containing CH-CL cross without charged residues (construct6.5). SEQ ID NO Construct Sequence SEQ ID NO: 120 Dimeric ligandREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL (71-248)-CLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV Fc knob chainFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 121Monomeric REGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPL ligand (71-248)-SWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYV CH1FFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC SEQ ID NO: 104 anti- See Table 31TnC(18D4) Fc hole chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4)light chain

Construct 6.6: Bivalent TnC (18D4) Targeted Split Trimeric 4-1BBLigand (71-248) Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand (71-248),separated by (G4S)2 (SEQ ID NO: 150) linkers was fused to theC-terminus of human IgG1 Fc hole chain, as depicted in FIG. 15A:human IgG1 Fc hole, (G4S)2 connector, human 4-1BB ligand, (G4S)2connector, human 4-1BB ligand.

A polypeptide containing one ectodomain of 4-1BB ligand (71-248)and fused to the C-terminus of human IgG1 Fc knob chain asdescribed in FIG. 15B: human IgG1 Fc knob, (G4S)2 connector, human4-1BB ligand.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole, the knob or theconstant light chain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (patent accessionnumber).

Combination of the anti-TnC huIgG1 hole dimeric ligand chaincontaining the Y349C/T366S/L368A/Y407V mutations, the anti-TnChuIgG1 knob monomeric ligand chain containing the S354C/T366Wmutations and the anti-TnC light chain allows generation of aheterodimer, which includes an assembled trimeric 4-1BB ligand andtwo TnC binding Fabs (FIG. 16).

Table 40 and Table 41 show, respectively, the cDNA and amino acidsequences of the bivalent TnC (18D4) targeted split trimeric 4-1BBligand (71-248) Fc (kih) fusion (construct 6.6).

TABLE-US-00041 TABLE 40 Base pair sequences of bivalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion(construct 6.6) SEQ ID NO Construct Sequence SEQ ID NO: 122 anti-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAG TnC(18D4) FcAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCT hole dimericCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGG ligand (71-248)TGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGG chainGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCCCAGCCGGGCTC SEQ ID NO: 123 anti-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAAG TnC(18D4) FcAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCCT knobCCGGAGGCACATTCAGCAGCTACGCTATAAGCTGGG monomeric (71-TGCGACAGGCCCCTGGACAAGGGCTCGAGTGGATGG 248) ligandGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAG ATTCCTGCCGGGCTC SEQ ID NO: 73anti- See Table 20 TnC(18D4) light chain

TABLE-US-00042 TABLE 41 Amino acid sequences of bivalent TnC(18D4)targeted split trimeric 4-1BB ligand (71-248) Fc (kih) fusion(construct 6.6) SEQ ID NO Construct Sequence SEQ ID NO: 124 anti-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR TnC(18D4) FcQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTS hole dimericTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTV ligand (71-248)TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE chainPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHA WQLTQGATVLGLFRVTPEIPAGL SEQID NO: 125 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVR TnC(18D4)Fc QAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTS knobTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQGTTV monomeric (71-TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE 248) ligandPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEAR ARHAWQLTQGATVLGLFRVTPEIPAGLSEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Constructs 6.11 and 6.12: Monovalent TnC (Knob) Targeted TrimericC-Terminal 4-1BB Ligand Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand, separatedby (G4S)2 (SEQ ID NO: 150) linkers was subcloned in frame to theC-terminus of human IgG1 Fc hole or knob chain (Merchant, Zhu etal. 1998), as depicted in FIG. 17A: human IgG1 Fc, (G4S)2connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BBligand. A polypeptide containing one ectodomain of 4-1BB ligand wassubcloned in frame to the C-terminus of human IgG1 Fc knob or holechain as described in FIG. 17B: human IgG1 Fc, (G4S)2 connector,human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the knob or the constant lightchain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (described inInternational Patent Appl. Publ. No. WO 2012/130831).

Combination of the huIgG1 Fc hole chain containing theY349C/T366S/L368A/Y407V mutations, the anti-TnC huIgG1 knob chaincontaining the S354C/T366W mutations and the anti-TnC light chainallows generation of a heterodimer, which includes an assembledtrimeric 4-1BB ligand and one TnC binding Fab (FIGS. 18A and18B).

Table 42 and Table 43 show respectively, the cDNA and amino acidsequences of the monovalent targeted TnC (18D4) split trimericC-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct6.11).

TABLE-US-00043 TABLE 42 Base pair sequences of bivalent TnC(18D4)split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion(construct 6.11) SEQ ID NO Construct Sequence SEQ ID NO: 126 Fchole dimeric GACAAAACTCACACATGCCCACCGTGCCCAGCACCT ligand (71-248)GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC chainCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACAC CTGAGATCCCAGCCGGGCTC SEQ IDNO: 123 anti- See Table 40 TnC(18D4) Fc knob monomeric ligand(71-248) chain SEQ ID NO: 73 anti- See Table 20 TnC(18D4) lightchain

TABLE-US-00044 TABLE 43 Amino acid sequences of bivalent TnC(18D4)split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih) fusion(construct 6.11) SEQ ID NO Construct Sequence SEQ ID NO: 127 Fchole dimeric DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV ligand(71-248) TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ chainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGL SEQ ID NO: 125anti- See Table 41 TnC(18D4) Fc knob monomeric ligand (71-248)chain SEQ ID NO: 77 anti- See Table 21 TnC(18D4) light chain

Table 44 and Table 45 show, respectively, the cDNA and amino acidsequences of the monovalent targeted TnC (18D4) split trimericC-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct6.12).

TABLE-US-00045 TABLE 44 Base pair sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.12) SEQ ID NO Construct Sequence SEQ ID NO: 128Fc hole GACAAAACTCACACATGCCCACCGTGCCCAGCACCT monomericGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC ligand (71-248)CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC chainCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCTGCCGGGCTC SEQ ID NO: 129 anti-CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) FcGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGC knob dimericCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTG ligand (71-248)GGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGA chainTGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCC CAGCCGGGCTC SEQ ID NO: 73anti- See Table 20 TnC(18D4) light chain

TABLE-US-00046 TABLE 45 Amino acid sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.12) SEQ ID NO Construct Sequence SEQ ID NO: 130Fc hole DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV monomericTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ ligand (71-248)YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP chainIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 131 anti-QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV TnC(18D4) FcRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKS knob dimericTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQG ligand (71-248)TTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD chainYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGL SEQ ID NO: 77 anti- See Table21 TnC(18D4) light chain

Constructs 6.13 and 6.14: Monovalent TnC (Hole) Targeted TrimericC-Terminal 4-1BB Ligand Fc (kih) Fusion

A polypeptide containing two ectodomains of 4-1BB ligand, separatedby (G4S)2 (SEQ ID NO: 150) linkers was subcloned in frame to theC-terminus of human IgG1 Fc hole or knob chain (Merchant, Zhu etal. 1998), as depicted in FIG. 17A: human IgG1 Fc, (G4S)2connector, human 4-1BB ligand, (G4S)2 connector, human 4-1BBligand. A polypeptide containing one ectodomain of 4-1BB ligand wassubcloned in frame to the C-terminus of human IgG1 Fc knob or holechain as described in FIG. 17B: human IgG1 Fc, (G4S)2 connector,human 4-1BB ligand.

The variable region of heavy and light chain DNA sequences encodinga binder specific for TnC, clone 18D4, were subcloned in frame witheither the constant heavy chain of the hole or the constant lightchain of human IgG1.

The Pro329Gly, Leu234Ala and Leu235Ala mutations have beenintroduced in the constant region of the knob and hole heavy chainsto abrogate binding to Fc gamma receptors (described inInternational Patent Appl. Publ. No. WO 2012/130831).

Combination of the anti-TnC huIgG1 Fc hole chain containing theY349C/T366S/L368A/Y407V mutations, the huIgG1 knob chain containingthe S354C/T366W mutations and the anti-TnC light chain allowsgeneration of a heterodimer, which includes an assembled trimeric4-1BB ligand and one TnC binding Fab (FIGS. 19A and 19B).

Table 46 and Table 47 show, respectively, the cDNA and amino acidsequences of the monovalent targeted TnC (18D4) split trimericC-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct6.13).

TABLE-US-00047 TABLE 46 Base pair sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.13). SEQ ID NO Construct Sequence SEQ ID NO:122 anti- See Table 40 TnC(18D4) Fc hole dimeric ligand (71-248)chain SEQ ID NO: 132 Fc knob GACAAAACTCACACATGCCCACCGTGCCCAGCACCTmonomeric GAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC ligand (71-248)CCAAAACCCAAGGACACCCTCATGATCTCCCGGACC chainCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCA GAGTGACCCCCGAGATTCCTGCCGGGCTCSEQ ID NO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE-US-00048 TABLE 47 Amino acid sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.13). SEQ ID NO Construct Sequence SEQ ID NO:124 anti- See Table 41 TnC(18D4) Fc hole dimeric ligand (71-248)chain SEQ ID NO: 133 Fc knobDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV monomericTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ ligand (71-248)YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAP chainIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTP EIPAGL SEQ ID NO: 77 anti- SeeTable 21 TnC(18D4) light chain

Table 48 and Table 49 show, respectively, the cDNA and amino acidsequences of the monovalent targeted TnC (18D4) split trimericC-terminal 4-1BB ligand (71-248) Fc (kih) fusion (construct6.14).

TABLE-US-00049 TABLE 48 Base pair sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.14). SEQ ID NO Construct Sequence SEQ ID NO:134 anti- CAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA TnC(18D4) FcGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGC holeCTCCGGAGGCACATTCAGCAGCTACGCTATAAGCTG monomericGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTGGA ligand (71-248)TGGGAGGGATCATCCCTATCTTTGGTACAGCAAACT chainACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGA TTCCTGCCGGGCTC SEQ ID NO: 135Fc knob GACAAAACTCACACATGCCCACCGTGCCCAGCACCT dimeric ligandGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCC (71-248) chainCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCTGATGATCCTGCCGGACTGCTGGACCTGCGGCAGGGAATGTTTGCCCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGATCTGCTGCTGGCGCCGCTGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCAGCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCAGGCCTGGGAGGCGGCGGATCTGGCGGCGGAGGATCTAGAGAAGGACCCGAGCTGTCCCCCGACGATCCCGCTGGGCTGCTGGATCTGAGACAGGGCATGTTCGCTCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCAGCAGGGGCTGCAGCACTGGCCCTGACTGTGGACCTGCCCCCAGCTTCTTCCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACA CCTGAGATCCCAGCCGGGCTC SEQ IDNO: 73 anti- See Table 20 TnC(18D4) light chain

TABLE-US-00050 TABLE 49 Amino acid sequences of monovalent targetedTnC(18D4) split trimeric C-terminal 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.14). SEQ ID NO Construct Sequence SEQ ID NO:136 anti- QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWV TnC(18D4) FcRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKS holeTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYWGQG monomericTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD ligand (71-248)YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV chainTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEI PAGL SEQ ID NO: 137 Fc knobDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEV dimeric ligandTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ (71-248) chainYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLF RVTPEIPAGL SEQ ID NO: 77 anti-See Table 21 TnC(18D4) light chain

Preparation of Untargeted Split Trimeric 4-1BB Ligand Fc Fusion andHuman IgG as Control Molecules

These control molecules were prepared as described above for theTnC targeted construct 6.1 (termed control B), 6.3 (termed controlC), 6.4 (termed control D) and 6.5 (termed control E) with the onlydifference that the anti-TnC binder (VH-VL) was replaced by agermline control, termed DP47, not binding to the antigen).

Table 50 shows, respectively, the cDNA and amino acid sequences ofthe monovalent DP47-untargeted split trimeric 4-1BB ligand (71-254)Fc (kih) fusion containing crossed CH-CL with charged residues,control B.

Table 51 shows, respectively, the cDNA and amino acid sequences ofthe bivalent DP47-untargeted split trimeric 4-1BB ligand (71-254)Fc (kih) fusion, control C.

Table 52 shows, respectively, the cDNA and amino acid sequences ofthe monovalent DP47-untargeted split trimeric 4-1BB ligand (71-248)Fc (kih) fusion containing CH-CL cross with charged residues,control D.

Table 53 shows, respectively, the cDNA and amino acid sequences ofthe monovalent DP47-untargeted split trimeric 4-1BB ligand (71-248)Fc (kih) fusion without charged residues in the CH-CL cross,control E.

TABLE-US-00051 TABLE 50 cDNA and amino acid sequences of monovalentDP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih)fusion with CH-CL cross and with charged residues (control B). SEQID NO: Description Sequence SEQ ID NO: 98 nucleotide see abovesequence dimeric hu 4- 1BBL (71-254)- CL* Fc knob chain SEQ ID NO:99 nucleotide see above sequence monomeric hu 4-1BBL (71- 254)-CH1*SEQ ID NO: 138 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTsequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc hole chainCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTC CCTGTCTCCGGGTAAA SEQ ID NO: 139nucleotide GAAATCGTGTTAACGCAGTCTCCAGGCACCCTGTC sequence DP47TTTGTCTCCAGGGGAAAGAGCCACCCTCTCTTGCA light chainGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGAGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGATCCGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGTAGCTCACCGCTGACGTTCGGCCAGGGGACCAAAGTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCT TCAACAGGGGAGAGTGT SEQ ID NO:102 Dimeric hu 4- see Table 31 1BBL (71-254)- CL* Fc knob chain SEQID NO: 103 Monomeric hu see Table 31 4-1BBL (71- 254)-CH1* SEQ IDNO: 140 DP47 Fc hole EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chainVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 141DP47 light chain EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC*charges residues

TABLE-US-00052 TABLE 51 cDNA and amino acid sequences of bivalentDP47 untargeted split trimeric human 4-1BB ligand (71-254) Fc (kih)fusion (control C). SEQ ID NO: Description Sequence SEQ ID NO: 142nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc hole chainCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGC fused to dimericTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT hu 4-1BBL (71-GGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA 254)TACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCCTCCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGTTCTGGCCTGTATAGCCTGAGCAGCGTGGTCACCGTGCCTTCTAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTCTCGTGCGCAGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAAGCGAAGGCGGAGGCGGATCTGGCGGCGGAGGATCTAGAGAGGGACCCGAACTGTCCCCTGACGATCCAGCCGGGCTGCTGGATCTGAGACAGGGAATGTTCGCCCAGCTGGTGGCTCAGAATGTGCTGCTGATTGACGGACCTCTGAGCTGGTACTCCGACCCAGGGCTGGCAGGGGTGTCCCTGACTGGGGGACTGTCCTACAAAGAAGATACAAAAGAACTGGTGGTGGCTAAAGCTGGGGTGTACTATGTGTTTTTTCAGCTGGAACTGAGGCGGGTGGTGGCTGGGGAGGGCTCAGGATCTGTGTCCCTGGCTCTGCATCTGCAGCCACTGCGCTCTGCTGCTGGCGCAGCTGCACTGGCTCTGACTGTGGACCTGCCACCAGCCTCTAGCGAGGCCAGAAACAGCGCCTTCGGGTTCCAAGGACGCCTGCTGCATCTGAGCGCCGGACAGCGCCTGGGAGTGCATCTGCATACTGAAGCCAGAGCCCGGCATGCTTGGCAGCTGACTCAGGGGGCAACTGTGCTGGGACTGTTTCGCGTGACACCTGAGATCC CTGCCGGACTGCCAAGCCCTAGATCAGAASEQ ID NO: 143 nucleotide GAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGTsequence DP47 ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG Fc knob chainCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGC fused toTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT monomeric huGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACA 4-1BBL (71-TACTACGCAGACTCCGTGAAGGGCCGGTTCACCAT 254)CTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCCTGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGTGGTGTCTGGTCAAGGGCTTCTACCCCAGCGATATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCTGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCGGAGGCGGCGGAAGCGGAGGAGGAGGATCCAGAGAGGGCCCTGAGCTGAGCCCCGATGATCCTGCTGGACTGCTGGACCTGCGGCAGGGCATGTTTGCTCAGCTGGTGGCCCAGAACGTGCTGCTGATCGATGGCCCCCTGTCCTGGTACAGCGATCCTGGACTGGCTGGCGTGTCACTGACAGGCGGCCTGAGCTACAAAGAGGACACCAAAGAACTGGTGGTGGCCAAGGCCGGCGTGTACTACGTGTTCTTTCAGCTGGAACTGCGGAGAGTGGTGGCCGGCGAAGGATCTGGCTCTGTGTCTCTGGCCCTGCATCTGCAGCCTCTGAGAAGCGCTGCTGGCGCTGCAGCTCTGGCACTGACAGTGGATCTGCCTCCTGCCAGCTCCGAGGCCCGGAATAGCGCATTTGGGTTTCAAGGCAGGCTGCTGCACCTGTCTGCCGGCCAGAGGCTGGGAGTGCATCTGCACACAGAGGCCAGGGCTAGACACGCCTGGCAGCTGACACAGGGCGCTACAGTGCTGGGCCTGTTCAGAGTGACCCCCGAGATTCCAGCCGGCCTGCCTTCTCCAAGAA GCGAA SEQ ID NO: 139 nucleotideSee Table 50 sequence DP47 light chain SEQ ID NO: 144 DP47 Fc holeEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain fused toVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISR dimeric hu 4-DNSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWG 1BBL (71-254)QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSEGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPR SE SEQ ID NO: 145 DP47 Fcknob EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain fused toVRQAPGKGLEWVSAIIGSGASTYYADSVKGRFTISRD monomeric huNSKNTLYLQMNSLRAEDTAVYYCAKGWFGGFNYW 4-1BBL (71-GQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC 254)LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYKEDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAW QLTQGATVLGLFRVTPEIPAGLPSPRSESEQ ID NO: 141 DP47 light chain See Table 50

TABLE-US-00053 TABLE 52 cDNA and amino acid sequences of monovalentDP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih)fusion with CH-CL cross and with charged residues (control D). SEQID NO: Description Sequence SEQ ID NO: 114 nucleotide see Table 36sequence dimeric hu 4- 1BBL (71-248) - CL* Fc knob chain SEQ ID NO:115 nucleotide see Table 36 sequence monomeric hu 4-1BBL (71- 248)- CH1* SEQ ID NO: 138 nucleotide see Table 50 sequence DP47 Fc holechain SEQ ID NO: 139 nucleotide see Table 50 sequence DP47 lightchain SEQ ID NO: 116 Dimeric hu 4- see Table 37 1BBL (71-248) - CL*Fc knob chain SEQ ID NO: 117 Monomeric hu see Table 37 4-1BBL (71-248) - CH1* SEQ ID NO: 140 DP47 Fc hole see Table 50 chain SEQ IDNO: 141 DP47 light chain see Table 50 *charged residues

TABLE-US-00054 TABLE 53 cDNA and amino acid sequences of monovalentDP47 untargeted split trimeric human 4-1BB ligand (71-248) Fc (kih)fusion with CH-CL cross and without charged residues (control E).SEQ ID NO: Description Sequence SEQ ID NO: 118 nucleotide see Table38 sequence dimeric hu 4- 1BBL (71-248) - CL Fc knob chain SEQ IDNO: 119 nucleotide see Table 38 sequence monomeric hu 4-1BBL (71-248) - CH1 SEQ ID NO: 138 nucleotide see Table 50 sequence DP47 Fchole chain SEQ ID NO: 139 nucleotide see Table 50 sequence DP47light chain SEQ ID NO: 120 Dimeric hu 4- see Table 39 1BBL (71-248)- CL Fc knob chain SEQ ID NO: 121 Monomeric hu see Table 39 4-1BBL(71- 248) - CH1 SEQ ID NO: 140 DP47 Fc hole see Table 50 chain SEQID NO: 141 DP47 light chain see Table 50

Two control human IgG1 molecules containing PGLALA were prepared.Table 54 shows the cDNA and amino acid sequences of germlinecontrol DP47 huIgG1 PGLALA (control F). Table 55 shows the cDNA andamino acid sequences of the anti-TnC (18D4) heavy chain (huIgG1PGLALA, i.e. control L).

TABLE-US-00055 TABLE 54 cDNA and amino acid sequences of germlinecontrol DP47 huIgG1 PGLALA (control F) SEQ ID NO: DescriptionSequence SEQ ID NO: 146 nucleotideGAGGTGCAATTGTTGGAGTCTGGGGGAGGCTTGGT sequence DP47ACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAG heavy chain (huCCTCCGGATTCACCTTTAGCAGTTATGCCATGAGCT IgG1 PGLALA)GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGCAGCGGATTTGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCGAGTGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC GGGTAAA SEQ ID NO: 139 DP47light chain see Table 50 SEQ ID NO: 147 DP47 heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW chain (hu IgG1VRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD PGLALA)NSKNTLYLQMNSLRAEDTAVYYCAKGSGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK SEQ ID NO: 141 DP47light chain see Table 50

TABLE-US-00056 TABLE 55 cDNA and amino acid sequences ofanti-TnC(18D4) heavy chain (huIgG1 PGLALA) (control L) SEQ ID NO:Description Sequence SEQ ID NO: 148 NucleotideCAGGTGCAATTGGTGCAGTCTGGGGCTGAGGTGAA sequence anti-GAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGG TnC(18D4)CCTCCGGAGGCACATTCAGCAGCTACGCTATAAGC heavy chainTGGGTGCGACAGGCCCCTGGACAAGGGCTCGAGTG (huIgG1GATGGGAGGGATCATCCCTATCTTTGGTACAGCAA PGLALA)ACTACGCACAGAAGTTCCAGGGCAGGGTCACCATTACTGCAGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACCGCCGTGTATTACTGTGCGAAAGGTAACTTCTACGGTGGTCTGGACTACTGGGGCCAAGGGACCACCGTGACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCTGCAGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCGGCGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTC TCCCTGTCTCCGGGTAAA SEQ ID NO:73 Nucleotide see Table 20 sequence 18D4 Light chain SEQ ID NO: 149anti-TnC(18D4) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISW heavy chainVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITAD (huIgG1KSTSTAYMELSSLRSEDTAVYYCAKGNFYGGLDYW PGLALA)GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO: 77 18D4Light see Table 21 chain

Example 9

Production of TnC-Targeted Split Trimeric C-Terminal 4-1BB LigandFc Fusion and their Controls

The targeted and untargeted trimeric 4-1BB ligand C-terminal Fc(kih) fusion encoding sequences were cloned into a plasmid vector,which drives expression of the insert from an MPSV promoter andcontains a synthetic polyA sequence located at the 3' end of theCDS. In addition, the vector contains an EBV OriP sequence forepisomal maintenance of the plasmid. The split trimeric 4-1BBligand Fc (kih) fusion molecules were produced by co-transfectingHEK293-EBNA cells with the mammalian expression vectors usingpolyethylenimine. The cells were transfected with the correspondingexpression vectors. For constructs 6.1, 6.2, 6.4, 6.6 and it'scontrol B, D and E, at a 1:1:1:1 ratio ("vector Fc holechain":"vector PD1 light chain":"vector Fc knob chain":"vector4-1BBL light chain"). For constructs 6.3, 6.6, 6.11, 6.12, 6.13,6.14 and it's control C, at a 1:1:1 ratio ("vector Fc holechain":"vector Fc knob chain":"vector anti-PD1 light chain"). HumanIgGs, used as control in the assay, were produced as for thebispecific construct (for transfection only a vector for light anda vector for heavy chain were used at a 1:1 ratio).

For production in 500 mL shake flasks, 300 million HEK293 EBNAcells were seeded 24 hours before transfection. For transfectioncells were centrifuged for 10 minutes at 210.times.g, and thesupernatant was replaced by 20 mL pre-warmed CD CHO medium.Expression vectors (200 .mu.g of total DNA) were mixed in 20 mL CDCHO medium. After addition of 540 .mu.L PEI, the solution wasvortexed for 15 seconds and incubated for 10 minutes at roomtemperature. Afterwards, cells were mixed with the DNA/PEIsolution, transferred to a 500 mL shake flask and incubated for 3hours at 37.degree. C. in an incubator with a 5% CO.sub.2atmosphere. After the incubation, 160 mL of Excell mediumsupplemented with 6 mM L-Glutamine, 5 g/L PEPSOY and 1.2 mMvalproic acid was added and cells were cultured for 24 hours. Oneday after transfection 12% Feed were added. After culturing for 7days, the supernatant was collected by centrifugation for 30-40minutes at least 400.times.g. The solution was sterile filtered(0.22 .mu.m filter), supplemented with sodium azide to a finalconcentration of 0.01% (w/v), and kept at 4.degree. C.

Secreted proteins were purified from cell culture supernatants byaffinity chromatography using Protein A, followed by size exclusionchromatography. For affinity chromatography, the supernatant wasloaded on a MabSelect Sure column (CV=5-15 mL, resin from GEHealthcare) equilibrated with Sodium Phosphate (20 mM), SodiumCitrate (20 mM) buffer (pH 7.5). Unbound protein was removed bywashing with at least 6 column volumes of the same buffer. Thebound protein was eluted using either a linear gradient (20 CV) ora step elution (8 CV) with 20 mM sodium citrate, 100 mM Sodiumchloride, 100 mM Glycine buffer (pH 2.5). For the linear gradientan additional 4 column volumes step elution was applied.

The pH of collected fractions was adjusted by adding 1/10 (v/v) of0.5M sodium phosphate, pH8.0. The protein was concentrated prior toloading on a HiLoad Superdex 200 column (GE Healthcare)equilibrated with 20 mM Histidine, 140 mM sodium chloride, 0.01%(v/v) Tween20 solution of pH 6.0.

The protein concentration was determined by measuring the opticaldensity (OD) at 280 nm, using a molar extinction coefficientcalculated on the basis of the amino acid sequence. Purity andmolecular weight of the targeted trimeric 4-1BB ligand Fc (kih)fusion was analyzed by SDS-PAGE in the presence and absence of areducing agent (5 mM 1,4-dithiotreitol) and staining with CoomassieSimpleBlue.TM. SafeStain (Invitrogen USA) or CE-SDS using CaliperLabChip GXII (Perkin Elmer). The aggregate content of samples wasanalyzed using a TSKgel G3000 SW XL analytical size-exclusioncolumn (Tosoh) equilibrated in 25 mM K2HPO4, 125 mM NaCl, 200 mML-Arginine Monohydrocloride, 0.02% (w/v) NaN3, pH 6.7 runningbuffer at 25.degree. C.

Table 56 summarizes the yield and final monomer content of the TnCtargeted split trimeric 4-1BB ligand Fc (kih) fusion.

TABLE-US-00057 TABLE 56 Biochemical analysis of TnC targeted splittrimeric 4-1BB ligand Fc (kih) fusion Monomer [%] Yield Construct(SEC) [mg/l] monovalent TnC(18D4) targeted 91.5 3.7 split trimeric4-1BB ligand (71-248) Fc (kih) fusion containing CH-CL crosswithout charged residues (construct 6.5) bivalent TnC(18D4)targeted 97 9.9 split trimeric 4-1BB ligand (71-248) Fc (kih)fusion (construct 6.6)

Example 10

Simultaneous Binding of TnC Targeted 4-1BB Split Trimeric Ligand FcFusion Antigen Binding Molecule by Surface Plasmon Resonance

The capacity of binding simultaneously human 4-1BB Fc(kih) andhuman TnC was assessed by surface plasmon resonance (SPR). All SPRexperiments were performed on a Biacore T200 at 25.degree. C. withHBS-EP as running buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mMEDTA, 0.005% Surfactant P20, Biacore, Freiburg/Germany).

Biotinylated TnC-antigen was directly coupled to a flow cell of astreptavidin (SA) sensor chip. Immobilization levels up to 500resonance units (RU) were used. The TnC-targeted 4-1BB ligand Fc(kih) fusion molecules were passed at a concentration range of 100nM with a flow of 30 .mu.L/minute through the flow cells over 90seconds and dissociation was set to zero sec. Human 4-1BB avi Hiswas injected as second analyte with a flow of 30 .mu.L/minutethrough the flow cells over 90 seconds at a concentration of 1000nM (FIGS. 20A and 20B). The dissociation was monitored for 120 sec.Bulk refractive index differences were corrected for by subtractingthe response obtained in a reference flow cell, where no proteinwas immobilized. All bispecific constructs could bindsimultaneously human 4-1BB and human TnC (FIGS. 20A and 20B).

Example 11

Affinity Determination of Monovalent TnC Targeted 4-1BB SplitTrimeric Ligand Fc Fusion Antigen Binding Molecule by SurfacePlasmon Resonance

The binding of TnC-targeted 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecules to recombinant TnC was assessed bysurface plasmon resonance (SPR). All SPR experiments were performedon a Biacore T100 at 25.degree. C. with HBS-EP as a running buffer(0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% SurfactantP20, Biacore, Freiburg/Germany).

For affinity measurement, direct coupling of around 500 resonanceunits (RU) of recombinant human, cynomolgus and murine TnC wasperformed on a CMS chip at pH 5.0 using the standard amine couplingkit (GE Healthcare). A dilution series (0.05-50 nM) of TnC-targeted4-1BB ligand trimer-containing Fc (kih) fusion antigen bindingmolecule was passed on both flow cells at 30 .mu.l/min for 120 secto record the association phase. The dissociation phase wasmonitored for 180 s and triggered by switching from the samplesolution to HBS-EP. The chip surface was regenerated after everycycle using a double injection of 60 sec 10 mM Glycine-HCl pH 2.1.Bulk refractive index differences were corrected for by subtractingthe response obtained on reference flow cell 1. For the interactionbetween the TnC-targeted 4-1BB ligand trimer-containing Fc (kih)fusion antigen binding molecule and recombinant TnC, the affinityconstants were derived from the rate constants by fitting to a 1:1Langmuir binding curve using the Biaeval software (GE Healthcare)(Table 57).

TABLE-US-00058 TABLE 57 Fittings to 1:1 Langmuir binding andAffinity constants Ligand Analyte ka (1/Ms) kd (1/s) KD (M)monovalent TnC(18D4) targeted Human 2.9E+06 2.0E-03 6.8E-10 splittrimeric 4-1BB ligand (71- TnC 248) Fc (kih) fusion containingCynomolgus 1.5E+06 6.9E-03 4.7E-09 CH-CL cross without charged TnCresidues Murine 5.9E+05 2.9E-03 5.0E-09 (construct 6.5) TnCbivalent TnC(18D4) targeted split Human 5.3E+06 1.1E-04 2.1E-11trimeric 4-1BB ligand (71-248) Fc TnC (kih) fusion Cynomolgus4.1E+06 1.1E-04 2.7E-11 (construct 6.6) TnC Murine 1.2E+06 3.0E-042.5E-10 TnC

Example 12

Functional Characterization of TnC-Targeted 4-1BB Split TrimericLigand Fc Fusion Antigen Binding Molecules

A) Binding on Fresh and Activated Human PBMCs of TnC-Targeted SplitTrimeric 4-1BB Ligand

Buffy coats were obtained from the Zurich blood donation center. Toisolate fresh peripheral blood mononuclear cells (PBMCs) the buffycoat was diluted with the same volume of DPBS (Gibco by LifeTechnologies, Cat. No. 14190 326). 50 mL polypropylene centrifugetubes (TPP, Cat.-No. 91050) were supplied with 15 mL Histopaque1077 (SIGMA Life Science, Cat.-No. 10771, polysucrose and sodiumdiatrizoate, adjusted to a density of 1.077 g/mL) and the dilutedbuffy coat contents were layered above the Histopaque 1077. Thetubes were centrifuged for 30 min at 450.times.g. PBMCs were thencollected from the interface, washed three times with DPBS andresuspended in T cell medium consisting of RPMI 1640 medium (Gibcoby Life Technology, Cat. No. 42401-042) supplied with 10% (v/v)Fetal Bovine Serum (FBS, US-origin, PAN biotech, P30-2009, LotP150307GI, gamma irradiated mycoplasma free, heat inactivated 35min 56.degree. C.), 1% (v/v) GlutaMAX I (GIBCO by LifeTechnologies, Cat. No. 35050 038), 1 mM Sodium-Pyruvat (SIGMA, Cat.No. S8636), 1% (v/v) MEM non-essential amino acids (SIGMA, Cat.-No.M7145) and 50 .mu.M .beta.-Mercaptoethanol (SIGMA, M3148).

PBMCs were used directly after isolation (binding on fresh humanPBMCs) or they were stimulated to induce 4-1BB expression on thecell surface of T cells (binding on activated human PBMCs). Toinduce 4-1BB upregulation on human T cells, PBMCs were cultured for3 days in RPMI 1640 supplied with 10% FBS, 1 mM Sodium-Pyruvat(SIGMA, Cat.-No. S8636), 1% Gluta-MAX-I, 1% MEM-non essential aminoacid Solution (SIGMA, Cat.-No. M7145), 50 .mu.Mbeta-Mercaptoethanol (Sigma M3148), 200 U/mL Proleukin (NovartisPharma Schweiz AG) and 2 .mu.g/mL PHA-L (SIGMA, Cat.-No. L2769) toa final concentration of 1.times.10.sup.6 cells/mL. Afterpre-activation cells were harvested and resuspended in RPMI 1640supplied with 10% PBS, 1 mM Sodium-Pyruvat (SIGMA, Cat.-No. S8636),1% Gluta-MAX-I, 1% MEM-non essential amino acid solution (SIGMA,Cat.-No. M7145), 50 .mu.M beta-Mercaptoethanol (Sigma M3148) andseeded in 6-well tissue culture plates (TTP, Cat.-No. 92006), whichhad been coated overnight at 4.degree. C. with 10 .mu.g/mLanti-human CD3 (clone OKT3, Mouse IgG2a, BioLegend, Cat.-No.317315) and 2 ug/mL anti-human CD28 (clone CD28.2, Mouse IgG1,BioLegend, Cat.-No. 302923). Cells were further incubated for 3days at 37.degree. C. and 5% CO.sub.2.

For binding assay 0.1.times.10.sup.6 fresh or activated PBMCs wereadded to each well of round-bottom suspension cell 96-well plates(greiner bio-one, cellstar, Cat. No. 650185). Plates werecentrifuged 5 minutes with 350.times.g and at 4.degree. C. andsupernatant were discarded. Afterwards cells were stained in 100.mu.L/well DPBS containing 1:5000 diluted fixable viability dyeeF660 (eBioscience, Cat.-No. 65-0864-18) for 30 minutes at4.degree. C. in the dark. Cells were washed once with 200 .mu.Lcold DPBS buffer. Next, 50 .mu.L/well of 4.degree. C. cold FACSbuffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8 (Amresco, Cat. No.E177) and 7.5 mM Sodium azide (Sigma-Aldrich, Cat.-No. S2002))containing titrated concentrations of construct 6.5 and construct6.6 and as controls control E, control F and control L were addedand cells were incubated for 60 minutes at 4.degree. C. and washedthree times with 200 .mu.L/well 4.degree. C. FACS buffer. Cellswere further resuspended in 50 .mu.L/well of 4.degree. C. cold FACSbuffer containing 0.67 .mu.g/mL anti-human CD45-AF488 (clone HI30,mouse igG1k, BioLegend, Cat.-No. 304019), 0.33 .mu.g/mL anti-humanCD8a-B V510 (clone SK1, mouse IgG1k, BioLegend, Cat.-No. 344732),0.23 .mu.g/mL anti-human CD4-BV421 (clone OKT4, mouse IgG2b.kappa.,BioLegend, Cat.-No. 317434), 0.67 .mu.g/mL anti-humanCD3-PerCP-Cy5.5 (clone UCHT1, mouse IgG1k, BioLegend, Cat.-No.300430), 0.67 .mu.g/mL anti-human CD19-PE/Cy7 (clone HIB19, mouseIgG1k, BioLegend, Cat.-No. 302216), 5 .mu.g/mL PE-conjugatedAffiniPure anti-human IgG F(ab')2-fragment-specific goat F(ab')2fragment (Jackson ImmunoResearch, Cat. No. 109 116 098) andincubated for 30 min at 4.degree. C. Cells were washed twice with200 .mu.L/well 4.degree. C. FACS buffer and then fixed with DPBSsupplied with 1% Formaldehyde (Sigma, HT501320-9.5L). Cells wereresuspended in 100 .mu.L/well FACS-buffer and acquired using theMACS Quant Analyzer 10 (Miltenyi Biotech). Data was analyzed usingFlowJo V10 (FlowJo, LLC) and GraphPad Prism 6.04 (GraphPadSoftware, Inc).

Gates were set on living CD45+ CD3+ CD4+ T cells, CD45+ CD3+ CD8+ Tcells, CD45+ CD3+ CD4neg CD8neg .gamma..delta. T cells and CD45+CD3neg CD19+ B cells and geo means of fluorescence intensity ofPE-conjugated AffiniPure anti-human IgG IgGFc.gamma.-fragment-specific goat F(ab')2 fragment were blottedagainst the titrated concentration of TnC-targeted orDP47-untargeted 4-1BB split trimeric ligand Fc fusion antigenbinding molecules and their controls. As shown in FIGS. 21A, 21B,21C, and 21D, none of the tested TnC-targeted split trimeric 4-1BBligand fusion molecules or their controls bind to cells of freshlyisolated PBMC.

As shown in FIGS. 22A, 22B, 22C, and 22D, after activation livingCD45+ CD3+CD4+ T cells, CD45+ CD3+ CD8+ T cells and CD45+ CD3+CD4neg CD8neg .gamma..delta. T cells express human 4-1BB. Theanti-TnC huIgG1 P329G LALA control L and the DP47 huIgG1 P329G LALAcontrol F did not bind to the activated human PBMCs. All moleculescontaining a 4-1BB split trimeric ligand as construct 6.5,construct 6.6 and control E bind to activated CD8+ T cells, CD4+ Tcells and .gamma..delta. T cells. In Table 58 the EC50 values aresummarized for the binding curves to activated human PBMCs.

TABLE-US-00059 TABLE 58 EC50 values of binding curves to activatedhuman PBMCs Control E construct 6.5 construct 6.6 EC50 [nM] onactivated CD4 0.23 1.11 8.56 T cells EC50 [nM] on activated CD80.26 1.28 1.29 T cells EC50 [nM] on activated .gamma..delta. 0.231.79 0.80 T cells

B) Binding of Human TnC Expressing Tumor Cells

For binding assays on TnC expressing tumor cells the humanglioblastoma cell line U87-MG (ATCC HTB-14) was used.

0.2.times.10.sup.6 tumor cells resuspended in cold DPBS (Gibco byLife Technologies, Cat. No. 14190 326) were added to each well of around-bottom suspension cell 96-well plates (greiner bio-one,cellstar, Cat. No. 650185). Cells were washed once with 200 .mu.LDPBS. Cells were resuspended in 100 .mu.L/well of 4.degree. C. coldDPBS buffer containing 1:5000 diluted Fixable Viability Dye eFluor660 (eBioscience, Cat. No. 65-0864-18) and plates were incubatedfor 30 minutes at 4.degree. C. Cells were washed once with 200.mu.L/well 4.degree. C. cold DPBS buffer and resuspended in 50.mu.L/well of 4.degree. C. cold FACS buffer (DPBS supplied with 2%FBS, 5 mM EDTA pH8 (Amresco, Cat. No. E177) and 7.5 mM Sodium azide(Sigma-Aldrich S2002)) containing TnC-targeted 4-1BB split trimericligand Fc fusion antigen binding molecules or their controls at aseries of concentrations, followed by incubation for 1 hour at4.degree. C. After extensive washing, cells were further stainedwith 50 .mu.L/well of 4.degree. C. cold FACS buffer containing 5.mu.g/mL PE-conjugated AffiniPure anti-human IgGF(ab')2-fragment-specific goat F(ab')2 fragment (JacksonImmunoResearch, Cat. No. 109 116 098) for 30 minutes at 4.degree.C. Cells were washed twice with 200 .mu.L/well 4.degree. C. FACSbuffer and cells were fixed in 50 .mu.L/well DPBS containing 1%Formaldehyde (Sigma, HT501320-9.5L). Cells were resuspended in 100.mu.L/well FACS-buffer and acquired using the MACS Quant Analyzer10 (Miltenyi Biotech). Data was analyzed using FlowJo V10 (FlowJo,LLC) and GraphPad Prism 6.04 (GraphPad Software, Inc).

Gates were set on living tumor cells and geo means of fluorescenceintensity of PE-conjugated AffiniPure anti-human IgG IgGFc.gamma.-fragment-specific goat F(ab')2 fragment were blottedagainst the titrated concentration of TnC-targeted 4-1BB splittrimeric ligand Fc fusion antigen binding molecules or theircontrols. As shown in FIG. 23 only the TnC-targeted moleculesconstruct 6.5, construct 6.6 and control L bound to TnC-expressingU87-MG whereas the not TnC-targeted controls control E and controlF did not bind to the tumor cells. In Table 59 the EC50 values ofthe curves are listed.

TABLE-US-00060 TABLE 59 EC50 values of binding curves toTnC-expressing tumor cells Construct 6.5 Construct 6.6 Control LEC50 [nM] on 73.99 12.75 4.24 U87MG

C) Biological Activity: NF-.kappa.B Activation of Human 4-1BBExpressing HeLa Reporter Cell Line

Generation of HeLa Reporter Cells Expressing Human 4-1BB andNF-.kappa.B-Luciferase

The human-papilloma-virus-18-induced cervix carcinoma cell lineHeLa (ATCC CCL-2) was transduced with a plasmid based on theexpression vector pETR10829, which contains the sequence of human4-1BB (Uniprot accession Q07011) under control of a CMV-promoterand a puromycin resistance gene. Cells were cultured in DMEM-medium(Gibco by Life Technologies Cat. No. 42430-025) supplied with 10%Fetal Bovine Serum (FBS, GIBCO by Life Technologies, Cat. No.16000-044, Lot. No. 941273, gamma-irradiated mycoplasma free, heatinactivated at 56.degree. C. for 35 minutes), 1% GlutaMAX-I (GIBCOby Life Technologies, Cat.-No. 35050-038) and 3 .mu.g/mL Puromycin(InvivoGen, Cat-No. ant-pr). 4-1BB-transduced HeLa cells weretested for 4-1BB expression by flow cytometry: 0.2.times.10.sup.6living cells were resuspended in 100 .mu.L FACS buffer (DPBSsupplied with 2% FBS, 5 mM EDTA pH 8 (Amresco, Cat. No. E177) and7.5 mM Sodium Azide (Sigma-Aldrich, Cat. No. S2002)) containing 0.1.mu.g PerCP/Cy5.5 conjugated anti-human 4-1BB mouse IgG1K clone4B4-1 (BioLegend Cat. No. 309814) or its isotype control(PerCP/Cy5.5 conjugated mouse IgG1K isotype control antibody cloneMOPC 21, BioLegend Cat. No. 400150) and incubated for 30 minutes at4.degree. C. Cells were washed twice with FACS buffer, resuspendedin 300 .mu.L FACS buffer containing 0.06 .mu.g DAPI (Santa CruzBiotec, Cat. No. Sc-3598) and acquired using a 5-laser LSR-Fortessa(BD Bioscience, DIVA software). Limited dilutions were performed togenerate single clones as following: Human-4-1BB transduced HeLacells were resuspended in medium to a concentration of 10, 5 and2.5 cells/mL and 200 .mu.L, of cell suspensions were transferred toround bottom tissue-culture treated 96-well plates (6 plates/cellconcentration, TPP Cat. No. 92697). Single clones were harvested,expanded and tested for 4-1BB expression as described above. Theclone with the highest expression of 4-1BB (clone 5) was chosen forsubsequent transfection with the NF.kappa.B-luciferaseexpression-vector 5495p Tranlucent HygB. The vector conferstransfected cells both with resistance to hygromycin B and capacityto express luciferase under control of NFkB-response element(Panomics, Cat. No. LR0051). Human-4-1BB HeLa clone 5 cells werecultured to 70% confluence. 50 .mu.g (40 .mu.L) linearized(restriction enzymes AseI and SalI) 5495p Tranlucent HygBexpression vector were added to a sterile 0.4 cm GenePulser/MicroPulser Cuvette (Biorad, Cat.-No, 165-2081).2.5.times.10.sup.6 human-4-1BB HeLa clone 5 cells in 400 .mu.lsupplement-free DMEM were added and mixed carefully with theplasmid solution. Transfection of cells was performed using a GenePulser Xcell total system (Biorad, Cat No. 165 2660) under thefollowing settings: exponential pulse, capacitance 500 .mu.F,voltage 160 V, resistance .infin.. Immediately after the pulsetransfected cells were transferred to a tissue culture flask 75cm.sup.2 (TPP, Cat. No. 90075) with 15 mL 37.degree. C. warmDMEM-Medium (Gibco by Life Technologies Cat. No. 42430-025)supplied with 10% FBS and 1% GlutaMAX I (GIBCO by LifeTechnologies, Cat. No. 35050 038). Next day, culture mediumcontaining 3 .mu.g/mL Puromycin (InvivoGen, Cat No. ant pr) and 200.mu.g/mL Hygromycin B (Roche, Cat. No. 10843555001) was added.Surviving cells were expanded and limited dilution was performed asdescribed above to generate single clones. Clones were tested for4-1BB expression as described above and for NF.kappa.B-Luciferaseactivity as following: Clones were harvested in selection mediumand counted using a Cell Counter Vi-cell xr 2.03 (Beckman Coulter,Cat. No. 731050). Cells were set to a cell density of0.33.times.10.sup.6 cells/mL and 150 .mu.L of this cell suspensionwere transferred to each well of a sterile white 96-well flatbottom tissue culture plate with lid (greiner bio one, Cat. No.655083) and as a control to normal 96 well flat bottom tissueculture plate (TPP Cat. No. 92096) to test survival and celldensity the next day. Cells were incubated at 37.degree. C. and 5%CO.sub.2 overnight. The next day 50 .mu.L of medium containingdifferent concentrations of recombinant human tumor necrosis factoralpha (rhTNF .alpha., PeproTech, Cat.-No. 300 01A) were added toeach well of a 96-well plate resulting in final concentration ofrhTNF .alpha. of 100, 50, 25, 12.5, 6.25 and 0 ng/well. Cells wereincubated for 6 hours at 37.degree. C. and 5% CO.sub.2 and thenwashed three times with 200 .mu.L/well DPBS. Reporter Lysis Buffer(Promega, Cat-No: E3971) was added to each well (30 .mu.l) and theplates were stored over night at -20.degree. C. The next day frozencell plates and Detection Buffer (Luciferase 1000 Assay System,Promega, Cat. No. E4550) were thawed to room temperature. 100 uL ofdetection buffer were added to each well and the plate was measuredas fast as possible using a SpectraMax M5/M5e microplate reader andthe SoftMax Pro Software (Molecular Devices). Measured URLs abovecontrol (no rhTNF-.alpha. added) were taken as luciferase activity.The NF.kappa.B-luc-4-1BB-HeLa clone 26 was chosen for further useexhibiting the highest luciferase activity and a considerable levelof 4-1BB-expression.

NF-.kappa.B Activation of HeLa Reporter Cells Expressing Human4-1BB Co-Cultured with TnC-Expressing U87MG and SkK-Me15 Cells

Adherent human glioblastoma cell line U87MG (ATCC HTB-14) werewashed with DPBS (Gibco by Life Technologies, Cat. No. 14190 326)and treated with enzyme-free, PBS-based cell dissociation buffer(Gibco by Life Technologies, Cat.-No. 13151-014) for 10 minutes at37.degree. C. Afterwards cell were harvested and counted using CellCounter Vi-cell xr 2.03. Cells were resuspended in DMEM medium(Gibco by Life Technologies Cat. No. 42430 025) supplied with 10%Fetal Calf Serum (FBS, US-origin, PAN biotech, P30-2009, LotP150307GI, gamma irradiated mycoplasma free, heat inactivated 35min 56.degree. C.) and 1% GlutaMAX-I (GIBCO by Life Technologies,Cat. No. 35050 038) to 1.times.10.sup.6 cells/mL and 100 .mu.L wereadded/well of sterile white 96-well flat bottom tissue cultureplates with lid (greiner bio one, Cat. No. 655083). For negativecontrols 100 .mu.L medium were added. Plates were incubatedovernight at 37.degree. C. and 5% CO.sub.2.

The next day different titrated split trimeric 4-1BB ligandcontaining fusion molecules and their controls were added.Afterwards NF.kappa.B-luc-4-1BB-HeLa clone 26 cells were washedwith DPBS and treated with 0.05% Trypsin EDTA (Gibco by LifeTechnologies Cat. No. 25300 054) for 5 min at 37.degree. C. Cellswere harvested and resuspended in DMEM medium supplied with 10%Fetal Calf Serum and 1% GlutaMAX-I. Cells were counted using CellCounter Vi-cell xr 2.03 (Beckman Coulter, Cat. No. 731050) and setto a cell density of 0.4.times.10.sup.6 cells/mL. 50 .mu.L(2.times.10.sup.4 cells) of this cell suspension were transferredto each well. Plates were incubated for 6 hours at 37.degree. C.and 5% CO.sub.2. White flat bottom 96-well plates were washed threetimes with 200 .mu.L/well DPBS. 40 .mu.l fresh prepared ReporterLysis Buffer (Promega, Cat-No: E3971) were added to each well andthe plate were stored over night at -20.degree. C. The next dayfrozen plates and detection buffer (Luciferase 1000 Assay System,Promega, Cat. No. E4550) were thawed to room temperature. 100 uL ofdetection buffer were added to each well and plates were measuredas fast as possible using the SpectraMax M5/M5e microplate readerand SoftMax Pro Software (Molecular Devices) with followingsettings: for luciferase (RLUs), 500 ms integration time, nofilter, collecting all wave length and top reading.

TABLE-US-00061 TABLE 60 EC50 values of NF.kappa.B-activation-induced Luciferase activity-curves construct 6.5 construct 6.6U87MG EC50 [nM] 0.33 0.70

D) Activation Assay of Human PBMCs in the Presence ofTnC-Expressing Tumor Cells

For TnC-binding-mediated crosslinking the TnC-expressing adherenthuman glioblastoma cell line U87MG (ATCC HTB-14) was used. U87MGcells were washed with DPBS (Gibco by Life Technologies, Cat. No.14190 326) and treated with enzyme-free, PBS-based CellDissociation Buffer (Gibco by Life Technologies, Cat.-No.13151-014) for 10 minutes at 37.degree. C. Cells were harvested andresuspended in T cell medium consisting of RPMI 1640 (GIBCO by LifeTechnologies, Cat.-No. 42401-042) supplied with 10% fetal bovineserum (FBS, US-origin, PAN biotech, P30-2009, Lot P150307GI, gammairradiated mycoplasma free, heat inactivated 35 min 56.degree. C.),1% L-GlutaMAX-I (GIBCO by Life Technologies, Cat-No. 35050-038), 1mM Sodium-Pyruvat (SIGMA-Aldrich, Cat.-No. S8636), 1% MEM-Nonessential Aminoacid Solution (SIGMA-Aldrich, Cat.-No. M7145), 50.mu.M .beta.-Mercaptoethanol (Sigma-Aldrich, Cyt.-No. M3148) andirradiated with 50 Gy (X-Ray Irradiator RS 2000, Rad source).2.times.10.sup.4 U87MG cells in 50 .mu.L T cell medium were seededto each well of a round bottom tissue culture 96-well plate (TTP,Cat.-No. 92697). 50 .mu.L, of T cell medium containing differenttitrated concentrations of construct 6.5 and 6.6 or the fittingcontrols (control F, control L and control E) were added. HumanPBMCs were labeled in 37.degree. C. warm DPBS containing 40 nMCFDA-SE (SIGMA-Aldrich, Cat.-No. 21888-25MG-F) for 15 min at37.degree. C. CFSE-labeling was stopped by adding FBS, PBMCs werewashed twice and resuspended in T cell medium to a finalconcentration of 1.5.times.10.sup.6 cells/mL. 50 .mu.L of this PBMCcell solution were seeded to each well e.g. 7.5.times.10.sup.4CFSE-labeled PBMCs were added to each well. Finally a stocksolution of T cell medium containing 8 nM agonistic anti-human CD3human IgG1 clone V9 was prepared and 50 .mu.L/well were added toeach well giving a final concentration of 2 nM anti-human CD3 humanIgG1 clone V9.

Plates were incubated for 4 days at 37.degree. C. Cells were washedwith DPBS and stained with 100 .mu.L/well DPBS containing 1:1000diluted LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (MolecularProbes by Life Technology, Cat.-No. L34957) for 30 min at 4.degree.C. Cells were washed once with 200 .mu.L/well DPBS and stained with50 .mu.L FACS buffer (DPBS supplied with 2% FBS, 5 mM EDTA pH8(Amresco, Cat. No. E177) and 7.5 mM Sodium azide (Sigma-AldrichS2002)) containing 0.1 .mu.g/mL PerCP-Cy5.5-conjugated anti-humanCD137 mouse IgG1 .kappa. (clone 4B4-1, BioLegend, Cat.-No. 309814),0.1 .mu.g/mL PE/Cy7-conjugated anti-human PD-1 mouse IgG1 .kappa.(clone EH12.2H7, BioLegend, Cat.-No. 329918), 0.03 .mu.g/mLAPC-conjugated anti-human CD25 mouse IgG1 .kappa. (clone BC96,BioLegend, Cat.-No. 302610), 0.06 .mu.g/mL APC/Cy7-conjugatedanti-human CD8 Mouse IgG1 .kappa. (clone RPA-T8, BioLegend,Cat.-No. 3301016), BV421-conjugated anti-human CD4 Mouse IgG1.kappa. (clone RPA-T4, BioLegend, Cat.-No. 300532) for 30 min at4.degree. C. Cells were washed twice with 200 .mu.L/well DPBS andincubated for 30 min at 4.degree. C. with 50 .mu.L/well freshlyprepared FoxP3 Perm/Fix buffer (eBioscience Cat.-No. 00-5123).Cells were washed twice with 200 .mu.L/well DPBS, resuspended in 50.mu.L/well freshly prepared Perm-buffer (eBioscience Cat.-No00-8333) supplied with PE-conjugated 1:250 diluted anti-humanGranzyme B mouse IgG1 .kappa. (clone GB11, Lot 4269803, BDPharmingen, Cat.-No. 561142) and incubated for 1 h at 4.degree. C.Plates were washed twice with 200 .mu.L/well DPBS and cells werefixed for 15 min with DPBS containing 1% Formaldehyde (Sigma,HT501320-9.5L). Cells were resuspended in 100 .mu.L/wellFACS-buffer and acquired using the MACS Quant Analyzer 10 (MiltenyiBiotech). Data was analyzed using FlowJo V10 (FlowJo, LLC) andGraphPad Prism 6.04 (GraphPad Software, Inc).

As shown in FIGS. 27A and 27B only the TnC-targeted split trimeric4-1BBL fusion molecules construct 6.6 and to a less extentconstruct 6.5 induced a concentration-dependent increased surfaceexpressed of CD25 and 4-1BB on CD8+ T cells. The negative controlmolecules (control F, control L and control E) did not induce thisupregulation of activation markers. The correlating EC50 values arelisted in Table 61.

TABLE-US-00062 TABLE 61 EC50 values of upregulation of CD25 and4-1BB (CD137) on activated CD8+ T cells Construct 6.5 Construct 6.6% CD25+ CD8 EC50 [nM] 0.13 0.29 % CD137 (4-1BB)+ CD8 EC50 [nM] 0.360.13

CITATIONS

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SEQUENCE LISTINGS

1

22513102DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotidehuTNC 1atgtccccta tactaggtta ttggaaaattaagggccttg tgcaacccac tcgacttctt 60ttggaatatc ttgaagaaaa atatgaagagcatttgtatg agcgcgatga aggtgataaa 120tggcgaaaca aaaagtttgaattgggtttg gagtttccca atcttcctta ttatattgat 180ggtgatgttaaattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac240atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaaggagcggttttg 300gatattagat acggtgtttc gagaattgca tatagtaaagactttgaaac tctcaaagtt 360gattttctta gcaagctacc tgaaatgctgaaaatgttcg aagatcgttt atgtcataaa 420acatatttaa atggtgatcatgtaacccat cctgacttca tgttgtatga cgctcttgat 480gttgttttatacatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa540aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaagtatatagca 600tggcctttgc agggctggca agccacgttt ggtggtggcgaccatcctcc aaaatcggat 660ggttcaacta gtggttctgg tcatcaccatcaccatcact ccgcgggtct ggtgccacgc 720ggtagtactg caattggtatgaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780gacagcccagatctgggtac cggtggtggc tccggtattg agggacgcgg gtccatggga840tatcggggat ccgagctgga cacccccaag gacctgcagg tgtccgagacagccgagaca 900agcctgaccc tgctgtggaa aacccccctg gccaagttcgaccggtacag actgaactac 960agcctgccca ctggacagtg ggtcggcgtgcagctgcccc ggaacaccac ctcctacgtg 1020ctgcggggcc tggaacccggccaggaatac aacgtcctgc tgacggccga gaagggccgg 1080cacaagagcaagcccgccag agtgaaggcc agcaccgagc aggcccccga gctggaaaac1140ctgaccgtga ccgaagtggg ctgggacggc ctgcggctga actggaccgcggctgaccag 1200gcctatgagc actttatcat tcaggtgcag gaggccaacaaggtggaggc agctcggaac 1260ctcaccgtgc ctggcagcct tcgggctgtggacataccgg gcctcaaggc tgctacgcct 1320tatacagtct ccatctatggggtgatccag ggctatagaa caccagtgct ctctgctgag 1380gcctccacaggcgaaacacc gaacctgggc gaagtggtgg tggcggaagt gggttgggat1440gcgctgaaac tgaactggac cgcgccggaa ggcgcgtatg aatattttttcatccaggtg 1500caggaagcgg ataccgttga agcggcgcag aacctgaccgttccgggcgg tctgcgtagc 1560accgatctgc cgggcctgaa agcggcgacccattatacca ttaccatccg tggggtgacc 1620caggacttct ctaccacccctctgagcgtg gaggtgctga ccgaggaggt acccgacatg 1680ggcaacctgaccgtgaccga ggtgtcctgg gacgccctgc ggctgaactg gaccaccccc1740gacggcacct acgaccagtt cacaatccag gtgcaggaag ccgaccaggtggaagaagca 1800cataatctga ccgttccggg tagcctgcgt agcatggaaattccgggtct gcgtgcaggc 1860accccgtata ccgttaccct gcatggtgaagttcgtggtc atagcacccg tccgctggca 1920gttgaagttg ttaccgaagatctgccgcag ctgggtgatc tggcagttag cgaagttggt 1980tgggatggtctgcgtctgaa ttggaccgca gcagataatg catatgaaca ttttgtgatc2040caggtgcaag aggtgaataa agttgaagca gcccagaatc tgaccctgcctggttcactg 2100cgtgcagttg atattccggg actcgaggca gcaaccccgtatcgtgttag catttatggt 2160gttattcgcg gttatcgtac accggttctgagcgcagaag caagcaccgc aaaagaaccg 2220gaaattggta atctgaacgtgagcgatatt acaccggaat catttaatct gagctggatg 2280gcaaccgatggtatttttga aacctttacc atcgagatca tcgatagcaa tcgtctgctg2340gaaaccgtgg aatataatat tagcggtgca gaacgtaccg cacatattagcggtctgcct 2400ccgagcaccg attttattgt ttatctgagc ggtctggcaccgagcattcg taccaaaacc 2460attagcgcaa ccgcaaccac cgaagcactgccgctgctgg aaaatctgac cattagcgat 2520attaacccgt atggttttaccgtttcatgg atggcaagcg aaaatgcatt tgatagcttt 2580ctggttacagttgtggatag cggtaaactg ctggacccgc aagaatttac cctgagcggc2640acccagcgca aactggaact gcgtggtctg attaccggta ttggttatgaagttatggtg 2700agcggtttta cccagggtca tcagaccaaa ccgctgcgtgcagaaattgt taccgaagca 2760atgggtagcc cgaaagaagt tattttttccgatatcaccg agaattcggc aaccgttagc 2820tggcgtgcac cgaccgcacaggttgaaagc tttcgtatta cctatgttcc gattaccggt 2880ggcaccccgagcatggttac agttgatggc accaaaaccc agacccgtct ggttaaactg2940attccgggtg ttgaatatct ggttagcatt attgccatga aaggctttgaagaaagcgaa 3000ccggttagcg gtagctttac cacagctagc ggcctgaacgacatcttcga ggctcagaaa 3060atcgaatggc acgaaggtac ccatcaccatcaccaccact aa 310222566DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotidemuTNC 2tatgtcccctatactaggtt attggaaaat taagggcctt gtgcaaccca ctcgacttct 60tttggaatatcttgaagaaa aatatgaaga gcatttgtat gagcgcgatg aaggtgataa120atggcgaaac aaaaagtttg aattgggttt ggagtttccc aatcttccttattatattga 180tggtgatgtt aaattaacac agtctatggc catcatacgttatatagctg acaagcacaa 240catgttgggt ggttgtccaa aagagcgtgcagagatttca atgcttgaag gagcggtttt 300ggatattaga tacggtgtttcgagaattgc atatagtaaa gactttgaaa ctctcaaagt 360tgattttcttagcaagctac ctgaaatgct gaaaatgttc gaagatcgtt tatgtcataa420aacatattta aatggtgatc atgtaaccca tcctgacttc atgttgtatgacgctcttga 480tgttgtttta tacatggacc caatgtgcct ggatgcgttcccaaaattag tttgttttaa 540aaaacgtatt gaagctatcc cacaaattgataagtacttg aaatccagca agtatatagc 600atggcctttg cagggctggcaagccacgtt tggtggtggc gaccatcctc caaaatcgga 660tggttcaactagtggttctg gtcatcacca tcaccatcac tccgcgggtc tggtgccacg720cggtagtact gcaattggta tgaaagaaac cgctgctgct aaattcgaacgccagcacat 780ggacagccca gatctgggta ccggtggtgg ctccggtattgagggacgcg ggtccatggg 840atatcgggga tccgagctgg acacccccaaggacctgcag gtgtccgaga cagccgagac 900aagcctgacc ctgctgtggaaaacccccct ggccaagttc gaccggtaca gactgaacta 960cagcctgcccactggacagt gggtcggcgt gcagctgccc cggaacacca cctcctacgt1020gctgcggggc ctggaacccg gccaggaata caacgtcctg ctgacggccgagaagggccg 1080gcacaagagc aagcccgcca gagtgaaggc cagcaccgaggaagtgccca gcctggaaaa 1140cctgaccgtg accgaggccg gctgggacggcctgcggctg aactggaccg ccgacgacct 1200ggcctacgag tacttcgtgatccaggtgca ggaagccaac aacgtcgaga cagcccacaa 1260cttcaccgtgcccggcaacc tgagagccgc cgacatcccc ggcctgaagg tggccacatc1320ctaccgggtg tccatctacg gcgtggccag gggctaccgg acccccgtgctgtccgccga 1380gacaagcacc ggcaccacgc cgaacctggg cgaagtgaccgtggcggaag tgggttggga 1440tgcgctgacc ctgaattgga ccgcaccggaaggcgcgtat aaaaactttt tcatccaggt 1500gctggaagcg gataccacccagaccgtgca gaacctgacc gtgccgggtg gtctgcgtag 1560cgtagatctgcctggtctga aagcagcaac ccgctattac attaccctgc gtggtgttac1620ccaggatttt ggcaccgcac cgctgagcgt tgaagttctg accgaggatctgccgcagct 1680gggtggtctg agcgttaccg aagttagttg ggatggtctgaccctgaatt ggaccaccga 1740tgatctggca tataaacatt ttgtggtgcaggttcaagag gccaataatg ttgaagcagc 1800acagaatctg accgttccgggtagcctgcg tgcagttgat attccgggac tgaaagccga 1860taccccgtatcgtgttagca tttatggtgt tattcagggt tatcgtaccc cgatgctgag1920caccgatgtt agcacagcac gtgaaccgga aattggtaat ctgaatgttagtgatgtgac 1980cccgaaatca tttaatctga gctggaccgc aaccgatggtatttttgata tgtttaccat 2040tgaaattatt gatagcaatc gcctgctgcagaccgcagaa cataacatta gcggtgcaga 2100acgtaccgca catattagcggtctgcctcc gagcaccgat tttattgttt atctgagcgg 2160tattgcaccgagcattcgta ccaaaaccat tagcaccacc gcaaccaccg aagcactgac2220cgcaatgggt agcccgaaag aagtgatttt tagcgatatt accgaaaatagcgccaccgt 2280ttcatggcgt gcaccgaccg cacaggttga aagctttcgtattacctatg ttccgattac 2340cggtggcacc ccgagcatgg ttaccgttgatggcaccaaa acccagaccc gtctggttaa 2400actgattccg ggtgttgaatatctggttag cattattgcc atgaaaggct ttgaagaaag 2460cgaaccggttagcggtagct ttaccacagc tagcggcctg aacgacatct tcgaggctca2520gaaaatcgaa tggcacgaag gtacccatca ccatcaccac cactaa256632565DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotidecynoTNC 3atgtccccta tactaggtta ttggaaaattaagggccttg tgcaacccac tcgacttctt 60ttggaatatc ttgaagaaaa atatgaagagcatttgtatg agcgcgatga aggtgataaa 120tggcgaaaca aaaagtttgaattgggtttg gagtttccca atcttcctta ttatattgat 180ggtgatgttaaattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac240atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaaggagcggttttg 300gatattagat acggtgtttc gagaattgca tatagtaaagactttgaaac tctcaaagtt 360gattttctta gcaagctacc tgaaatgctgaaaatgttcg aagatcgttt atgtcataaa 420acatatttaa atggtgatcatgtaacccat cctgacttca tgttgtatga cgctcttgat 480gttgttttatacatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa540aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaagtatatagca 600tggcctttgc agggctggca agccacgttt ggtggtggcgaccatcctcc aaaatcggat 660ggttcaacta gtggttctgg tcatcaccatcaccatcact ccgcgggtct ggtgccacgc 720ggtagtactg caattggtatgaaagaaacc gctgctgcta aattcgaacg ccagcacatg 780gacagcccagatctgggtac cggtggtggc tccggtattg agggacgcgg gtccatggga840tatcggggat ccgaactgga taccccgaaa gatctgcgtg ttagcgaaaccgcagaaacc 900agcctgaccc tgttttggaa aacaccgctg gcaaaatttgatcgttatcg tctgaattat 960agcctgccga ccggtcagtg ggttggtgttcagctgcctc gtaataccac cagttatgtt 1020ctgcgtggtc tggaaccgggtcaagaatat aacgttctgc tgaccgcaga aaaaggtcgt 1080cataaaagcaaaccggcacg tgttaaagca agcaccgaac aggcaccgga actggaaaat1140ctgaccgtta ccgaagttgg ctgggatggc ctgcgcctga actggacggctgcggaccag 1200gcctacgaac acttcgttat ccaggtgcaa gaagccaacaaagtagaagc cgctcagaat 1260ctgacggttc cgggaaatct gcgtgcagttgatattccgg gtctgaaagc agcaaccccg 1320tataccgtta gcatttatggtgttattcag ggttatcgta caccggttct gagtgccgaa 1380gccagcaccggtgaaacccc gaatctgggt gaagttatgg ttagcgaagt gggctgggat1440gcactgaaac tgaattggac agttccggaa ggtgcctatg aatactttttcattcaggtt 1500caagaagcgg ataccgttga agccgctcag aatcataccgttccgggtgg tctgcgtagc 1560accgatctgc ctggcctgaa agccgctacccattacacca ttaccattcg tggtgttacc 1620caggatttta gcaccacaccgctgagcgtt gaagttctga cagaagaact gccgcagctg 1680ggtgatctggcagttagcga agttggttgg gatggtctgc gtctgaattg gaccgcagca1740gatcaggcat atgaacattt tgttatccag gtgcaagaag tgaacaaagttgaagcagca 1800cagaatctga ccgttccggg tagcctgcgt gcagttgatattccgggtct gaaagcagca 1860accccgtata ccgttagcat ttatggtgttattcgcggtt atcgtacacc ggttctgagc 1920gcagaagcaa gcaccgcaaaagaaccggaa attggtaatc tgaacgtgag cgatattaca 1980ccggaaagttttagcctgag ctggaccgca accgatggta tttttgaaac ctttaccatc2040gagatcatcg atagcaatcg tctgctggaa atcgtggaat ataacattagcggtgcagaa 2100cgtaccgcac atattagcgg tctgcctccg agcaccgattttattgttta tctgagcggt 2160ctggcaccga gctttcgtac caaaaccattagcgcaaccg caaccaccga agcactgacc 2220gcaatgggta gcccgaaagaagtgattttt agcgatatta ccgaaaatag cgccaccgtt 2280tcatggcgtgcaccgaccgc acaggttgaa agctttcgta ttacctatgt tccgattacc2340ggtggcaccc cgagcatggt taccgtggat ggcaccaaaa cccagacccgtctggttaaa 2400ctggttccgg gtgttgaata tctggtgaat atcattgccatgaaaggctt tgaagaaagc 2460gaaccggtta gcggtagctt taccaccgctagcggcctga acgacatctt cgaggctcag 2520aaaatcgaat ggcacgaaggtacccatcac catcaccacc actaa 256541033PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptidehuTNC 4Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys GlyLeu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu LysTyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp ArgAsn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro TyrTyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala Ile IleArg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly Cys ProLys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val Leu AspIle Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys Asp PheGlu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125MetLeu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala LeuAsp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp AlaPhe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala IlePro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr Ile AlaTrp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly Gly AspHis Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser Gly HisHis His His His His Ser Ala Gly Leu Val Pro Arg225 230 235 240GlySer Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu 245 250255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly260 265 270Ile Glu Gly Arg Gly Ser Met Gly Tyr Arg Gly Ser Glu LeuAsp Thr 275 280 285Pro Lys Asp Leu Gln Val Ser Glu Thr Ala Glu ThrSer Leu Thr Leu 290 295 300Leu Trp Lys Thr Pro Leu Ala Lys Phe AspArg Tyr Arg Leu Asn Tyr305 310 315 320Ser Leu Pro Thr Gly Gln TrpVal Gly Val Gln Leu Pro Arg Asn Thr 325 330 335Thr Ser Tyr Val LeuArg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val 340 345 350Leu Leu ThrAla Glu Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val 355 360 365LysAla Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr 370 375380Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala AspGln385 390 395 400Ala Tyr Glu His Phe Ile Ile Gln Val Gln Glu AlaAsn Lys Val Glu 405 410 415Ala Ala Arg Asn Leu Thr Val Pro Gly SerLeu Arg Ala Val Asp Ile 420 425 430Pro Gly Leu Lys Ala Ala Thr ProTyr Thr Val Ser Ile Tyr Gly Val 435 440 445Ile Gln Gly Tyr Arg ThrPro Val Leu Ser Ala Glu Ala Ser Thr Gly 450 455 460Glu Thr Pro AsnLeu Gly Glu Val Val Val Ala Glu Val Gly Trp Asp465 470 475 480AlaLeu Lys Leu Asn Trp Thr Ala Pro Glu Gly Ala Tyr Glu Tyr Phe 485 490495Phe Ile Gln Val Gln Glu Ala Asp Thr Val Glu Ala Ala Gln Asn Leu500 505 510Thr Val Pro Gly Gly Leu Arg Ser Thr Asp Leu Pro Gly LeuLys Ala 515 520 525Ala Thr His Tyr Thr Ile Thr Ile Arg Gly Val ThrGln Asp Phe Ser 530 535 540Thr Thr Pro Leu Ser Val Glu Val Leu ThrGlu Glu Val Pro Asp Met545 550 555 560Gly Asn Leu Thr Val Thr GluVal Ser Trp Asp Ala Leu Arg Leu Asn 565 570 575Trp Thr Thr Pro AspGly Thr Tyr Asp Gln Phe Thr Ile Gln Val Gln 580 585 590Glu Ala AspGln Val Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser 595 600 605LeuArg Ser Met Glu Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr 610 615620Val Thr Leu His Gly Glu Val Arg Gly His Ser Thr Arg Pro LeuAla625 630 635 640Val Glu Val Val Thr Glu Asp Leu Pro Gln Leu GlyAsp Leu Ala Val 645 650 655Ser Glu Val Gly Trp Asp Gly Leu Arg LeuAsn Trp Thr Ala Ala Asp 660 665 670Asn Ala Tyr Glu His Phe Val IleGln Val Gln Glu Val Asn Lys Val 675 680 685Glu Ala Ala Gln Asn LeuThr Leu Pro Gly Ser Leu Arg Ala Val Asp 690 695 700Ile Pro Gly LeuGlu Ala Ala Thr Pro Tyr Arg Val Ser Ile Tyr Gly705 710 715 720ValIle Arg Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr 725 730735Ala Lys Glu Pro Glu Ile Gly Asn Leu Asn Val Ser Asp Ile Thr Pro740 745 750Glu Ser Phe Asn Leu Ser Trp Met Ala Thr Asp Gly Ile PheGlu Thr 755 760 765Phe Thr Ile Glu Ile Ile Asp Ser Asn Arg Leu LeuGlu Thr Val Glu 770 775 780Tyr Asn Ile Ser Gly Ala Glu Arg Thr AlaHis Ile Ser Gly Leu Pro785 790 795 800Pro Ser Thr Asp Phe Ile ValTyr Leu Ser Gly Leu Ala Pro Ser Ile 805 810 815Arg Thr Lys Thr IleSer Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu 820 825 830Leu Glu AsnLeu Thr Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val 835 840 845SerTrp Met Ala Ser Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val 850 855860Val Asp Ser Gly Lys Leu Leu Asp Pro Gln Glu Phe Thr Leu SerGly865 870 875 880Thr Gln Arg Lys Leu Glu Leu Arg Gly Leu Ile ThrGly Ile Gly Tyr 885 890 895Glu Val Met Val Ser Gly Phe Thr Gln GlyHis Gln Thr Lys Pro Leu 900 905 910Arg Ala Glu Ile Val Thr Glu AlaMet Gly Ser Pro Lys Glu Val Ile 915 920 925Phe Ser Asp Ile Thr GluAsn Ser Ala Thr Val Ser Trp Arg Ala Pro 930 935 940Thr Ala Gln ValGlu Ser Phe Arg Ile Thr Tyr Val Pro Ile Thr Gly945 950 955 960GlyThr Pro Ser Met Val Thr Val Asp Gly Thr Lys Thr Gln Thr Arg 965 970975Leu Val Lys Leu Ile Pro Gly Val Glu Tyr Leu Val Ser Ile Ile Ala980 985 990Met Lys Gly Phe Glu Glu Ser Glu Pro Val Ser Gly Ser PheThr Thr 995 1000 1005Ala Ser Gly Leu Asn Asp Ile Phe Glu Ala GlnLys Ile Glu Trp 1010 1015 1020His Glu Gly Thr His His His His HisHis 1025 10305854PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptidemuTNC 5Met Ser Pro Ile Leu Gly TyrTrp Lys Ile

Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu GluGlu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp LysTrp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn LeuPro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met AlaIle Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly GlyCys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala ValLeu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110LysAsp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp AlaLeu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu AspAla Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu AlaIle Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr IleAla Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly GlyAsp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser GlyHis His His His His His Ser Ala Gly Leu Val Pro Arg225 230 235240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu245 250 255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly GlySer Gly 260 265 270Ile Glu Gly Arg Gly Ser Met Gly Tyr Arg Gly SerGlu Leu Asp Thr 275 280 285Pro Lys Asp Leu Gln Val Ser Glu Thr AlaGlu Thr Ser Leu Thr Leu 290 295 300Leu Trp Lys Thr Pro Leu Ala LysPhe Asp Arg Tyr Arg Leu Asn Tyr305 310 315 320Ser Leu Pro Thr GlyGln Trp Val Gly Val Gln Leu Pro Arg Asn Thr 325 330 335Thr Ser TyrVal Leu Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val 340 345 350LeuLeu Thr Ala Glu Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val 355 360365Lys Ala Ser Thr Glu Glu Val Pro Ser Leu Glu Asn Leu Thr Val Thr370 375 380Glu Ala Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala AspAsp Leu385 390 395 400Ala Tyr Glu Tyr Phe Val Ile Gln Val Gln GluAla Asn Asn Val Glu 405 410 415Thr Ala His Asn Phe Thr Val Pro GlyAsn Leu Arg Ala Ala Asp Ile 420 425 430Pro Gly Leu Lys Val Ala ThrSer Tyr Arg Val Ser Ile Tyr Gly Val 435 440 445Ala Arg Gly Tyr ArgThr Pro Val Leu Ser Ala Glu Thr Ser Thr Gly 450 455 460Thr Thr ProAsn Leu Gly Glu Val Thr Val Ala Glu Val Gly Trp Asp465 470 475480Ala Leu Thr Leu Asn Trp Thr Ala Pro Glu Gly Ala Tyr Lys Asn Phe485 490 495Phe Ile Gln Val Leu Glu Ala Asp Thr Thr Gln Thr Val GlnAsn Leu 500 505 510Thr Val Pro Gly Gly Leu Arg Ser Val Asp Leu ProGly Leu Lys Ala 515 520 525Ala Thr Arg Tyr Tyr Ile Thr Leu Arg GlyVal Thr Gln Asp Phe Gly 530 535 540Thr Ala Pro Leu Ser Val Glu ValLeu Thr Glu Asp Leu Pro Gln Leu545 550 555 560Gly Gly Leu Ser ValThr Glu Val Ser Trp Asp Gly Leu Thr Leu Asn 565 570 575Trp Thr ThrAsp Asp Leu Ala Tyr Lys His Phe Val Val Gln Val Gln 580 585 590GluAla Asn Asn Val Glu Ala Ala Gln Asn Leu Thr Val Pro Gly Ser 595 600605Leu Arg Ala Val Asp Ile Pro Gly Leu Lys Ala Asp Thr Pro Tyr Arg610 615 620Val Ser Ile Tyr Gly Val Ile Gln Gly Tyr Arg Thr Pro MetLeu Ser625 630 635 640Thr Asp Val Ser Thr Ala Arg Glu Pro Glu IleGly Asn Leu Asn Val 645 650 655Ser Asp Val Thr Pro Lys Ser Phe AsnLeu Ser Trp Thr Ala Thr Asp 660 665 670Gly Ile Phe Asp Met Phe ThrIle Glu Ile Ile Asp Ser Asn Arg Leu 675 680 685Leu Gln Thr Ala GluHis Asn Ile Ser Gly Ala Glu Arg Thr Ala His 690 695 700Ile Ser GlyLeu Pro Pro Ser Thr Asp Phe Ile Val Tyr Leu Ser Gly705 710 715720Ile Ala Pro Ser Ile Arg Thr Lys Thr Ile Ser Thr Thr Ala Thr Thr725 730 735Glu Ala Leu Thr Ala Met Gly Ser Pro Lys Glu Val Ile PheSer Asp 740 745 750Ile Thr Glu Asn Ser Ala Thr Val Ser Trp Arg AlaPro Thr Ala Gln 755 760 765Val Glu Ser Phe Arg Ile Thr Tyr Val ProIle Thr Gly Gly Thr Pro 770 775 780Ser Met Val Thr Val Asp Gly ThrLys Thr Gln Thr Arg Leu Val Lys785 790 795 800Leu Ile Pro Gly ValGlu Tyr Leu Val Ser Ile Ile Ala Met Lys Gly 805 810 815Phe Glu GluSer Glu Pro Val Ser Gly Ser Phe Thr Thr Ala Ser Gly 820 825 830LeuAsn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Thr 835 840845His His His His His His 8506854PRTArtificial SequenceDescriptionof Artificial Sequence Synthetic polypeptidecynoTNC 6Met Ser ProIle Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr ArgLeu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30TyrGlu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 4045Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys HisAsn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile SerMet Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser ArgIle Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp PheLeu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp ArgLeu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr HisPro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val LeuTyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175ValCys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly SerThr Ser 210 215 220Gly Ser Gly His His His His His His Ser Ala GlyLeu Val Pro Arg225 230 235 240Gly Ser Thr Ala Ile Gly Met Lys GluThr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln His Met Asp Ser ProAsp Leu Gly Thr Gly Gly Gly Ser Gly 260 265 270Ile Glu Gly Arg GlySer Met Gly Tyr Arg Gly Ser Glu Leu Asp Thr 275 280 285Pro Lys AspLeu Arg Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu 290 295 300PheTrp Lys Thr Pro Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr305 310315 320Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln Leu Pro Arg AsnThr 325 330 335Thr Ser Tyr Val Leu Arg Gly Leu Glu Pro Gly Gln GluTyr Asn Val 340 345 350Leu Leu Thr Ala Glu Lys Gly Arg His Lys SerLys Pro Ala Arg Val 355 360 365Lys Ala Ser Thr Glu Gln Ala Pro GluLeu Glu Asn Leu Thr Val Thr 370 375 380Glu Val Gly Trp Asp Gly LeuArg Leu Asn Trp Thr Ala Ala Asp Gln385 390 395 400Ala Tyr Glu HisPhe Val Ile Gln Val Gln Glu Ala Asn Lys Val Glu 405 410 415Ala AlaGln Asn Leu Thr Val Pro Gly Asn Leu Arg Ala Val Asp Ile 420 425430Pro Gly Leu Lys Ala Ala Thr Pro Tyr Thr Val Ser Ile Tyr Gly Val435 440 445Ile Gln Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala SerThr Gly 450 455 460Glu Thr Pro Asn Leu Gly Glu Val Met Val Ser GluVal Gly Trp Asp465 470 475 480Ala Leu Lys Leu Asn Trp Thr Val ProGlu Gly Ala Tyr Glu Tyr Phe 485 490 495Phe Ile Gln Val Gln Glu AlaAsp Thr Val Glu Ala Ala Gln Asn His 500 505 510Thr Val Pro Gly GlyLeu Arg Ser Thr Asp Leu Pro Gly Leu Lys Ala 515 520 525Ala Thr HisTyr Thr Ile Thr Ile Arg Gly Val Thr Gln Asp Phe Ser 530 535 540ThrThr Pro Leu Ser Val Glu Val Leu Thr Glu Glu Leu Pro Gln Leu545 550555 560Gly Asp Leu Ala Val Ser Glu Val Gly Trp Asp Gly Leu Arg LeuAsn 565 570 575Trp Thr Ala Ala Asp Gln Ala Tyr Glu His Phe Val IleGln Val Gln 580 585 590Glu Val Asn Lys Val Glu Ala Ala Gln Asn LeuThr Val Pro Gly Ser 595 600 605Leu Arg Ala Val Asp Ile Pro Gly LeuLys Ala Ala Thr Pro Tyr Thr 610 615 620Val Ser Ile Tyr Gly Val IleArg Gly Tyr Arg Thr Pro Val Leu Ser625 630 635 640Ala Glu Ala SerThr Ala Lys Glu Pro Glu Ile Gly Asn Leu Asn Val 645 650 655Ser AspIle Thr Pro Glu Ser Phe Ser Leu Ser Trp Thr Ala Thr Asp 660 665670Gly Ile Phe Glu Thr Phe Thr Ile Glu Ile Ile Asp Ser Asn Arg Leu675 680 685Leu Glu Ile Val Glu Tyr Asn Ile Ser Gly Ala Glu Arg ThrAla His 690 695 700Ile Ser Gly Leu Pro Pro Ser Thr Asp Phe Ile ValTyr Leu Ser Gly705 710 715 720Leu Ala Pro Ser Phe Arg Thr Lys ThrIle Ser Ala Thr Ala Thr Thr 725 730 735Glu Ala Leu Thr Ala Met GlySer Pro Lys Glu Val Ile Phe Ser Asp 740 745 750Ile Thr Glu Asn SerAla Thr Val Ser Trp Arg Ala Pro Thr Ala Gln 755 760 765Val Glu SerPhe Arg Ile Thr Tyr Val Pro Ile Thr Gly Gly Thr Pro 770 775 780SerMet Val Thr Val Asp Gly Thr Lys Thr Gln Thr Arg Leu Val Lys785 790795 800Leu Val Pro Gly Val Glu Tyr Leu Val Asn Ile Ile Ala Met LysGly 805 810 815Phe Glu Glu Ser Glu Pro Val Ser Gly Ser Phe Thr ThrAla Ser Gly 820 825 830Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile GluTrp His Glu Gly Thr 835 840 845His His His His His His85071033PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideGST huTNC fn5 A1234 BC fn6 B 7Met Ser Pro IleLeu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg LeuLeu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr GluArg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45GlyLeu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 5560Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn6570 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met LeuGlu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile AlaTyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu SerLys Leu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu CysHis Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro AspPhe Met Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr MetAsp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys PheLys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190LeuLys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser210 215 220Gly Ser Gly His His His His His His Ser Ala Gly Leu ValPro Arg225 230 235 240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr AlaAla Ala Lys Phe Glu 245 250 255Arg Gln His Met Asp Ser Pro Asp LeuGly Thr Gly Gly Gly Ser Gly 260 265 270Ile Glu Gly Arg Gly Ser MetGly Tyr Arg Gly Ser Glu Leu Asp Thr 275 280 285Pro Lys Asp Leu GlnVal Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu 290 295 300Leu Trp LysThr Pro Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr305 310 315320Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln Leu Pro Arg Asn Thr325 330 335Thr Ser Tyr Val Leu Arg Gly Leu Glu Pro Gly Gln Glu TyrAsn Val 340 345 350Leu Leu Thr Ala Glu Lys Gly Arg His Lys Ser LysPro Ala Arg Val 355 360 365Lys Ala Ser Thr Glu Gln Ala Pro Glu LeuGlu Asn Leu Thr Val Thr 370 375 380Glu Val Gly Trp Asp Gly Leu ArgLeu Asn Trp Thr Ala Ala Asp Gln385 390 395 400Ala Tyr Glu His PheIle Ile Gln Val Gln Glu Ala Asn Lys Val Glu 405 410 415Ala Ala ArgAsn Leu Thr Val Pro Gly Ser Leu Arg Ala Val Asp Ile 420 425 430ProGly Leu Lys Ala Ala Thr Pro Tyr Thr Val Ser Ile Tyr Gly Val 435 440445Ile Gln Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr Gly450 455 460Glu Thr Pro Asn Leu Gly Glu Val Val Val Ala Glu Val GlyTrp Asp465 470 475 480Ala Leu Lys Leu Asn Trp Thr Ala Pro Glu GlyAla Tyr Glu Tyr Phe 485 490 495Phe Ile Gln Val Gln Glu Ala Asp ThrVal Glu Ala Ala Gln Asn Leu 500 505 510Thr Val Pro Gly Gly Leu ArgSer Thr Asp Leu Pro Gly Leu Lys Ala 515 520 525Ala Thr His Tyr ThrIle Thr Ile Arg Gly Val Thr Gln Asp Phe Ser 530 535 540Thr Thr ProLeu Ser Val Glu Val Leu Thr Glu Glu Val Pro Asp Met545 550 555560Gly Asn Leu Thr Val Thr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn565 570 575Trp Thr Thr Pro Asp Gly Thr Tyr Asp Gln Phe Thr Ile GlnVal Gln 580 585 590Glu Ala Asp Gln Val Glu Glu Ala His Asn Leu ThrVal Pro Gly Ser 595 600 605Leu Arg Ser Met Glu Ile Pro Gly Leu ArgAla Gly Thr Pro Tyr Thr 610 615 620Val Thr Leu His Gly Glu Val ArgGly His Ser Thr Arg Pro Leu Ala625 630 635 640Val Glu Val Val ThrGlu Asp Leu Pro Gln Leu Gly Asp Leu Ala Val 645 650 655Ser Glu ValGly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp 660 665 670AsnAla Tyr Glu His Phe Val Ile Gln Val Gln Glu Val Asn Lys Val 675 680685Glu Ala Ala Gln Asn Leu Thr Leu Pro Gly Ser Leu Arg Ala Val Asp690 695 700Ile Pro Gly Leu Glu Ala Ala Thr Pro Tyr Arg Val Ser IleTyr Gly705 710 715 720Val Ile Arg Gly Tyr Arg Thr Pro Val Leu SerAla Glu Ala Ser Thr 725 730 735Ala Lys Glu Pro Glu Ile Gly Asn LeuAsn Val Ser Asp Ile Thr Pro

740 745 750Glu Ser Phe Asn Leu Ser Trp Met Ala Thr Asp Gly Ile PheGlu Thr 755 760 765Phe Thr Ile Glu Ile Ile Asp Ser Asn Arg Leu LeuGlu Thr Val Glu 770 775 780Tyr Asn Ile Ser Gly Ala Glu Arg Thr AlaHis Ile Ser Gly Leu Pro785 790 795 800Pro Ser Thr Asp Phe Ile ValTyr Leu Ser Gly Leu Ala Pro Ser Ile 805 810 815Arg Thr Lys Thr IleSer Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu 820 825 830Leu Glu AsnLeu Thr Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val 835 840 845SerTrp Met Ala Ser Glu Asn Ala Phe Asp Ser Phe Leu Val Thr Val 850 855860Val Asp Ser Gly Lys Leu Leu Asp Pro Gln Glu Phe Thr Leu SerGly865 870 875 880Thr Gln Arg Lys Leu Glu Leu Arg Gly Leu Ile ThrGly Ile Gly Tyr 885 890 895Glu Val Met Val Ser Gly Phe Thr Gln GlyHis Gln Thr Lys Pro Leu 900 905 910Arg Ala Glu Ile Val Thr Glu AlaMet Gly Ser Pro Lys Glu Val Ile 915 920 925Phe Ser Asp Ile Thr GluAsn Ser Ala Thr Val Ser Trp Arg Ala Pro 930 935 940Thr Ala Gln ValGlu Ser Phe Arg Ile Thr Tyr Val Pro Ile Thr Gly945 950 955 960GlyThr Pro Ser Met Val Thr Val Asp Gly Thr Lys Thr Gln Thr Arg 965 970975Leu Val Lys Leu Ile Pro Gly Val Glu Tyr Leu Val Ser Ile Ile Ala980 985 990Met Lys Gly Phe Glu Glu Ser Glu Pro Val Ser Gly Ser PheThr Thr 995 1000 1005Ala Ser Gly Leu Asn Asp Ile Phe Glu Ala GlnLys Ile Glu Trp 1010 1015 1020His Glu Gly Thr His His His His HisHis 1025 10308854PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptideGST huTNCfn5 mu A124 BC hu fn6 B 8MetSer Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 1015Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe GluLeu 35 40 45Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly AspVal Lys 50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala AspLys His Asn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala GluIle Ser Met Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly ValSer Arg Ile Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys ValAsp Phe Leu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys Met Phe GluAsp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His ValThr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160ValVal Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr180 185 190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly TrpGln Ala 195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser AspGly Ser Thr Ser 210 215 220Gly Ser Gly His His His His His His SerAla Gly Leu Val Pro Arg225 230 235 240Gly Ser Thr Ala Ile Gly MetLys Glu Thr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln His Met AspSer Pro Asp Leu Gly Thr Gly Gly Gly Ser Gly 260 265 270Ile Glu GlyArg Gly Ser Met Gly Tyr Arg Gly Ser Glu Leu Asp Thr 275 280 285ProLys Asp Leu Gln Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu 290 295300Leu Trp Lys Thr Pro Leu Ala Lys Phe Asp Arg Tyr Arg Leu AsnTyr305 310 315 320Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln LeuPro Arg Asn Thr 325 330 335Thr Ser Tyr Val Leu Arg Gly Leu Glu ProGly Gln Glu Tyr Asn Val 340 345 350Leu Leu Thr Ala Glu Lys Gly ArgHis Lys Ser Lys Pro Ala Arg Val 355 360 365Lys Ala Ser Thr Glu GluVal Pro Ser Leu Glu Asn Leu Thr Val Thr 370 375 380Glu Ala Gly TrpAsp Gly Leu Arg Leu Asn Trp Thr Ala Asp Asp Leu385 390 395 400AlaTyr Glu Tyr Phe Val Ile Gln Val Gln Glu Ala Asn Asn Val Glu 405 410415Thr Ala His Asn Phe Thr Val Pro Gly Asn Leu Arg Ala Ala Asp Ile420 425 430Pro Gly Leu Lys Val Ala Thr Ser Tyr Arg Val Ser Ile TyrGly Val 435 440 445Ala Arg Gly Tyr Arg Thr Pro Val Leu Ser Ala GluThr Ser Thr Gly 450 455 460Thr Thr Pro Asn Leu Gly Glu Val Thr ValAla Glu Val Gly Trp Asp465 470 475 480Ala Leu Thr Leu Asn Trp ThrAla Pro Glu Gly Ala Tyr Lys Asn Phe 485 490 495Phe Ile Gln Val LeuGlu Ala Asp Thr Thr Gln Thr Val Gln Asn Leu 500 505 510Thr Val ProGly Gly Leu Arg Ser Val Asp Leu Pro Gly Leu Lys Ala 515 520 525AlaThr Arg Tyr Tyr Ile Thr Leu Arg Gly Val Thr Gln Asp Phe Gly 530 535540Thr Ala Pro Leu Ser Val Glu Val Leu Thr Glu Asp Leu Pro GlnLeu545 550 555 560Gly Gly Leu Ser Val Thr Glu Val Ser Trp Asp GlyLeu Thr Leu Asn 565 570 575Trp Thr Thr Asp Asp Leu Ala Tyr Lys HisPhe Val Val Gln Val Gln 580 585 590Glu Ala Asn Asn Val Glu Ala AlaGln Asn Leu Thr Val Pro Gly Ser 595 600 605Leu Arg Ala Val Asp IlePro Gly Leu Lys Ala Asp Thr Pro Tyr Arg 610 615 620Val Ser Ile TyrGly Val Ile Gln Gly Tyr Arg Thr Pro Met Leu Ser625 630 635 640ThrAsp Val Ser Thr Ala Arg Glu Pro Glu Ile Gly Asn Leu Asn Val 645 650655Ser Asp Val Thr Pro Lys Ser Phe Asn Leu Ser Trp Thr Ala Thr Asp660 665 670Gly Ile Phe Asp Met Phe Thr Ile Glu Ile Ile Asp Ser AsnArg Leu 675 680 685Leu Gln Thr Ala Glu His Asn Ile Ser Gly Ala GluArg Thr Ala His 690 695 700Ile Ser Gly Leu Pro Pro Ser Thr Asp PheIle Val Tyr Leu Ser Gly705 710 715 720Ile Ala Pro Ser Ile Arg ThrLys Thr Ile Ser Thr Thr Ala Thr Thr 725 730 735Glu Ala Leu Thr AlaMet Gly Ser Pro Lys Glu Val Ile Phe Ser Asp 740 745 750Ile Thr GluAsn Ser Ala Thr Val Ser Trp Arg Ala Pro Thr Ala Gln 755 760 765ValGlu Ser Phe Arg Ile Thr Tyr Val Pro Ile Thr Gly Gly Thr Pro 770 775780Ser Met Val Thr Val Asp Gly Thr Lys Thr Gln Thr Arg Leu ValLys785 790 795 800Leu Ile Pro Gly Val Glu Tyr Leu Val Ser Ile IleAla Met Lys Gly 805 810 815Phe Glu Glu Ser Glu Pro Val Ser Gly SerPhe Thr Thr Ala Ser Gly 820 825 830Leu Asn Asp Ile Phe Glu Ala GlnLys Ile Glu Trp His Glu Gly Thr 835 840 845His His His His His His8509668PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideGST TNC hu fn5 B-C fn6 B 9Met Ser Pro Ile LeuGly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu LeuLeu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu ArgAsp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly LeuGlu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60LeuThr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 7580Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala TyrSer 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser LysLeu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys HisLys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr His Pro Asp PheMet Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met AspPro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe LysLys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu LysSer Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser210 215 220Gly Ser Gly His His His His His His Ser Ala Gly Leu ValPro Arg225 230 235 240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr AlaAla Ala Lys Phe Glu 245 250 255Arg Gln His Met Asp Ser Pro Asp LeuGly Thr Gly Gly Gly Ser Gly 260 265 270Ile Glu Gly Arg Gly Ser MetGly Tyr Arg Gly Ser Glu Leu Asp Thr 275 280 285Pro Lys Asp Leu GlnVal Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu 290 295 300Leu Trp LysThr Pro Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr305 310 315320Ser Leu Pro Thr Gly Gln Trp Val Gly Val Gln Leu Pro Arg Asn Thr325 330 335Thr Ser Tyr Val Leu Arg Gly Leu Glu Pro Gly Gln Glu TyrAsn Val 340 345 350Leu Leu Thr Ala Glu Lys Gly Arg His Lys Ser LysPro Ala Arg Val 355 360 365Lys Ala Ser Thr Ala Lys Glu Pro Glu IleGly Asn Leu Asn Val Ser 370 375 380Asp Ile Thr Pro Glu Ser Phe AsnLeu Ser Trp Met Ala Thr Asp Gly385 390 395 400Ile Phe Glu Thr PheThr Ile Glu Ile Ile Asp Ser Asn Arg Leu Leu 405 410 415Glu Thr ValGlu Tyr Asn Ile Ser Gly Ala Glu Arg Thr Ala His Ile 420 425 430SerGly Leu Pro Pro Ser Thr Asp Phe Ile Val Tyr Leu Ser Gly Leu 435 440445Ala Pro Ser Ile Arg Thr Lys Thr Ile Ser Ala Thr Ala Thr Thr Glu450 455 460Ala Leu Pro Leu Leu Glu Asn Leu Thr Ile Ser Asp Ile AsnPro Tyr465 470 475 480Gly Phe Thr Val Ser Trp Met Ala Ser Glu AsnAla Phe Asp Ser Phe 485 490 495Leu Val Thr Val Val Asp Ser Gly LysLeu Leu Asp Pro Gln Glu Phe 500 505 510Thr Leu Ser Gly Thr Gln ArgLys Leu Glu Leu Arg Gly Leu Ile Thr 515 520 525Gly Ile Gly Tyr GluVal Met Val Ser Gly Phe Thr Gln Gly His Gln 530 535 540Thr Lys ProLeu Arg Ala Glu Ile Val Thr Ala Met Gly Ser Pro Lys545 550 555560Glu Val Ile Phe Ser Asp Ile Thr Glu Asn Ser Ala Thr Val Ser Trp565 570 575Arg Ala Pro Thr Ala Gln Val Glu Ser Phe Arg Ile Thr TyrVal Pro 580 585 590Ile Thr Gly Gly Thr Pro Ser Met Val Thr Val AspGly Thr Lys Thr 595 600 605Gln Thr Arg Leu Val Lys Leu Ile Pro GlyVal Glu Tyr Leu Val Ser 610 615 620Ile Ile Ala Met Lys Gly Phe GluGlu Ser Glu Pro Val Ser Gly Ser625 630 635 640Phe Thr Thr Ala SerGly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile 645 650 655Glu Trp HisGlu Gly Thr His His His His His His 660 66510853PRTArtificialSequenceDescription of Artificial Sequence Synthetic polypeptideGSThuTNC fn5 A1234 fn6 B 10Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile LysGly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu Tyr Leu Glu GluLys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu Gly Asp Lys TrpArg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe Pro Asn Leu ProTyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln Ser Met Ala IleIle Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met Leu Gly Gly CysPro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95Gly Ala Val LeuAsp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110Lys AspPhe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp AlaLeu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met Cys Leu AspAla Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu AlaIle Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser Lys Tyr IleAla Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe Gly Gly GlyAsp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220Gly Ser GlyHis His His His His His Ser Ala Gly Leu Val Pro Arg225 230 235240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu245 250 255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly Gly GlySer Gly 260 265 270Ile Glu Gly Arg Gly Ser Met Gly Tyr Arg Gly SerGlu Leu Asp Thr 275 280 285Pro Lys Asp Leu Gln Val Ser Glu Thr AlaGlu Thr Ser Leu Thr Leu 290 295 300Leu Trp Lys Thr Pro Leu Ala LysPhe Asp Arg Tyr Arg Leu Asn Tyr305 310 315 320Ser Leu Pro Thr GlyGln Trp Val Gly Val Gln Leu Pro Arg Asn Thr 325 330 335Thr Ser TyrVal Leu Arg Gly Leu Glu Pro Gly Gln Glu Tyr Asn Val 340 345 350LeuLeu Thr Ala Glu Lys Gly Arg His Lys Ser Lys Pro Ala Arg Val 355 360365Lys Ala Ser Thr Glu Gln Ala Pro Glu Leu Glu Asn Leu Thr Val Thr370 375 380Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr Ala AlaAsp Gln385 390 395 400Ala Tyr Glu His Phe Ile Ile Gln Val Gln GluAla Asn Lys Val Glu 405 410 415Ala Ala Arg Asn Leu Thr Val Pro GlySer Leu Arg Ala Val Asp Ile 420 425 430Pro Gly Leu Lys Ala Ala ThrPro Tyr Thr Val Ser Ile Tyr Gly Val 435 440 445Ile Gln Gly Tyr ArgThr Pro Val Leu Ser Ala Glu Ala Ser Thr Gly 450 455 460Glu Thr ProAsn Leu Gly Glu Val Val Val Ala Glu Val Gly Trp Asp465 470 475480Ala Leu Lys Leu Asn Trp Thr Ala Pro Glu Gly Ala Tyr Glu Tyr Phe485 490 495Phe Ile Gln Val Gln Glu Ala Asp Thr Val Glu Ala Ala GlnAsn Leu 500 505 510Thr Val Pro Gly Gly Leu Arg Ser Thr Asp Leu ProGly Leu Lys Ala 515 520 525Ala Thr His Tyr Thr Ile Thr Ile Arg GlyVal Thr Gln Asp Phe Ser 530 535 540Thr Thr Pro Leu Ser Val Glu ValLeu Thr Glu Glu Val Pro Asp Met545 550 555 560Gly Asn Leu Thr ValThr Glu Val Ser Trp Asp Ala Leu Arg Leu Asn 565 570 575Trp Thr ThrPro Asp Gly Thr Tyr Asp Gln Phe Thr Ile Gln Val Gln 580 585 590GluAla Asp Gln Val Glu Glu Ala His Asn Leu Thr Val Pro Gly Ser 595 600605Leu Arg Ser Met Glu Ile Pro Gly Leu Arg Ala Gly Thr Pro Tyr Thr610 615

620Val Thr Leu His Gly Glu Val Arg Gly His Ser Thr Arg Pro LeuAla625 630 635 640Val Glu Val Val Thr Glu Asp Leu Pro Gln Leu GlyAsp Leu Ala Val 645 650 655Ser Glu Val Gly Trp Asp Gly Leu Arg LeuAsn Trp Thr Ala Ala Asp 660 665 670Asn Ala Tyr Glu His Phe Val IleGln Val Gln Glu Val Asn Lys Val 675 680 685Glu Ala Ala Gln Asn LeuThr Leu Pro Gly Ser Leu Arg Ala Val Asp 690 695 700Ile Pro Gly LeuGlu Ala Ala Thr Pro Tyr Arg Val Ser Ile Tyr Gly705 710 715 720ValIle Arg Gly Tyr Arg Thr Pro Val Leu Ser Ala Glu Ala Ser Thr 725 730735Ala Lys Glu Ala Met Gly Ser Pro Lys Glu Val Ile Phe Ser Asp Ile740 745 750Thr Glu Asn Ser Ala Thr Val Ser Trp Arg Ala Pro Thr AlaGln Val 755 760 765Glu Ser Phe Arg Ile Thr Tyr Val Pro Ile Thr GlyGly Thr Pro Ser 770 775 780Met Val Thr Val Asp Gly Thr Lys Thr GlnThr Arg Leu Val Lys Leu785 790 795 800Ile Pro Gly Val Glu Tyr LeuVal Ser Ile Ile Ala Met Lys Gly Phe 805 810 815Glu Glu Ser Glu ProVal Ser Gly Ser Phe Thr Thr Ala Ser Gly Leu 820 825 830Asn Asp IlePhe Glu Ala Gln Lys Ile Glu Trp His Glu Gly Thr His 835 840 845HisHis His His His 85011400PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptidehuTNC A4 B 11Met Ser ProIle Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr ArgLeu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30TyrGlu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 4045Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys50 55 60Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys HisAsn65 70 75 80Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile SerMet Leu Glu 85 90 95Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser ArgIle Ala Tyr Ser 100 105 110Lys Asp Phe Glu Thr Leu Lys Val Asp PheLeu Ser Lys Leu Pro Glu 115 120 125Met Leu Lys Met Phe Glu Asp ArgLeu Cys His Lys Thr Tyr Leu Asn 130 135 140Gly Asp His Val Thr HisPro Asp Phe Met Leu Tyr Asp Ala Leu Asp145 150 155 160Val Val LeuTyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175ValCys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185190Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala195 200 205Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Gly SerThr Ser 210 215 220Gly Ser Gly His His His His His His Ser Ala GlyLeu Val Pro Arg225 230 235 240Gly Ser Thr Ala Ile Gly Met Lys GluThr Ala Ala Ala Lys Phe Glu 245 250 255Arg Gln His Met Asp Ser ProAsp Leu Gly Thr Gly Gly Gly Ser Gly 260 265 270Ile Glu Gly Arg GlySer Met Gly Tyr Arg Gly Ser Glu Asp Leu Pro 275 280 285Gln Leu GlyAsp Leu Ala Val Ser Glu Val Gly Trp Asp Gly Leu Arg 290 295 300LeuAsn Trp Thr Ala Ala Asp Asn Ala Tyr Glu His Phe Val Ile Gln305 310315 320Val Gln Glu Val Asn Lys Val Glu Ala Ala Gln Asn Leu Thr LeuPro 325 330 335Gly Ser Leu Arg Ala Val Asp Ile Pro Gly Leu Glu AlaAla Thr Pro 340 345 350Tyr Arg Val Ser Ile Tyr Gly Val Ile Arg GlyTyr Arg Thr Pro Val 355 360 365Leu Ser Ala Glu Ala Ser Thr Ala SerGly Leu Asn Asp Ile Phe Glu 370 375 380Ala Gln Lys Ile Glu Trp HisGlu Gly Thr His His His His His His385 390 39540012400PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptidehuTNC A1 B 12Met Ser Pro Ile Leu Gly Tyr TrpLys Ile Lys Gly Leu Val Gln Pro1 5 10 15Thr Arg Leu Leu Leu Glu TyrLeu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30Tyr Glu Arg Asp Glu GlyAsp Lys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45Gly Leu Glu Phe ProAsn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60Leu Thr Gln SerMet Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn65 70 75 80Met LeuGly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95GlyAla Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105110Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu115 120 125Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr TyrLeu Asn 130 135 140Gly Asp His Val Thr His Pro Asp Phe Met Leu TyrAsp Ala Leu Asp145 150 155 160Val Val Leu Tyr Met Asp Pro Met CysLeu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg IleGlu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190Leu Lys Ser Ser LysTyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205Thr Phe GlyGly Gly Asp His Pro Pro Lys Ser Asp Gly Ser Thr Ser 210 215 220GlySer Gly His His His His His His Ser Ala Gly Leu Val Pro Arg225 230235 240Gly Ser Thr Ala Ile Gly Met Lys Glu Thr Ala Ala Ala Lys PheGlu 245 250 255Arg Gln His Met Asp Ser Pro Asp Leu Gly Thr Gly GlyGly Ser Gly 260 265 270Ile Glu Gly Arg Gly Ser Met Gly Tyr Arg GlySer Glu Gln Ala Pro 275 280 285Glu Leu Glu Asn Leu Thr Val Thr GluVal Gly Trp Asp Gly Leu Arg 290 295 300Leu Asn Trp Thr Ala Ala AspGln Ala Tyr Glu His Phe Ile Ile Gln305 310 315 320Val Gln Glu AlaAsn Lys Val Glu Ala Ala Arg Asn Leu Thr Val Pro 325 330 335Gly SerLeu Arg Ala Val Asp Ile Pro Gly Leu Lys Ala Ala Thr Pro 340 345350Tyr Thr Val Ser Ile Tyr Gly Val Ile Gln Gly Tyr Arg Thr Pro Val355 360 365Leu Ser Ala Glu Ala Ser Thr Ala Ser Gly Leu Asn Asp IlePhe Glu 370 375 380Ala Gln Lys Ile Glu Trp His Glu Gly Thr His HisHis His His His385 390 395 400131614DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotidepRJH33 library template DP88-4 library; complete Fabcoding region comprising PelB leader sequence + Vk1_5 kappaV-domain + CL constant domain for light chain and PelB + VH1_69V-domain + CH1 constant domain for heavy chain) 13atgaaatacctattgcctac ggcagccgct ggattgttat tactcgcggc ccagccggcc 60atggccgacatccagatgac ccagtctcct tccaccctgt ctgcatctgt aggagaccgt120gtcaccatca cttgccgtgc cagtcagagt attagtagct ggttggcctggtatcagcag 180aaaccaggga aagcccctaa gctcctgatc tatgatgcctccagtttgga aagtggggtc 240ccatcacgtt tcagcggcag tggatccgggacagaattca ctctcaccat cagcagcttg 300cagcctgatg attttgcaacttattactgc caacagtata atagttattc tacgtttggc 360cagggcaccaaagtcgagat caagcgtacg gtggctgcac catctgtctt catcttcccg420ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgctgaataacttc 480tatcccagag aggccaaagt acagtggaag gtggataacgccctccaatc gggtaactcc 540caggagagtg tcacagagca ggacagcaaggacagcacct acagcctcag cagcaccctg 600acgctgagca aagcagactacgagaaacac aaagtctacg cctgcgaagt cacccatcag 660ggcctgagctcgcccgtcac aaagagcttc aacaggggag agtgtggagc cgcagaacaa720aaactcatct cagaagagga tctgaatgga gccgcagact acaaggacgacgacgacaag 780ggtgccgcat aataaggcgc gccaattcta tttcaaggagacagtcatat gaaatacctg 840ctgccgaccg ctgctgctgg tctgctgctcctcgctgccc agccggcgat ggcccaggtg 900caattggtgc agtctggggctgaggtgaag aagcctgggt cctcggtgaa ggtctcctgc 960aaggcctccggaggcacatt cagcagctac gctataagct gggtgcgaca ggcccctgga1020caagggctcg agtggatggg agggatcatc cctatctttg gtacagcaaactacgcacag 1080aagttccagg gcagggtcac cattactgca gacaaatccacgagcacagc ctacatggag 1140ctgagcagcc tgagatctga ggacaccgccgtgtattact gtgcgagact atccccaggc 1200ggttactatg ttatggatgcctggggccaa gggaccaccg tgaccgtctc ctcagctagc 1260accaaaggcccatcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca1320gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggtgtcgtggaac 1380tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctgtcctacagtc ctcaggactc 1440tactccctca gcagcgtggt gaccgtgccctccagcagct tgggcaccca gacctacatc 1500tgcaacgtga atcacaagcccagcaacacc aaagtggaca agaaagttga gcccaaatct 1560tgtgacgcggccgcaagcac tagtgcccat caccatcacc atcacgccgc ggca161414723DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideFab light chain V1_5 14gacatccagatgacccagtc tccttccacc ctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgccgtgccagtca gagtattagt agctggttgg cctggtatca gcagaaacca120gggaaagccc ctaagctcct gatctatgat gcctccagtt tggaaagtggggtcccatca 180cgtttcagcg gcagtggatc cgggacagaa ttcactctcaccatcagcag cttgcagcct 240gatgattttg caacttatta ctgccaacagtataatagtt attctacgtt tggccagggc 300accaaagtcg agatcaagcgtacggtggct gcaccatctg tcttcatctt cccgccatct 360gatgagcagttgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc420agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaactcccaggag 480agtgtcacag agcaggacag caaggacagc acctacagcctcagcagcac cctgacgctg 540agcaaagcag actacgagaa acacaaagtctacgcctgcg aagtcaccca tcagggcctg 600agctcgcccg tcacaaagagcttcaacagg ggagagtgtg gagccgcaga acaaaaactc 660atctcagaagaggatctgaa tggagccgca gactacaagg acgacgacga caagggtgcc 720gca72315720DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideFab heavy chain VH1_69 15caggtgcaattggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaaggcctccggagg cacattcagc agctacgcta taagctgggt gcgacaggcc120cctggacaag ggctcgagtg gatgggaggg atcatcccta tctttggtacagcaaactac 180gcacagaagt tccagggcag ggtcaccatt actgcagacaaatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggacaccgccgtgt attactgtgc gagactatcc 300ccaggcggtt actatgttatggatgcctgg ggccaaggga ccaccgtgac cgtctcctca 360gctagcaccaaaggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg420ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggtgacggtgtcg 480tggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca 540ggactctact ccctcagcag cgtggtgaccgtgccctcca gcagcttggg cacccagacc 600tacatctgca acgtgaatcacaagcccagc aacaccaaag tggacaagaa agttgagccc 660aaatcttgtgacgcggccgc aagcactagt gcccatcacc atcaccatca cgccgcggca72016241PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideFab light chain Vk1_5 16Asp Ile Gln Met ThrGln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val ThrIle Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala TrpTyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr AspAla Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60SerGly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 7580Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Ser Thr85 90 95Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala AlaPro 100 105 110Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu LysSer Gly Thr 115 120 125Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro Arg Glu Ala Lys 130 135 140Val Gln Trp Lys Val Asp Asn Ala LeuGln Ser Gly Asn Ser Gln Glu145 150 155 160Ser Val Thr Glu Gln AspSer Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175Thr Leu Thr LeuSer Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200205Asn Arg Gly Glu Cys Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu210 215 220Asp Leu Asn Gly Ala Ala Asp Tyr Lys Asp Asp Asp Asp LysGly Ala225 230 235 240Ala17240PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptideFab heavy chain VH1_69(DP88) 17Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys ProGly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr PheSer Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln GlyLeu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala AsnTyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp LysSer Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Leu Ser Pro Gly GlyTyr Tyr Val Met Asp Ala Trp Gly Gln 100 105 110Gly Thr Thr Val ThrVal Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro LeuAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140LeuGly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile CysAsn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys LysVal Glu Pro Lys Ser Cys Asp 210 215 220Ala Ala Ala Ser Thr Ser AlaHis His His His His His Ala Ala Ala225 230 235 2401826DNAArtificialSequenceDescription of Artificial Sequence SyntheticoligonucleotideLMB3 18caggaaacag ctatgaccat gattac261971DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideVk1_5_L3r_Smisc_feature(28)..(28)60% givensequence and 40% mmisc_feature(29)..(30)60% given sequence and 40%nmisc_feature(31)..(31)60% given sequence and 40%mmisc_feature(32)..(33)60% given sequence and 40%nmisc_feature(34)..(34)60% given sequence and 40%mmisc_feature(35)..(36)60% given sequence and 40%nmisc_feature(37)..(37)60% given sequence and 40%mmisc_feature(38)..(39)60% given sequence and 40%nmisc_feature(40)..(40)60% given sequence and 40%mmisc_feature(41)..(42)60% given sequence and 40% n 19ctcgactttggtgccctggc caaacgtsba atacgaatta tactgttggc agtaataagt 60tgcaaaatcat 712074DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideVk1_5_L3r_SYmisc_feature(28)..(28)60%given sequence and 40% mmisc_feature(29)..(30)60% given sequenceand 40% nmisc_feature(31)..(31)60% given sequence and 40%mmisc_feature(32)..(33)60% given sequence and 40%nmisc_feature(34)..(34)60% given sequence and 40%mmisc_feature(35)..(36)60% given sequence and 40%nmisc_feature(37)..(37)60% given sequence and 40%mmisc_feature(38)..(39)60% given sequence and 40%nmisc_feature(40)..(40)60% given sequence and 40%mmisc_feature(41)..(42)60% given sequence and 40%nmisc_feature(43)..(43)60% given sequence and 40%mmisc_feature(44)..(45)60% given sequence and 40% n 20ctcgactttggtgccctggc caaacgtmhr sgratacgaa ttatactgtt ggcagtaata 60agttgcaaaatcat 742177DNAArtificial SequenceDescription of Artificial SequenceSynthetic

oligonucleotideVk1_5_L3r_SPYmisc_feature(28)..(28)60% givensequence and 40% mmisc_feature(29)..(30)60% given sequence and 40%nmisc_feature(31)..(31)60% given sequence and 40%mmisc_feature(32)..(33)60% given sequence and 40%nmisc_feature(34)..(34)60% given sequence and 40%mmisc_feature(35)..(36)60% given sequence and 40%nmisc_feature(37)..(37)60% given sequence and 40%mmisc_feature(38)..(39)60% given sequence and 40%nmisc_feature(40)..(40)60% given sequence and 40%mmisc_feature(41)..(42)60% given sequence and 40%nmisc_feature(43)..(43)60% given sequence and 40%mmisc_feature(44)..(45)60% given sequence and 40%nmisc_feature(46)..(46)60% given sequence and 40%mmisc_feature(47)..(48)60% given sequence and 40% n 21ctcgactttggtgccctggc caaacgtmhh msssgratac gaattatact gttggcagta 60ataagttgcaaaatcat 772227DNAArtificial SequenceDescription of ArtificialSequence Synthetic oligonucleotideRJH31 22acgtttggcc agggcaccaaagtcgag 272328DNAArtificial SequenceDescription of ArtificialSequence Synthetic oligonucleotideRJH32 23tctcgcacag taatacacggcggtgtcc 282479DNAArtificial SequenceDescription of ArtificialSequence SyntheticoligonucleotideDP88-v4-4modified_base(29)..(31)a, c, t, orgmisc_feature(29)..(31)This region encodes G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y=5%modified_base(32)..(34)a, c, t, orgmisc_feature(32)..(34)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/A/Y = 20%, P/W/S/D/T =8%modified_base(41)..(43)a, c, t, or gmisc_feature(41)..(43)Thisregion encodes F = 46%, L/M = 15%, G/I/Y = 8% 24ggacaccgccgtgtattact gtgcgagann nnnnnnnnnn nnngactact ggggccaagg 60gaccaccgtgaccgtctcc 792585DNAArtificial SequenceDescription of ArtificialSequence SyntheticoligonucleotideDP88-v4-6modified_base(29)..(31)a, c, t, orgmisc_feature(29)..(31)This region encodes G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y=5%modified_base(32)..(34)a, c, t, orgmisc_feature(32)..(34)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(41)..(43)a, c, t, orgmisc_feature(41)..(43)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(44)..(46)a, c, t, orgmisc_feature(44)..(46)This region encodes G/A/Y = 20%, P/W/S/D/T =8%modified_base(47)..(49)a, c, t, or gmisc_feature(47)..(49)Thisregion encodes F = 46%, L/M = 15%, G/I/Y = 8% 25ggacaccgccgtgtattact gtgcgagann nnnnnnnnnn nnnnnnnnng actactgggg 60ccaagggaccaccgtgaccg tctcc 852691DNAArtificial SequenceDescription ofArtificial Sequence SyntheticoligonucleotideDP88-v4-8modified_base(29)..(31)a, c, t, orgmisc_feature(29)..(31)This region encodes G/D = 20%, E/V/S = 10%,A/P/R/L/T/Y=5%modified_base(32)..(34)a, c, t, orgmisc_feature(32)..(34)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(41)..(43)a, c, t, orgmisc_feature(41)..(43)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(44)..(46)a, c, t, orgmisc_feature(44)..(46)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(47)..(49)a, c, t, orgmisc_feature(47)..(49)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E = 4,6%modified_base(50)..(52)a, c, t, orgmisc_feature(50)..(52)This region encodes G/A/Y = 20%, P/W/S/D/T =8%modified_base(53)..(55)a, c, t, or gmisc_feature(53)..(55)Thisregion encodes F = 46%, L/M = 15%, G/I/Y = 8% 26ggacaccgccgtgtattact gtgcgagann nnnnnnnnnn nnnnnnnnnn nnnnngacta 60ctggggccaagggaccaccg tgaccgtctc c 912730DNAArtificial SequenceDescription ofArtificial Sequence Synthetic oligonucleotidefdseqlong 27gacgttagtaaatgaatttt ctgtatgagg 30281605DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotidepRJH53 library templateof lambda-DP47 library Vl3_19/VH3_23 28atgaaatacc tattgcctacggcagccgct ggattgttat tactcgcggc ccagccggcc 60atggcctcgt ctgagctgactcaggaccct gctgtgtctg tggccttggg acagacagtc 120aggatcacatgccaaggaga cagcctcaga agttattatg caagctggta ccagcagaag180ccaggacagg cccctgtact tgtcatctat ggtaaaaaca accggccctcagggatccca 240gaccgattct ctggctccag ctcaggaaac acagcttccttgaccatcac tggggctcag 300gcggaagatg aggctgacta ttactgtaactcccgtgata gtagcggtaa tcatgtggta 360ttcggcggag ggaccaagctgaccgtccta ggacaaccca aggctgcccc cagcgtgacc 420ctgttcccccccagcagcga ggaattgcag gccaacaagg ccaccctggt ctgcctgatc480agcgacttct acccaggcgc cgtgaccgtg gcctggaagg ccgacagcagccccgtgaag 540gccggcgtgg agaccaccac ccccagcaag cagagcaacaacaagtacgc cgccagcagc 600tacctgagcc tgacccccga gcagtggaagagccacaggt cctacagctg ccaggtgacc 660cacgagggca gcaccgtggagaaaaccgtg gcccccaccg agtgcagcgg agccgcagaa 720caaaaactcatctcagaaga ggatctgaat ggagccgcag actacaagga cgacgacgac780aagggtgccg cataataagg cgcgccaatt ctatttcaag gagacagtcatatgaaatac 840ctgctgccga ccgctgctgc tggtctgctg ctcctcgctgcccagccggc gatggccgag 900gtgcaattgc tggagtctgg gggaggcttggtacagcctg gggggtccct gagactctcc 960tgtgcagcct ccggattcacctttagcagt tatgccatga gctgggtccg ccaggctcca 1020gggaaggggctggagtgggt ctcagctatt agtggtagtg gtggtagcac atactacgca1080gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacacgctgtatctg 1140cagatgaaca gcctgagagc cgaggacacg gccgtatattactgtgcgaa accgtttccg 1200tattttgact actggggcca aggaaccctggtcaccgtct cgagtgctag caccaaaggc 1260ccatcggtct tccccctggcaccctcctcc aagagcacct ctgggggcac agcggccctg 1320ggctgcctggtcaaggacta cttccccgaa ccggtgacgg tgtcgtggaa ctcaggcgcc1380ctgaccagcg gcgtgcacac cttcccggct gtcctacagt cctcaggactctactccctc 1440agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacccagacctacat ctgcaacgtg 1500aatcacaagc ccagcaacac caaagtggacaagaaagttg agcccaaatc ttgtgacgcg 1560gccgcaagca ctagtgcccatcaccatcac catcacgccg cggca 160529726DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotideFab light chain Vl3_19 29tcgtctgagc tgactcaggaccctgctgtg tctgtggcct tgggacagac agtcaggatc 60acatgccaag gagacagcctcagaagttat tatgcaagct ggtaccagca gaagccagga 120caggcccctgtacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga180ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggctcaggcggaa 240gatgaggctg actattactg taactcccgt gatagtagcggtaatcatgt ggtattcggc 300ggagggacca agctgaccgt cctaggacaacccaaggctg cccccagcgt gaccctgttc 360ccccccagca gcgaggaattgcaggccaac aaggccaccc tggtctgcct gatcagcgac 420ttctacccaggcgccgtgac cgtggcctgg aaggccgaca gcagccccgt gaaggccggc480gtggagacca ccacccccag caagcagagc aacaacaagt acgccgccagcagctacctg 540agcctgaccc ccgagcagtg gaagagccac aggtcctacagctgccaggt gacccacgag 600ggcagcaccg tggagaaaac cgtggcccccaccgagtgca gcggagccgc agaacaaaaa 660ctcatctcag aagaggatctgaatggagcc gcagactaca aggacgacga cgacaagggt 720gccgca72630708DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideFab heavy chain VH3_23 30gaggtgcaattgctggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcagcctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct120ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtagcacatactac 180gcagactccg tgaagggccg gttcaccatc tccagagacaattccaagaa cacgctgtat 240ctgcagatga acagcctgag agccgaggacacggccgtat attactgtgc gaaaccgttt 300ccgtattttg actactggggccaaggaacc ctggtcaccg tctcgagtgc tagcaccaaa 360ggcccatcggtcttccccct ggcaccctcc tccaagagca cctctggggg cacagcggcc420ctgggctgcc tggtcaagga ctacttcccc gaaccggtga cggtgtcgtggaactcaggc 480gccctgacca gcggcgtgca caccttcccg gctgtcctacagtcctcagg actctactcc 540ctcagcagcg tggtgaccgt gccctccagcagcttgggca cccagaccta catctgcaac 600gtgaatcaca agcccagcaacaccaaagtg gacaagaaag ttgagcccaa atcttgtgac 660gcggccgcaagcactagtgc ccatcaccat caccatcacg ccgcggca 70831242PRTArtificialSequenceDescription of Artificial Sequence Synthetic polypeptideFablight chain Vl3_19 31Ser Ser Glu Leu Thr Gln Asp Pro Ala Val SerVal Ala Leu Gly Gln1 5 10 15Thr Val Arg Ile Thr Cys Gln Gly Asp SerLeu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln Gln Lys Pro Gly GlnAla Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn Asn Arg Pro Ser GlyIle Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser Gly Asn Thr Ala SerLeu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80Asp Glu Ala Asp TyrTyr Cys Asn Ser Arg Asp Ser Ser Gly Asn His 85 90 95Val Val Phe GlyGly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110Ala AlaPro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120125Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly130 135 140Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val LysAla Gly145 150 155 160Val Glu Thr Thr Thr Pro Ser Lys Gln Ser AsnAsn Lys Tyr Ala Ala 165 170 175Ser Ser Tyr Leu Ser Leu Thr Pro GluGln Trp Lys Ser His Arg Ser 180 185 190Tyr Ser Cys Gln Val Thr HisGlu Gly Ser Thr Val Glu Lys Thr Val 195 200 205Ala Pro Thr Glu CysSer Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu 210 215 220Glu Asp LeuAsn Gly Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys Gly225 230 235240Ala Ala32236PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptideFab heavy chain VH3_23 (DP47) 32GluVal Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 1015Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu TrpVal 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala AspSer Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys AsnThr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95Ala Lys Pro Phe Pro Tyr Phe Asp Tyr TrpGly Gln Gly Thr Leu Val 100 105 110Thr Val Ser Ser Ala Ser Thr LysGly Pro Ser Val Phe Pro Leu Ala 115 120 125Pro Ser Ser Lys Ser ThrSer Gly Gly Thr Ala Ala Leu Gly Cys Leu 130 135 140Val Lys Asp TyrPhe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly145 150 155 160AlaLeu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 165 170175Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu180 185 190Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro SerAsn Thr 195 200 205Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys AspAla Ala Ala Ser 210 215 220Thr Ser Ala His His His His His His AlaAla Ala225 230 2353385DNAArtificial SequenceDescription ofArtificial Sequence SyntheticoligonucleotideVl_3_19_L3r_Vmisc_feature(32)..(32)60% original baseand 40% randomization as mmisc_feature(33)..(34)60% original baseand 40% randomization as nmisc_feature(35)..(35)60% original baseand 40% randomization as mmisc_feature(36)..(37)60% original baseand 40% randomization as nmisc_feature(38)..(38)60% original baseand 40% randomization as mmisc_feature(39)..(40)60% original baseand 40% randomization as nmisc_feature(41)..(41)60% original baseand 40% randomization as mmisc_feature(42)..(43)60% original baseand 40% randomization as nmisc_feature(44)..(44)60% original baseand 40% randomization as mmisc_feature(45)..(46)60% original baseand 40% randomization as nmisc_feature(47)..(47)60% original baseand 40% randomization as mmisc_feature(48)..(49)60% original baseand 40% randomization as nmisc_feature(50)..(50)60% original baseand 40% randomization as mmisc_feature(51)..(52)60% original baseand 40% randomization as n 33ggacggtcag cttggtccct ccgccgaatacvhvattacc gctactatca cgggagttac 60agtaatagtc agcctcatct tccgc853488DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideVl_3_19_L3r_HVmisc_feature(32)..(32)60%original base and 40% randomization as mmisc_feature(33)..(34)60%original base and 40% randomization as nmisc_feature(35)..(35)60%original base and 40% randomization as mmisc_feature(36)..(37)60%original base and 40% randomization as nmisc_feature(38)..(38)60%original base and 40% randomization as mmisc_feature(39)..(40)60%original base and 40% randomization as nmisc_feature(41)..(41)60%original base and 40% randomization as mmisc_feature(42)..(43)60%original base and 40% randomization as nmisc_feature(44)..(44)60%original base and 40% randomization as mmisc_feature(45)..(46)60%original base and 40% randomization as nmisc_feature(47)..(47)60%original base and 40% randomization as mmisc_feature(48)..(49)60%original base and 40% randomization as nmisc_feature(50)..(50)60%original base and 40% randomization as mmisc_feature(51)..(52)60%original base and 40% randomization as nmisc_feature(53)..(53)60%original base and 40% randomization as mmisc_feature(54)..(55)60%original base and 40% randomization as n 34ggacggtcag cttggtccctccgccgaata ccmmatgatt accgctacta tcacgggagt 60tacagtaata gtcagcctcatcttccgc 883591DNAArtificial SequenceDescription of ArtificialSequence SyntheticoligonucleotideVl_3_19_L3r_HLVmisc_feature(32)..(32)60% originalbase and 40% randomization as mmisc_feature(33)..(34)60% originalbase and 40% randomization as nmisc_feature(35)..(35)60% originalbase and 40% randomization as mmisc_feature(36)..(37)60% originalbase and 40% randomization as nmisc_feature(38)..(38)60% originalbase and 40% randomization as mmisc_feature(39)..(40)60% originalbase and 40% randomization as nmisc_feature(41)..(41)60% originalbase and 40% randomization as mmisc_feature(42)..(43)60% originalbase and 40% randomization as nmisc_feature(44)..(44)60% originalbase and 40% randomization as mmisc_feature(45)..(46)60% originalbase and 40% randomization as nmisc_feature(47)..(47)60% originalbase and 40% randomization as mmisc_feature(48)..(49)60% originalbase and 40% randomization as nmisc_feature(50)..(50)60% originalbase and 40% randomization as mmisc_feature(51)..(52)60% originalbase and 40% randomization as nmisc_feature(53)..(53)60% originalbase and 40% randomization as mmisc_feature(54)..(55)60% originalbase and 40% randomization as nmisc_feature(56)..(56)60% originalbase and 40% randomization as mmisc_feature(57)..(58)60% originalbase and 40% randomization as n 35ggacggtcag cttggtccct ccgccgaatacrhmvwgatg attaccgcta ctatcacggg 60agttacagta atagtcagcc tcatcttccgc 913628DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideRJH80 36ttcggcggag ggaccaagct gaccgtcc283725DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideDP47CDR3_ba (mod.) 37cgcacagtaa tatacggccgtgtcc 253882DNAArtificial SequenceDescription of ArtificialSequence SyntheticoligonucleotideDP47-v4-4modified_base(29)..(31)a, c, t, or

gmisc_feature(29)..(31)This region encodes K=70%,R=30%modified_base(32)..(34)a, c, t, or gmisc_feature(32)..(34)Thisregion encodes G/D=20%, E/V/S=10%,A/P/R/L/T/Y=5%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(41)..(43)a, c, t, orgmisc_feature(41)..(43)This region encodes G/A/Y=20,P/W/S/D/T=8%modified_base(44)..(46)a, c, t, orgmisc_feature(44)..(46)This region encodes F=46%, L/M=15%, G/I/Y=8%38cgaggacacg gccgtatatt actgtgcgnn nnnnnnnnnn nnnnnngact actggggcca60aggaaccctg gtcaccgtct cg 823988DNAArtificial SequenceDescriptionof Artificial Sequence SyntheticoligonucleotideDP47-v4-6modified_base(29)..(31)a, c, t, orgmisc_feature(29)..(31)This region encodes K=70%,R=30%modified_base(32)..(34)a, c, t, or gmisc_feature(32)..(34)Thisregion encodes G/D=20%, E/V/S=10%,A/P/R/L/T/Y=5%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(41)..(43)a, c, t, orgmisc_feature(41)..(43)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(44)..(46)a, c, t, orgmisc_feature(44)..(46)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(47)..(49)a, c, t, orgmisc_feature(47)..(49)This region encodes G/A/Y=20,P/W/S/D/T=8%modified_base(50)..(52)a, c, t, orgmisc_feature(50)..(52)This region encodes F=46%, L/M=15%, G/I/Y=8%39cgaggacacg gccgtatatt actgtgcgnn nnnnnnnnnn nnnnnnnnnn nngactactg60gggccaagga accctggtca ccgtctcg 884094DNAArtificialSequenceDescription of Artificial Sequence SyntheticoligonucleotideDP47-v4-8modified_base(29)..(31)a, c, t, orgmisc_feature(29)..(31)This region encodes K=70%,R=30%modified_base(32)..(34)a, c, t, or gmisc_feature(32)..(34)Thisregion encodes G/D=20%, E/V/S=10%,A/P/R/L/T/Y=5%modified_base(35)..(37)a, c, t, orgmisc_feature(35)..(37)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(38)..(40)a, c, t, orgmisc_feature(38)..(40)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(41)..(43)a, c, t, orgmisc_feature(41)..(43)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(44)..(46)a, c, t, orgmisc_feature(44)..(46)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(47)..(49)a, c, t, orgmisc_feature(47)..(49)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(50)..(52)a, c, t, orgmisc_feature(50)..(52)This region encodes G/Y/S=15%,A/D/T/R/P/L/V/N/W/F/I/E=4,6%modified_base(53)..(55)a, c, t, orgmisc_feature(53)..(55)This region encodes G/A/Y=20,P/W/S/D/T=8%modified_base(56)..(58)a, c, t, orgmisc_feature(56)..(58)This region encodes F=46%, L/M=15%, G/I/Y=8%40cgaggacacg gccgtatatt actgtgcgnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnga60ctactggggc caaggaaccc tggtcaccgt ctcg 9441324DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotide18D4 VL 41gacatccaga tgacccagtc tccatccacc ctgtctgcatctgtaggaga ccgtgtcacc 60atcacttgcc gtgccagtca gagtattagt agctggttggcctggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgatgcctccagtt tggaaagtgg ggtcccatca 180cgtttcagcg gcagtggatccgggacagaa ttcactctca ccatcagcag cttgcagcct 240gatgattttgcaacttatta ctgccaacag aataagaagt ttccttcggg gacgtttggc300cagggcacca aagtcgagat caag 32442354DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotide18D4 VH 42caggtgcaat tggtgcagtc tggggctgag gtgaagaagcctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgctataagctgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggagggatcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcagggtcaccatt actgcagaca aatccacgag cacagcctac 240atggagctgagcagcctgag atctgaggac accgccgtgt attactgtgc gaaaggtaac300ttctacggtg gtctggacta ctggggccaa gggaccaccg tgaccgtctc ctca35443321DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotide11C7 VL 43tcgtctgagc tgactcagga ccctgctgtgtctgtggcct tgggacagac agtcagggtc 60acatgccaag gagacagcct cagaagttattatgcaagct ggtaccagca gaagccagga 120caggcccctg tacttgtcatctatggtaaa aacaaccggc cctcagggat cccagaccga 180ttctctggctccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa240gatgaggctg actattactg taactccatt aatagtactc gtaatgaggtattcggcgga 300gggaccaagc tgaccgtcct a 32144354DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotide11C7 VH 44gaggtgcaat tgttggagtc tgggggaggc ttggtacagcctggggggtc cctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgccatgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagctattagcggta gtggtggtag cacatactac 180gcagactccg tgaagggccggttcaccatc tccaaagaca attccaagaa cacgctgtat 240ctgcagatgaacagcctgag agccgaggac acggccgtat attactgtgc gaaaacttct300ccgcgtgttc cgctggacta ctggggccaa ggaaccctgg tcaccgtctc gagt35445108PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptide18D4 VL 45Asp Ile Gln Met Thr Gln Ser Pro SerThr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys ArgAla Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr Gln Gln LysPro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala Ser Ser LeuGlu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly ThrGlu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80Asp Asp PheAla Thr Tyr Tyr Cys Gln Gln Asn Lys Lys Phe Pro Ser 85 90 95Gly ThrPhe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 10546118PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptide18D4 VH 46Gln Val Gln Leu Val Gln Ser Gly Ala Glu ValLys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser GlyGly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala ProGly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe GlyThr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile ThrAla Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser SerLeu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly AsnPhe Tyr Gly Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr ValThr Val Ser Ser 11547107PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptide11C7 VL 47Ser Ser Glu LeuThr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val ArgVal Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser TrpTyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45GlyLys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 5560Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu6570 75 80Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Ile Asn Ser Thr Arg AsnGlu 85 90 95Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 10010548118PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptide11C7 VH 48Glu Val Gln Leu Leu Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg GlnAla Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly SerGly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe ThrIle Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln MetAsn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala LysThr Ser Pro Arg Val Pro Leu Asp Tyr Trp Gly Gln Gly Thr 100 105110Leu Val Thr Val Ser Ser 1154933DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic oligonucleotide18D4 LCDR149cgtgccagtc agagtattag tagctggttg gcc 335021DNAArtificialSequenceDescription of Artificial Sequence Syntheticoligonucleotide18D4 LCDR2 50gatgcctcca gtttggaaag t215130DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotide18D4 LCDR3 51caacagaata agaagtttccttcggggacg 305233DNAArtificial SequenceDescription of ArtificialSequence Synthetic oligonucleotide11C7 LCDR1 52caaggagacagcctcagaag ttattatgca agc 335321DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic oligonucleotide11C7 LCDR253ggtaaaaaca accggccctc a 215430DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic oligonucleotide11C7 LCDR354aactccatta atagtactcg taatgaggta 305511PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptide18D4LCDR1 55Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala1 510567PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide18D4 LCDR2 56Asp Ala Ser Ser Leu Glu Ser155710PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide18D4 LCDR3 57Gln Gln Asn Lys Lys Phe Pro Ser GlyThr1 5 105811PRTArtificial SequenceDescription of ArtificialSequence Synthetic peptide11C7 LCDR1 58Gln Gly Asp Ser Leu Arg SerTyr Tyr Ala Ser1 5 10597PRTArtificial SequenceDescription ofArtificial Sequence Synthetic peptide11C7 LCDR2 59Gly Lys Asn AsnArg Pro Ser1 56010PRTArtificial SequenceDescription of ArtificialSequence Synthetic peptide11C7 LCDR3 60Asn Ser Ile Asn Ser Thr ArgAsn Glu Val1 5 106115DNAArtificial SequenceDescription ofArtificial Sequence Synthetic oligonucleotide18D4 HCDR161agctacgcta taagc 156251DNAArtificial SequenceDescription ofArtificial Sequence Synthetic oligonucleotide18D4 HCDR262gggatcatcc ctatctttgg tacagcaaac tacgcacaga agttccaggg c516327DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotide18D4 HCDR3 63ggtaacttct acggtggtct ggactac276430DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotide11C7 HCDR1 64ggattcacct ttagcagttatgccatgagc 306551DNAArtificial SequenceDescription of ArtificialSequence Synthetic oligonucleotide11C7 HCDR2 65gctattagcggtagtggtgg tagcacatac tacgcagact ccgtgaaggg c 516627DNAArtificialSequenceDescription of Artificial Sequence Syntheticoligonucleotide11C7 HCDR3 66acttctccgc gtgttccgct ggactac27675PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide18D4 HCDR1 67Ser Tyr Ala Ile Ser156817PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide18D4 HCDR2 68Gly Ile Ile Pro Ile Phe Gly Thr AlaAsn Tyr Ala Gln Lys Phe Gln1 5 10 15Gly699PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptide18D4HCDR3 69Gly Asn Phe Tyr Gly Gly Leu Asp Tyr1 57010PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptide11C7HCDR1 70Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser1 5107117PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide11C7 HCDR2 71Ala Ile Ser Gly Ser Gly Gly Ser ThrTyr Tyr Ala Asp Ser Val Lys1 5 10 15Gly729PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptide11C7HCDR3 72Thr Ser Pro Arg Val Pro Leu Asp Tyr1 573645DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotide18D4 Light chain 73gacatccaga tgacccagtc tccatccaccctgtctgcat ctgtaggaga ccgtgtcacc 60atcacttgcc gtgccagtca gagtattagtagctggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcctgatctatgat gcctccagtt tggaaagtgg ggtcccatca 180cgtttcagcggcagtggatc cgggacagaa ttcactctca ccatcagcag cttgcagcct240gatgattttg caacttatta ctgccaacag aataagaagt ttccttcggggacgtttggc 300cagggcacca aagtcgagat caagcgtacg gtggctgcaccatctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaactgcctctgttg tgtgcctgct gaataacttc 420tatcccagag aggccaaagtacagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtgtcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg540acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagtcacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt645741344DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotide18D4 Heavy chain 74caggtgcaat tggtgcagtctggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggaggcacattcagc agctacgcta taagctgggt gcgacaggcc 120cctggacaagggctcgagtg gatgggaggg atcatcccta tctttggtac agcaaactac180gcacagaagt tccagggcag ggtcaccatt actgcagaca aatccacgagcacagcctac 240atggagctga gcagcctgag atctgaggac accgccgtgtattactgtgc gaaaggtaac 300ttctacggtg gtctggacta ctggggccaagggaccaccg tgaccgtctc ctcagctagc 360accaagggcc catcggtcttccccctggca ccctcctcca agagcacctc tgggggcaca 420gcggccctgggctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtcctcaggactc 540tactccctca gcagcgtggt gaccgtgccc tccagcagcttgggcaccca gacctacatc 600tgcaacgtga atcacaagcc cagcaacaccaaggtggaca agaaagttga gcccaaatct 660tgtgacaaaa ctcacacatgcccaccgtgc ccagcacctg aactcctggg gggaccgtca 720gtcttcctcttccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaactggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca aagccgcgggaggagcagta caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctgcaccaggact ggctgaatgg caaggagtac 960aagtgcaagg tctccaacaaagccctccca gcccccatcg agaaaaccat ctccaaagcc 1020aaagggcagccccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcgacatcgccgtg 1140gagtgggaga gcaatgggca gccggagaac aactacaagaccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct ctacagcaagctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct tctcatgctccgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320agcctctccctgtctccggg taaa 134475639DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotide11C7 Light chain75tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcagggtc60acatgccaag gagacagcct cagaagttat tatgcaagct ggtaccagca gaagccagga120caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggatcccagaccga 180ttctctggct ccagctcagg aaacacagct tccttgaccatcactggggc tcaggcggaa 240gatgaggctg actattactg taactccattaatagtactc gtaatgaggt attcggcgga 300gggaccaagc tgaccgtcctaggtcaaccc aaggctgccc ccagcgtgac cctgttcccc 360cccagcagcgaggaactgca ggccaacaag gccaccctgg tctgcctgat cagcgacttc420tacccaggcg ccgtgaccgt ggcctggaag gccgacagca gccccgtgaaggccggcgtg 480gagaccacca cccccagcaa gcagagcaac aacaagtacgccgccagcag ctacctgagc 540ctgacccccg agcagtggaa gagccacaggtcctacagct gccaggtgac ccacgagggc 600agcaccgtgg agaaaaccgtggcccccacc gagtgcagc 639761344DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotide11C7 Heavy chain76gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc60tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggt ccgccaggct120ccagggaagg ggctggagtg ggtctcagct

attagcggta gtggtggtag cacatactac 180gcagactccg tgaagggccggttcaccatc tccaaagaca attccaagaa cacgctgtat 240ctgcagatgaacagcctgag agccgaggac acggccgtat attactgtgc gaaaacttct300ccgcgtgttc cgctggacta ctggggccaa ggaaccctgg tcaccgtctcgagtgctagc 360accaagggcc catcggtctt ccccctggca ccctcctccaagagcacctc tgggggcaca 420gcggccctgg gctgcctggt caaggactacttccccgaac cggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcggcgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540tactccctcagcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc600tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttgagcccaaatct 660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctgaactcctggg gggaccgtca 720gtcttcctct tccccccaaa acccaaggacaccctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatggcaaggagtac 960aagtgcaagg tctccaacaa agccctccca gcccccatcgagaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtacaccctgcccc catcccggga tgagctgacc 1080aagaaccagg tcagcctgacctgcctggtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggagagcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1200tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcaggtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgcacaaccacta cacgcagaag 1320agcctctccc tgtctccggg taaa134477215PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptide18D4 Light chain 77Asp Ile Gln Met Thr Gln SerPro Ser Thr Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile ThrCys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30Leu Ala Trp Tyr GlnGln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Asp Ala SerSer Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly SerGly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80AspAsp Phe Ala Thr Tyr Tyr Cys Gln Gln Asn Lys Lys Phe Pro Ser 85 9095Gly Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln LeuLys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn PheTyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp Asn AlaLeu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr Glu GlnAsp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu ThrLeu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr Ala CysGlu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205SerPhe Asn Arg Gly Glu Cys 210 21578448PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptide18D4 Heavy chain 78Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys AlaSer Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg GlnAla Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro IlePhe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val ThrIle Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala LysGly Asn Phe Tyr Gly Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrVal Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr LysVal Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220HisThr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser225 230235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly ValGlu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys ValSer Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu TrpGlu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn ValPhe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44044579213PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptide11C7 Light chain 79Ser Ser Glu Leu Thr Gln AspPro Ala Val Ser Val Ala Leu Gly Gln1 5 10 15Thr Val Arg Val Thr CysGln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30Ser Trp Tyr Gln GlnLys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45Gly Lys Asn AsnArg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60Ser Ser GlyAsn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu65 70 75 80AspGlu Ala Asp Tyr Tyr Cys Asn Ser Ile Asn Ser Thr Arg Asn Glu 85 9095Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys Ala100 105 110Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu LeuGln Ala 115 120 125Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp PheTyr Pro Gly Ala 130 135 140Val Thr Val Ala Trp Lys Ala Asp Ser SerPro Val Lys Ala Gly Val145 150 155 160Glu Thr Thr Thr Pro Ser LysGln Ser Asn Asn Lys Tyr Ala Ala Ser 165 170 175Ser Tyr Leu Ser LeuThr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 180 185 190Ser Cys GlnVal Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 195 200 205ProThr Glu Cys Ser 21080448PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptide11C7 Heavy chain 80Glu ValGln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp SerVal 50 55 60Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn ThrLeu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr AlaVal Tyr Tyr Cys 85 90 95Ala Lys Thr Ser Pro Arg Val Pro Leu Asp TyrTrp Gly Gln Gly Thr 100 105 110Leu Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys SerThr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys AspTyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His LysPro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro GluLeu Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val ThrCys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285LysThr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluTyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala ProIle Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400SerAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser ProGly Lys 435 440 445811344DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotide18D4 Heavy chain PGLALA81caggtgcaat tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc60tcctgcaagg cctccggagg cacattcagc agctacgcta taagctgggt gcgacaggcc120cctggacaag ggctcgagtg gatgggaggg atcatcccta tctttggtacagcaaactac 180gcacagaagt tccagggcag ggtcaccatt actgcagacaaatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggacaccgccgtgt attactgtgc gaaaggtaac 300ttctacggtg gtctggactactggggccaa gggaccaccg tgaccgtctc ctcagctagc 360accaagggcccatcggtctt ccccctggca ccctcctcca agagcacctc tgggggcaca420gcggccctgg gctgcctggt caaggactac ttccccgaac cggtgacggtgtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctgtcctacagtc ctcaggactc 540tactccctca gcagcgtggt gaccgtgccctccagcagct tgggcaccca gacctacatc 600tgcaacgtga atcacaagcccagcaacacc aaggtggaca agaaagttga gcccaaatct 660tgtgacaaaactcacacatg cccaccgtgc ccagcacctg aagctgcagg gggaccgtca720gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggacccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaa gaccctgaggtcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataa tgccaagacaaagccgcggg aggagcagta caacagcacg 900taccgtgtgg tcagcgtcctcaccgtcctg caccaggact ggctgaatgg caaggagtac 960aagtgcaaggtctccaacaa agccctcggc gcccccatcg agaaaaccat ctccaaagcc1020aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccgggatgagctgacc 1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttctatcccagcga catcgccgtg 1140gagtgggaga gcaatgggca gccggagaacaactacaaga ccacgcctcc cgtgctggac 1200tccgacggct ccttcttcctctacagcaag ctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtcttctcatgctc cgtgatgcat gaggctctgc acaaccacta cacgcagaag1320agcctctccc tgtctccggg taaa 1344821344DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotide11C7 Heavy chain PGLALA 82gaggtgcaat tgttggagtctgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctccggattcacctttagc agttatgcca tgagctgggt ccgccaggct 120ccagggaaggggctggagtg ggtctcagct attagcggta gtggtggtag cacatactac180gcagactccg tgaagggccg gttcaccatc tccaaagaca attccaagaacacgctgtat 240ctgcagatga acagcctgag agccgaggac acggccgtatattactgtgc gaaaacttct 300ccgcgtgttc cgctggacta ctggggccaaggaaccctgg tcaccgtctc gagtgctagc 360accaagggcc catcggtcttccccctggca ccctcctcca agagcacctc tgggggcaca 420gcggccctgggctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac480tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtcctcaggactc 540tactccctca gcagcgtggt gaccgtgccc tccagcagcttgggcaccca gacctacatc 600tgcaacgtga atcacaagcc cagcaacaccaaggtggaca agaaagttga gcccaaatct 660tgtgacaaaa ctcacacatgcccaccgtgc ccagcacctg aagctgcagg gggaccgtca 720gtcttcctcttccccccaaa acccaaggac accctcatga tctcccggac ccctgaggtc780acatgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaactggtacgtg 840gacggcgtgg aggtgcataa tgccaagaca aagccgcgggaggagcagta caacagcacg 900taccgtgtgg tcagcgtcct caccgtcctgcaccaggact ggctgaatgg caaggagtac 960aagtgcaagg tctccaacaaagccctcggc gcccccatcg agaaaaccat ctccaaagcc 1020aaagggcagccccgagaacc acaggtgtac accctgcccc catcccggga tgagctgacc1080aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcgacatcgccgtg 1140gagtgggaga gcaatgggca gccggagaac aactacaagaccacgcctcc cgtgctggac 1200tccgacggct ccttcttcct ctacagcaagctcaccgtgg acaagagcag gtggcagcag 1260gggaacgtct tctcatgctccgtgatgcat gaggctctgc acaaccacta cacgcagaag 1320agcctctccctgtctccggg taaa 134483448PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptide18D4 Heavy chain PGLALA83Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser15 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser SerTyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr AlaGln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser ThrSer Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Phe Tyr Gly Gly LeuAsp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser AlaSer Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser SerLys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu ValLys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu ProLys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro AlaPro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe ProPro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly LysGlu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Gly AlaPro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu LeuThr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly PheTyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370

375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Lys 435 440 44584448PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptide11C7 Heavy chain PGLALA 84Glu Val Gln Leu Leu Glu SerGly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile SerGly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly ArgPhe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095Ala Lys Thr Ser Pro Arg Val Pro Leu Asp Tyr Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser ValPhe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrAla Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu ProVal Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205AsnThr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly ProSer225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val AspVal Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr ValAsp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu ThrVal Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320LysCys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 325 330335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser LeuThr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe LeuTyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln GlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His AsnHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44044585163PRThom*o sapiens 85Leu Gln Asp Pro Cys Ser Asn Cys Pro AlaGly Thr Phe Cys Asp Asn1 5 10 15Asn Arg Asn Gln Ile Cys Ser Pro CysPro Pro Asn Ser Phe Ser Ser 20 25 30Ala Gly Gly Gln Arg Thr Cys AspIle Cys Arg Gln Cys Lys Gly Val 35 40 45Phe Arg Thr Arg Lys Glu CysSer Ser Thr Ser Asn Ala Glu Cys Asp 50 55 60Cys Thr Pro Gly Phe HisCys Leu Gly Ala Gly Cys Ser Met Cys Glu65 70 75 80Gln Asp Cys LysGln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95Cys Cys PheGly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105 110TrpThr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120125Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro130 135 140Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu ProGly His145 150 155 160Ser Pro Gln86163PRTMacaca fascicularis 86LeuGln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn1 5 1015Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser20 25 30Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys GlyVal 35 40 45Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala GluCys Asp 50 55 60Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys SerMet Cys Glu65 70 75 80Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr LysLys Gly Cys Lys Asp 85 90 95Cys Cys Phe Gly Thr Phe Asn Asp Gln LysArg Gly Ile Cys Arg Pro 100 105 110Trp Thr Asn Cys Ser Leu Asp GlyLys Ser Val Leu Val Asn Gly Thr 115 120 125Lys Glu Arg Asp Val ValCys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135 140Gly Ala Ser SerAla Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His145 150 155 160SerPro Gln87164PRTMus musculus 87Val Gln Asn Ser Cys Asp Asn Cys GlnPro Gly Thr Phe Cys Arg Lys1 5 10 15Tyr Asn Pro Val Cys Lys Ser CysPro Pro Ser Thr Phe Ser Ser Ile 20 25 30Gly Gly Gln Pro Asn Cys AsnIle Cys Arg Val Cys Ala Gly Tyr Phe 35 40 45Arg Phe Lys Lys Phe CysSer Ser Thr His Asn Ala Glu Cys Glu Cys 50 55 60Ile Glu Gly Phe HisCys Leu Gly Pro Gln Cys Thr Arg Cys Glu Lys65 70 75 80Asp Cys ArgPro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr Cys 85 90 95Ser LeuGly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg Pro 100 105110Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly Thr115 120 125Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser PheSer Pro 130 135 140Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly ProGly Gly His Ser145 150 155 160Leu Gln Val Leu88681DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotideFc hole chain 88gacaaaactc acacatgccc accgtgcccagcacctgaac tcctgggggg accgtcagtc 60ttcctcttcc ccccaaaacc caaggacaccctcatgatct cccggacccc tgaggtcaca 120tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggac 180ggcgtggaggtgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac240cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaaggagtacaag 300tgcaaggtct ccaacaaagc cctcccagcc cccatcgagaaaaccatctc caaagccaaa 360gggcagcccc gagaaccaca ggtgtgcaccctgcccccat cccgggatga gctgaccaag 420aaccaggtca gcctctcgtgcgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 480tgggagagcaatgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc540gacggctcct tcttcctcgt gagcaagctc accgtggaca agagcaggtggcagcagggg 600aacgtcttct catgctccgt gatgcatgag gctctgcacaaccactacac gcagaagagc 660ctctccctgt ctccgggtaa a681891266DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotidehuman 4-1BB antigen Fc knob chain89ctgcaggacc cctgcagcaa ctgccctgcc ggcaccttct gcgacaacaa ccggaaccag60atctgcagcc cctgcccccc caacagcttc agctctgccg gcggacagcg gacctgcgac120atctgcagac agtgcaaggg cgtgttcaga acccggaaag agtgcagcagcaccagcaac 180gccgagtgcg actgcacccc cggcttccat tgtctgggagccggctgcag catgtgcgag 240caggactgca agcagggcca ggaactgaccaagaagggct gcaaggactg ctgcttcggc 300accttcaacg accagaagcggggcatctgc cggccctgga ccaactgtag cctggacggc 360aagagcgtgctggtcaacgg caccaaagaa cgggacgtcg tgtgcggccc cagccctgct420gatctgtctc ctggggccag cagcgtgacc cctcctgccc ctgccagagagcctggccac 480tctcctcagg tcgacgaaca gttatatttt cagggcggctcacccaaatc tgcagacaaa 540actcacacat gcccaccgtg cccagcacctgaactcctgg ggggaccgtc agtcttcctc 600ttccccccaa aacccaaggacaccctcatg atctcccgga cccctgaggt cacatgcgtg 660gtggtggacgtgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg720gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcacgtaccgtgtg 780gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatggcaaggagta caagtgcaag 840gtctccaaca aagccctccc agcccccatcgagaaaacca tctccaaagc caaagggcag 900ccccgagaac cacaggtgtacaccctgccc ccatgccggg atgagctgac caagaaccag 960gtcagcctgtggtgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag1020agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctggactccgacggc 1080tccttcttcc tctacagcaa gctcaccgtg gacaagagcaggtggcagca ggggaacgtc 1140ttctcatgct ccgtgatgca tgaggctctgcacaaccact acacgcagaa gagcctctcc 1200ctgtctccgg gtaaatccggaggcctgaac gacatcttcg aggcccagaa gattgaatgg 1260cacgag1266901266DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotidecynomolgus 4-1BB antigen Fc knob chain90ttgcaggatc tgtgtagtaa ctgcccagct ggtacattct gtgataataa caggagtcag60atttgcagtc cctgtcctcc aaatagtttc tccagcgcag gtggacaaag gacctgtgac120atatgcaggc agtgtaaagg tgttttcaag accaggaagg agtgttcctccaccagcaat 180gcagagtgtg actgcatttc agggtatcac tgcctgggggcagagtgcag catgtgtgaa 240caggattgta aacaaggtca agaattgacaaaaaaaggtt gtaaagactg ttgctttggg 300acatttaatg accagaaacgtggcatctgt cgcccctgga caaactgttc tttggatgga 360aagtctgtgcttgtgaatgg gacgaaggag agggacgtgg tctgcggacc atctccagcc420gacctctctc caggagcatc ctctgcgacc ccgcctgccc ctgcgagagagccaggacac 480tctccgcagg tcgacgaaca gttatatttt cagggcggctcacccaaatc tgcagacaaa 540actcacacat gcccaccgtg cccagcacctgaactcctgg ggggaccgtc agtcttcctc 600ttccccccaa aacccaaggacaccctcatg atctcccgga cccctgaggt cacatgcgtg 660gtggtggacgtgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg720gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcacgtaccgtgtg 780gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatggcaaggagta caagtgcaag 840gtctccaaca aagccctccc agcccccatcgagaaaacca tctccaaagc caaagggcag 900ccccgagaac cacaggtgtacaccctgccc ccatgccggg atgagctgac caagaaccag 960gtcagcctgtggtgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag1020agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctggactccgacggc 1080tccttcttcc tctacagcaa gctcaccgtg gacaagagcaggtggcagca ggggaacgtc 1140ttctcatgct ccgtgatgca tgaggctctgcacaaccact acacgcagaa gagcctctcc 1200ctgtctccgg gtaaatccggaggcctgaac gacatcttcg aggcccagaa gattgaatgg 1260cacgag1266911269DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotidemurine 4-1BB antigen Fc knob chain91gtgcagaaca gctgcgacaa ctgccagccc ggcaccttct gccggaagta caaccccgtg60tgcaagagct gcccccccag caccttcagc agcatcggcg gccagcccaa ctgcaacatc120tgcagagtgt gcgccggcta cttccggttc aagaagttct gcagcagcacccacaacgcc 180gagtgcgagt gcatcgaggg cttccactgc ctgggcccccagtgcaccag atgcgagaag 240gactgcagac ccggccagga actgaccaagcagggctgta agacctgcag cctgggcacc 300ttcaacgacc agaacgggaccggcgtgtgc cggccttgga ccaattgcag cctggacggg 360agaagcgtgctgaaaaccgg caccaccgag aaggacgtcg tgtgcggccc tcccgtggtg420tccttcagcc ctagcaccac catcagcgtg acccctgaag gcggccctggcggacactct 480ctgcaggtcc tggtcgacga acagttatat tttcagggcggctcacccaa atctgcagac 540aaaactcaca catgcccacc gtgcccagcacctgaactcc tggggggacc gtcagtcttc 600ctcttccccc caaaacccaaggacaccctc atgatctccc ggacccctga ggtcacatgc 660gtggtggtggacgtgagcca cgaagaccct gaggtcaagt tcaactggta cgtggacggc720gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agtacaacagcacgtaccgt 780gtggtcagcg tcctcaccgt cctgcaccag gactggctgaatggcaagga gtacaagtgc 840aaggtctcca acaaagccct cccagcccccatcgagaaaa ccatctccaa agccaaaggg 900cagccccgag aaccacaggtgtacaccctg cccccatgcc gggatgagct gaccaagaac 960caggtcagcctgtggtgcct ggtcaaaggc ttctatccca gcgacatcgc cgtggagtgg1020gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgctggactccgac 1080ggctccttct tcctctacag caagctcacc gtggacaagagcaggtggca gcaggggaac 1140gtcttctcat gctccgtgat gcatgaggctctgcacaacc actacacgca gaagagcctc 1200tccctgtctc cgggtaaatccggaggcctg aacgacatct tcgaggccca gaagattgaa 1260tggcacgag126992227PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideFc hole chain 92Asp Lys Thr His Thr Cys ProPro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 9095Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProGln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr LysAsn Gln Val Ser 130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser AspGly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205HisGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215220Pro Gly Lys22593422PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptidehuman 4-1BB antigen Fcknob chain 93Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr PheCys Asp Asn1 5 10 15Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro AsnSer Phe Ser Ser 20 25 30Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys ArgGln Cys Lys Gly Val 35 40 45Phe Arg Thr Arg Lys Glu Cys Ser Ser ThrSer Asn Ala Glu Cys Asp 50 55 60Cys Thr Pro Gly Phe His Cys Leu GlyAla Gly Cys Ser Met Cys Glu65 70 75 80Gln Asp Cys Lys Gln Gly GlnGlu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95Cys Cys Phe Gly Thr PheAsn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105 110Trp Thr Asn CysSer Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120 125Lys GluArg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135140Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro GlyHis145 150 155 160Ser Pro Gln Val Asp Glu Gln Leu Tyr Phe Gln GlyGly Ser Pro Lys 165 170 175Ser Ala Asp Lys Thr His Thr Cys Pro ProCys Pro Ala Pro Glu Leu 180 185 190Leu Gly Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr 195 200 205Leu Met Ile Ser Arg ThrPro Glu Val Thr Cys Val Val Val Asp Val 210 215 220Ser His Glu AspPro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val225 230 235 240GluVal His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 245 250255Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu260 265 270Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala LeuPro Ala 275 280 285Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro 290 295 300Gln Val Tyr Thr Leu Pro Pro Cys Arg AspGlu Leu Thr Lys Asn Gln305 310 315 320Val Ser Leu Trp Cys Leu ValLys Gly Phe Tyr Pro Ser Asp Ile Ala 325 330 335Val Glu Trp Glu SerAsn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

340 345 350Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu 355 360 365Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn ValPhe Ser Cys Ser 370 375 380Val Met His Glu Ala Leu His Asn His TyrThr Gln Lys Ser Leu Ser385 390 395 400Leu Ser Pro Gly Lys Ser GlyGly Leu Asn Asp Ile Phe Glu Ala Gln 405 410 415Lys Ile Glu Trp HisGlu 42094422PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptidecynomolgus 4-1BB antigen Fc knobchain 94Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys AspAsn1 5 10 15Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser PheSer Ser 20 25 30Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln CysLys Gly Val 35 40 45Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser AsnAla Glu Cys Asp 50 55 60Cys Ile Ser Gly Tyr His Cys Leu Gly Ala GluCys Ser Met Cys Glu65 70 75 80Gln Asp Cys Lys Gln Gly Gln Glu LeuThr Lys Lys Gly Cys Lys Asp 85 90 95Cys Cys Phe Gly Thr Phe Asn AspGln Lys Arg Gly Ile Cys Arg Pro 100 105 110Trp Thr Asn Cys Ser LeuAsp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120 125Lys Glu Arg AspVal Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135 140Gly AlaSer Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His145 150 155160Ser Pro Gln Val Asp Glu Gln Leu Tyr Phe Gln Gly Gly Ser Pro Lys165 170 175Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala ProGlu Leu 180 185 190Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr 195 200 205Leu Met Ile Ser Arg Thr Pro Glu Val ThrCys Val Val Val Asp Val 210 215 220Ser His Glu Asp Pro Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val225 230 235 240Glu Val His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 245 250 255Thr Tyr ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 260 265 270AsnGly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 275 280285Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro290 295 300Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr LysAsn Gln305 310 315 320Val Ser Leu Trp Cys Leu Val Lys Gly Phe TyrPro Ser Asp Ile Ala 325 330 335Val Glu Trp Glu Ser Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr 340 345 350Pro Pro Val Leu Asp Ser AspGly Ser Phe Phe Leu Tyr Ser Lys Leu 355 360 365Thr Val Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 370 375 380Val Met HisGlu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser385 390 395400Leu Ser Pro Gly Lys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala Gln405 410 415Lys Ile Glu Trp His Glu 42095423PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidemurine 4-1BB antigen Fc knob chain 95Val Gln Asn Ser CysAsp Asn Cys Gln Pro Gly Thr Phe Cys Arg Lys1 5 10 15Tyr Asn Pro ValCys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser Ile 20 25 30Gly Gly GlnPro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr Phe 35 40 45Arg PheLys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu Cys 50 55 60IleGlu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu Lys65 70 7580Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr Cys85 90 95Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys ArgPro 100 105 110Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu LysThr Gly Thr 115 120 125Thr Glu Lys Asp Val Val Cys Gly Pro Pro ValVal Ser Phe Ser Pro 130 135 140Ser Thr Thr Ile Ser Val Thr Pro GluGly Gly Pro Gly Gly His Ser145 150 155 160Leu Gln Val Leu Val AspGlu Gln Leu Tyr Phe Gln Gly Gly Ser Pro 165 170 175Lys Ser Ala AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 180 185 190Leu LeuGly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 195 200205Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp210 215 220Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr ValAsp Gly225 230 235 240Val Glu Val His Asn Ala Lys Thr Lys Pro ArgGlu Glu Gln Tyr Asn 245 250 255Ser Thr Tyr Arg Val Val Ser Val LeuThr Val Leu His Gln Asp Trp 260 265 270Leu Asn Gly Lys Glu Tyr LysCys Lys Val Ser Asn Lys Ala Leu Pro 275 280 285Ala Pro Ile Glu LysThr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 290 295 300Pro Gln ValTyr Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn305 310 315320Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile325 330 335Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn TyrLys Thr 340 345 350Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe PheLeu Tyr Ser Lys 355 360 365Leu Thr Val Asp Lys Ser Arg Trp Gln GlnGly Asn Val Phe Ser Cys 370 375 380Ser Val Met His Glu Ala Leu HisAsn His Tyr Thr Gln Lys Ser Leu385 390 395 400Ser Leu Ser Pro GlyLys Ser Gly Gly Leu Asn Asp Ile Phe Glu Ala 405 410 415Gln Lys IleGlu Trp His Glu 42096594DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotidehuman 4-1BB His96ctgcaggacc cctgcagcaa ctgccctgcc ggcaccttct gcgacaacaa ccggaaccag60atctgcagcc cctgcccccc caacagcttc agctctgccg gcggacagcg gacctgcgac120atctgcagac agtgcaaggg cgtgttcaga acccggaaag agtgcagcagcaccagcaac 180gccgagtgcg actgcacccc cggcttccat tgtctgggagccggctgcag catgtgcgag 240caggactgca agcagggcca ggaactgaccaagaagggct gcaaggactg ctgcttcggc 300accttcaacg accagaagcggggcatctgc cggccctgga ccaactgtag cctggacggc 360aagagcgtgctggtcaacgg caccaaagaa cgggacgtcg tgtgcggccc cagccctgct420gatctgtctc ctggggccag cagcgtgacc cctcctgccc ctgccagagagcctggccac 480tctcctcagg tcgacgaaca gttatatttt cagggcggctcaggcctgaa cgacatcttc 540gaggcccaga agatcgagtg gcacgaggctcgagctcacc accatcacca tcac 59497198PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidehuman 4-1BB His 97Leu Gln Asp Pro Cys Ser Asn Cys ProAla Gly Thr Phe Cys Asp Asn1 5 10 15Asn Arg Asn Gln Ile Cys Ser ProCys Pro Pro Asn Ser Phe Ser Ser 20 25 30Ala Gly Gly Gln Arg Thr CysAsp Ile Cys Arg Gln Cys Lys Gly Val 35 40 45Phe Arg Thr Arg Lys GluCys Ser Ser Thr Ser Asn Ala Glu Cys Asp 50 55 60Cys Thr Pro Gly PheHis Cys Leu Gly Ala Gly Cys Ser Met Cys Glu65 70 75 80Gln Asp CysLys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95Cys CysPhe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105110Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr115 120 125Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp LeuSer Pro 130 135 140Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala ArgGlu Pro Gly His145 150 155 160Ser Pro Gln Val Asp Glu Gln Leu TyrPhe Gln Gly Gly Ser Gly Leu 165 170 175Asn Asp Ile Phe Glu Ala GlnLys Ile Glu Trp His Glu Ala Arg Ala 180 185 190His His His His HisHis 195982166DNAArtificial SequenceDescription of ArtificialSequence Synthetic polynucleotideDimeric ligand (71-254)- CL* Fcknob chain 98agagagggcc ctgagctgag ccccgatgat cctgctggac tgctggacctgcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcg atggccccctgtcctggtac 120agcgatcctg gactggctgg cgtgtcactg acaggcggcctgagctacaa agaggacacc 180aaagaactgg tggtggccaa ggccggcgtgtactacgtgt tctttcagct ggaactgcgg 240agagtggtgg ccggcgaaggatctggctct gtgtctctgg ccctgcatct gcagcctctg 300agaagcgctgctggcgctgc agctctggca ctgacagtgg atctgcctcc tgccagctcc360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgc tgcacctgtctgccggccag 420aggctgggag tgcatctgca cacagaggcc agggctagacacgcctggca gctgacacag 480ggcgctacag tgctgggcct gttcagagtgacccccgaga ttccagccgg cctgccttct 540ccaagaagcg aaggcggaggcggatctggc ggcggaggat ctagagaggg acccgaactg 600tcccctgacgatccagccgg gctgctggat ctgagacagg gaatgttcgc ccagctggtg660gctcagaatg tgctgctgat tgacggacct ctgagctggt actccgacccagggctggca 720ggggtgtccc tgactggggg actgtcctac aaagaagatacaaaagaact ggtggtggct 780aaagctgggg tgtactatgt gttttttcagctggaactga ggcgggtggt ggctggggag 840ggctcaggat ctgtgtccctggctctgcat ctgcagccac tgcgctctgc tgctggcgca 900gctgcactggctctgactgt ggacctgcca ccagcctcta gcgaggccag aaacagcgcc960ttcgggttcc aaggacgcct gctgcatctg agcgccggac agcgcctgggagtgcatctg 1020catactgaag ccagagcccg gcatgcttgg cagctgactcagggggcaac tgtgctggga 1080ctgtttcgcg tgacacctga gatccctgccggactgccaa gccctagatc agaagggggc 1140ggaggttccg gagggggaggatctcgtacg gtggctgcac catctgtctt tatcttccca 1200cccagcgaccggaagctgaa gtctggcaca gccagcgtcg tgtgcctgct gaataacttc1260tacccccgcg aggccaaggt gcagtggaag gtggacaatg ccctgcagagcggcaacagc 1320caggaaagcg tgaccgagca ggacagcaag gactccacctacagcctgag cagcaccctg 1380accctgagca aggccgacta cgagaagcacaaggtgtacg cctgcgaagt gacccaccag 1440ggcctgtcta gccccgtgaccaagagcttc aaccggggcg agtgcgacaa gacccacacc 1500tgtcctccatgccctgcccc tgaagctgct ggcggcccta gcgtgttcct gttcccccca1560aagcccaagg acaccctgat gatcagccgg acccctgaag tgacctgcgtggtggtggat 1620gtgtcccacg aggaccctga agtgaagttc aattggtacgtggacggcgt ggaagtgcac 1680aatgccaaga ccaagccgcg ggaggagcagtacaacagca cgtaccgtgt ggtcagcgtc 1740ctcaccgtcc tgcaccaggactggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1800aaagccctcggcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa1860ccacaggtgt acaccctgcc cccatgccgg gatgagctga ccaagaaccaggtcagcctg 1920tggtgcctgg tcaaaggctt ctatcccagc gacatcgccgtggagtggga gagcaatggg 1980cagccggaga acaactacaa gaccacgcctcccgtgctgg actccgacgg ctccttcttc 2040ctctacagca agctcaccgtggacaagagc aggtggcagc aggggaacgt cttctcatgc 2100tccgtgatgcatgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 2160ggtaaa216699891DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideMonomeric ligand (71-254)-CH1* 99agagagggccctgagctgag ccccgatgat cctgctggac tgctggacct gcggcagggc 60atgtttgctcagctggtggc ccagaacgtg ctgctgatcg atggccccct gtcctggtac120agcgatcctg gactggctgg cgtgtcactg acaggcggcc tgagctacaaagaggacacc 180aaagaactgg tggtggccaa ggccggcgtg tactacgtgttctttcagct ggaactgcgg 240agagtggtgg ccggcgaagg atctggctctgtgtctctgg ccctgcatct gcagcctctg 300agaagcgctg ctggcgctgcagctctggct ctgacagtgg atctgcctcc tgccagctcc 360gaggcccggaatagcgcatt tgggtttcaa ggccggctgc tgcacctgtc tgccggccag420agactgggag tgcatctgca cacagaggcc agagccaggc acgcctggcagctgacacag 480ggcgctacag tgctgggcct gttcagagtg acccccgagattcctgccgg cctgcctagc 540cctagatctg aaggcggcgg aggttccggaggcggaggat ctgctagcac aaagggcccc 600agcgtgttcc ctctggcccctagcagcaag agcacatctg gcggaacagc cgccctgggc 660tgcctggtggaagattactt ccccgagccc gtgaccgtgt cctggaattc tggcgccctg720acaagcggcg tgcacacctt tccagccgtg ctgcagagca gcggcctgtactctctgagc 780agcgtcgtga cagtgcccag cagctctctg ggcacccagacctacatctg caacgtgaac 840cacaagccca gcaacaccaa ggtggacgagaaggtggaac ccaagtcctg c 8911001344DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic polynucleotideanti-TnC(18D4) Fchole chain 100caggtgcaat tggtgcagtc tggggctgag gtgaagaagcctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgctataagctgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggagggatcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcagggtcaccatt actgcagaca aatccacgag cacagcctac 240atggagctgagcagcctgag atctgaggac accgccgtgt attactgtgc gaaaggtaac300ttctacggtg gtctggacta ctggggccaa gggaccaccg tgaccgtctcctcagctagc 360accaagggcc cctccgtgtt ccccctggcc cccagcagcaagagcaccag cggcggcaca 420gccgctctgg gctgcctggt caaggactacttccccgagc ccgtgaccgt gtcctggaac 480agcggagccc tgacctccggcgtgcacacc ttccccgccg tgctgcagag ttctggcctg 540tatagcctgagcagcgtggt caccgtgcct tctagcagcc tgggcaccca gacctacatc600tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtggagcccaagagc 660tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctgaagctgcagg gggaccgtca 720gtcttcctct tccccccaaa acccaaggacaccctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatggcaaggagtac 960aagtgcaagg tctccaacaa agccctcggc gcccccatcgagaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtgcaccctgcccc catcccggga tgagctgacc 1080aagaaccagg tcagcctctcgtgcgcagtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggagagcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1200tccgacggct ccttcttcct cgtgagcaag ctcaccgtgg acaagagcaggtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgcacaaccacta cacgcagaag 1320agcctctccc tgtctccggg taaa134410110DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideRandomized sequence1modified_base(1)..(10)a, c, g, or t 101nnnnnnnnnn10102722PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideDimeric ligand (71-254) - CL* Fc knob chain102Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp15 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val LeuLeu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu AlaGly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr LysGlu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala GlyAla Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala SerSer Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg LeuLeu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu HisThr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala165 170 175Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser GlyGly Gly 180 185 190Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly Leu 195 200 205Leu Asp Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val 210 215 220Leu Leu Ile Asp Gly Pro Leu SerTrp Tyr Ser Asp Pro Gly Leu Ala225 230 235 240Gly Val Ser Leu ThrGly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 245 250 255Leu Val ValAla Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu 260 265 270LeuArg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala 275 280285Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala290 295 300Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg AsnSer Ala305 310 315 320Phe Gly Phe Gln Gly Arg Leu Leu His Leu SerAla Gly Gln Arg Leu 325 330 335Gly Val His Leu

His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu 340 345 350Thr GlnGly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile 355 360365Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly370 375 380Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe IlePhe Pro385 390 395 400Pro Ser Asp Arg Lys Leu Lys Ser Gly Thr AlaSer Val Val Cys Leu 405 410 415Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys Val Gln Trp Lys Val Asp 420 425 430Asn Ala Leu Gln Ser Gly AsnSer Gln Glu Ser Val Thr Glu Gln Asp 435 440 445Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 450 455 460Ala Asp TyrGlu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln465 470 475480Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp485 490 495Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala AlaGly Gly 500 505 510Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile 515 520 525Ser Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser His Glu 530 535 540Asp Pro Glu Val Lys Phe Asn TrpTyr Val Asp Gly Val Glu Val His545 550 555 560Asn Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 565 570 575Val Val SerVal Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 580 585 590GluTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 595 600605Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr610 615 620Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln ValSer Leu625 630 635 640Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser AspIle Ala Val Glu Trp 645 650 655Glu Ser Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val 660 665 670Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp 675 680 685Lys Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His 690 695 700Glu Ala LeuHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro705 710 715720Gly Lys103297PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptideMonomeric ligand (71-254)-CH1* 103ArgGlu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 1015Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala GlyVal 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys GluLeu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln LeuGlu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser ValSer Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly AlaAla Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser SerGlu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu LeuHis Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His ThrGlu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160GlyAla Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170175Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly180 185 190Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu AlaPro Ser 195 200 205Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu GlyCys Leu Val Glu 210 215 220Asp Tyr Phe Pro Glu Pro Val Thr Val SerTrp Asn Ser Gly Ala Leu225 230 235 240Thr Ser Gly Val His Thr PhePro Ala Val Leu Gln Ser Ser Gly Leu 245 250 255Tyr Ser Leu Ser SerVal Val Thr Val Pro Ser Ser Ser Leu Gly Thr 260 265 270Gln Thr TyrIle Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 275 280 285AspGlu Lys Val Glu Pro Lys Ser Cys 290 295104448PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptideanti-TnC(18D4) Fc hole chain 104Gln Val Gln Leu Val GlnSer Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val SerCys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser TrpVal Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly IleIle Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln GlyArg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 7580Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Gly Asn Phe Tyr Gly Gly Leu Asp Tyr Trp Gly Gln GlyThr 100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro SerVal Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly GlyThr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro GluPro Val Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr SerGly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly LeuTyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser LeuGly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly GlyPro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp ThrLeu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val ValAsp Val Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp TyrVal Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro ArgGlu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val LeuThr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315320Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val CysThr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln ValSer Leu Ser Cys 355 360 365Ala Val Lys Gly Phe Tyr Pro Ser Asp IleAla Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn TyrLys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser PhePhe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430LeuHis Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 44044510511DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideRandomized sequence2modified_base(1)..(11)a, c, g, or t 105nnnnnnnnnn n111062166DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideDimeric ligand (71-254)- CL Fc knob chain106agagagggcc ctgagctgag ccccgatgat cctgctggac tgctggacctgcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcg atggccccctgtcctggtac 120agcgatcctg gactggctgg cgtgtcactg acaggcggcctgagctacaa agaggacacc 180aaagaactgg tggtggccaa ggccggcgtgtactacgtgt tctttcagct ggaactgcgg 240agagtggtgg ccggcgaaggatctggctct gtgtctctgg ccctgcatct gcagcctctg 300agaagcgctgctggcgctgc agctctggca ctgacagtgg atctgcctcc tgccagctcc360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgc tgcacctgtctgccggccag 420aggctgggag tgcatctgca cacagaggcc agggctagacacgcctggca gctgacacag 480ggcgctacag tgctgggcct gttcagagtgacccccgaga ttccagccgg cctgccttct 540ccaagaagcg aaggcggaggcggatctggc ggcggaggat ctagagaggg acccgaactg 600tcccctgacgatccagccgg gctgctggat ctgagacagg gaatgttcgc ccagctggtg660gctcagaatg tgctgctgat tgacggacct ctgagctggt actccgacccagggctggca 720ggggtgtccc tgactggggg actgtcctac aaagaagatacaaaagaact ggtggtggct 780aaagctgggg tgtactatgt gttttttcagctggaactga ggcgggtggt ggctggggag 840ggctcaggat ctgtgtccctggctctgcat ctgcagccac tgcgctctgc tgctggcgca 900gctgcactggctctgactgt ggacctgcca ccagcctcta gcgaggccag aaacagcgcc960ttcgggttcc aaggacgcct gctgcatctg agcgccggac agcgcctgggagtgcatctg 1020catactgaag ccagagcccg gcatgcttgg cagctgactcagggggcaac tgtgctggga 1080ctgtttcgcg tgacacctga gatccctgccggactgccaa gccctagatc agaagggggc 1140ggaggttccg gagggggaggatctcgtacg gtggccgctc cctccgtgtt tatctttccc 1200ccatccgatgaacagctgaa aagcggcacc gcctccgtcg tgtgtctgct gaacaatttt1260taccctaggg aagctaaagt gcagtggaaa gtggataacg cactgcagtccggcaactcc 1320caggaatctg tgacagaaca ggactccaag gacagcacctactccctgtc ctccaccctg 1380acactgtcta aggctgatta tgagaaacacaaagtctacg cctgcgaagt cacccatcag 1440ggcctgagct cgcccgtcacaaagagcttc aacaggggag agtgtgacaa gacccacacc 1500tgtcccccttgtcctgcccc tgaagctgct ggcggccctt ctgtgttcct gttcccccca1560aagcccaagg acaccctgat gatcagccgg acccccgaag tgacctgcgtggtggtggat 1620gtgtcccacg aggaccctga agtgaagttc aattggtacgtggacggcgt ggaagtgcac 1680aatgccaaga ccaagccgcg ggaggagcagtacaacagca cgtaccgtgt ggtcagcgtc 1740ctcaccgtcc tgcaccaggactggctgaat ggcaaggagt acaagtgcaa ggtctccaac 1800aaagccctcggcgcccccat cgagaaaacc atctccaaag ccaaagggca gccccgagaa1860ccacaggtgt acaccctgcc cccatgccgg gatgagctga ccaagaaccaggtcagcctg 1920tggtgcctgg tcaaaggctt ctatcccagc gacatcgccgtggagtggga gagcaatggg 1980cagccggaga acaactacaa gaccacgcctcccgtgctgg actccgacgg ctccttcttc 2040ctctacagca agctcaccgtggacaagagc aggtggcagc aggggaacgt cttctcatgc 2100tccgtgatgcatgaggctct gcacaaccac tacacgcaga agagcctctc cctgtctccg 2160ggtaaa2166107891DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideMonomeric ligand (71-254)-CH1 107agagagggccctgagctgag ccccgatgat cctgctggac tgctggacct gcggcagggc 60atgtttgctcagctggtggc ccagaacgtg ctgctgatcg atggccccct gtcctggtac120agcgatcctg gactggctgg cgtgtcactg acaggcggcc tgagctacaaagaggacacc 180aaagaactgg tggtggccaa ggccggcgtg tactacgtgttctttcagct ggaactgcgg 240agagtggtgg ccggcgaagg atctggctctgtgtctctgg ccctgcatct gcagcctctg 300agaagcgctg ctggcgctgcagctctggct ctgacagtgg atctgcctcc tgccagctcc 360gaggcccggaatagcgcatt tgggtttcaa ggccggctgc tgcacctgtc tgccggccag420agactgggag tgcatctgca cacagaggcc agagccaggc acgcctggcagctgacacag 480ggcgctacag tgctgggcct gttcagagtg acccccgagattcctgccgg cctgcctagc 540cctagatctg aaggcggcgg aggttccggaggcggaggat ctgctagcac caaaggccct 600tccgtgtttc ctctggctcctagctccaag tccacctctg gaggcaccgc tgctctcgga 660tgcctcgtgaaggattattt tcctgagcct gtgacagtgt cctggaatag cggagcactg720acctctggag tgcatacttt ccccgctgtg ctgcagtcct ctggactgtacagcctgagc 780agcgtggtga cagtgcccag cagcagcctg ggcacccagacctacatctg caacgtgaac 840cacaagccca gcaacaccaa ggtggacaagaaggtggaac ccaagtcttg t 891108722PRTArtificial SequenceDescriptionof Artificial Sequence Synthetic polypeptideDimeric ligand (71-254)- CL Fc knob chain 108Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp ProAla Gly Leu Leu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu ValAla Gln Asn Val Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr SerAsp Pro Gly Leu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser TyrLys Glu Asp Thr Lys Glu Leu Val 50 55 60Val Ala Lys Ala Gly Val TyrTyr Val Phe Phe Gln Leu Glu Leu Arg65 70 75 80Arg Val Val Ala GlyGlu Gly Ser Gly Ser Val Ser Leu Ala Leu His 85 90 95Leu Gln Pro LeuArg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr 100 105 110Val AspLeu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly 115 120125Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val130 135 140His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln LeuThr Gln145 150 155 160Gly Ala Thr Val Leu Gly Leu Phe Arg Val ThrPro Glu Ile Pro Ala 165 170 175Gly Leu Pro Ser Pro Arg Ser Glu GlyGly Gly Gly Ser Gly Gly Gly 180 185 190Gly Ser Arg Glu Gly Pro GluLeu Ser Pro Asp Asp Pro Ala Gly Leu 195 200 205Leu Asp Leu Arg GlnGly Met Phe Ala Gln Leu Val Ala Gln Asn Val 210 215 220Leu Leu IleAsp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala225 230 235240Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu245 250 255Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu 260 265 270Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala 275 280 285Leu His Leu Gln Pro Leu Arg Ser Ala AlaGly Ala Ala Ala Leu Ala 290 295 300Leu Thr Val Asp Leu Pro Pro AlaSer Ser Glu Ala Arg Asn Ser Ala305 310 315 320Phe Gly Phe Gln GlyArg Leu Leu His Leu Ser Ala Gly Gln Arg Leu 325 330 335Gly Val HisLeu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu 340 345 350ThrGln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile 355 360365Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly370 375 380Gly Gly Gly Ser Arg Thr Val Ala Ala Pro Ser Val Phe IlePhe Pro385 390 395 400Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr AlaSer Val Val Cys Leu 405 410 415Leu Asn Asn Phe Tyr Pro Arg Glu AlaLys Val Gln Trp Lys Val Asp 420 425 430Asn Ala Leu Gln Ser Gly AsnSer Gln Glu Ser Val Thr Glu Gln Asp 435 440 445Ser Lys Asp Ser ThrTyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 450 455 460Ala Asp TyrGlu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln465 470 475480Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Asp485 490 495Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala AlaGly Gly 500 505 510Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys AspThr Leu Met Ile 515 520 525Ser Arg Thr Pro Glu Val Thr Cys Val ValVal Asp Val Ser His Glu 530 535 540Asp Pro Glu Val Lys Phe Asn TrpTyr Val Asp Gly Val Glu Val His545 550 555 560Asn Ala Lys Thr LysPro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 565 570 575Val Val SerVal Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 580 585 590GluTyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu 595 600605Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr610 615 620Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln ValSer Leu625 630 635 640Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser AspIle Ala Val Glu Trp 645 650 655Glu Ser Asn Gly Gln Pro Glu Asn AsnTyr Lys Thr Thr Pro Pro Val 660 665 670Leu Asp Ser Asp Gly Ser PhePhe Leu Tyr Ser Lys Leu Thr Val Asp 675 680 685Lys Ser Arg Trp GlnGln Gly Asn Val Phe Ser Cys Ser Val Met His 690

695 700Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu SerPro705 710 715 720Gly Lys109297PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptideMonomeric ligand (71-254)-CH1 109Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu LeuAsp1 5 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn ValLeu Leu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly LeuAla Gly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp ThrLys Glu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe PheGln Leu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser GlySer Val Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala AlaGly Ala Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro AlaSer Ser Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly ArgLeu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His LeuHis Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala165 170 175Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser GlyGly Gly 180 185 190Gly Ser Ala Ser Thr Lys Gly Pro Ser Val Phe ProLeu Ala Pro Ser 195 200 205Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly Cys Leu Val Lys 210 215 220Asp Tyr Phe Pro Glu Pro Val ThrVal Ser Trp Asn Ser Gly Ala Leu225 230 235 240Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu 245 250 255Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 260 265 270GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 275 280285Asp Lys Lys Val Glu Pro Lys Ser Cys 290 2951102505DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotideanti-TnC(18D4) Fc hole dimeric ligand (71-254) chain110caggtgcaat tggtgcagtc tggggctgag gtgaagaagc ctgggtcctcggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgcta taagctgggtgcgacaggcc 120cctggacaag ggctcgagtg gatgggaggg atcatccctatctttggtac agcaaactac 180gcacagaagt tccagggcag ggtcaccattactgcagaca aatccacgag cacagcctac 240atggagctga gcagcctgagatctgaggac accgccgtgt attactgtgc gaaaggtaac 300ttctacggtggtctggacta ctggggccaa gggaccaccg tgaccgtctc ctcagctagc360accaagggcc cctccgtgtt ccccctggcc cccagcagca agagcaccagcggcggcaca 420gccgctctgg gctgcctggt caaggactac ttccccgagcccgtgaccgt gtcctggaac 480agcggagccc tgacctccgg cgtgcacaccttccccgccg tgctgcagag ttctggcctg 540tatagcctga gcagcgtggtcaccgtgcct tctagcagcc tgggcaccca gacctacatc 600tgcaacgtgaaccacaagcc cagcaacacc aaggtggaca agaaggtgga gcccaagagc660tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagggggaccgtca 720gtcttcctct tccccccaaa acccaaggac accctcatgatctcccggac ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaagaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataatgccaagaca aagccgcggg aggagcagta caacagcacg 900taccgtgtggtcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac960aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccatctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtgc accctgcccccatcccggga tgagctgacc 1080aagaaccagg tcagcctctc gtgcgcagtcaaaggcttct atcccagcga catcgccgtg 1140gagtgggaga gcaatgggcagccggagaac aactacaaga ccacgcctcc cgtgctggac 1200tccgacggctccttcttcct cgtgagcaag ctcaccgtgg acaagagcag gtggcagcag1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccactacacgcagaag 1320agcctctccc tgtctccggg tggaggcggc ggaagcggaggaggaggatc cagagagggc 1380cctgagctga gccccgatga tcctgctggactgctggacc tgcggcaggg catgtttgct 1440cagctggtgg cccagaacgtgctgctgatc gatggccccc tgtcctggta cagcgatcct 1500ggactggctggcgtgtcact gacaggcggc ctgagctaca aagaggacac caaagaactg1560gtggtggcca aggccggcgt gtactacgtg ttctttcagc tggaactgcggagagtggtg 1620gccggcgaag gatctggctc tgtgtctctg gccctgcatctgcagcctct gagaagcgct 1680gctggcgctg cagctctggc actgacagtggatctgcctc ctgccagctc cgaggcccgg 1740aatagcgcat ttgggtttcaaggcaggctg ctgcacctgt ctgccggcca gaggctggga 1800gtgcatctgcacacagaggc cagggctaga cacgcctggc agctgacaca gggcgctaca1860gtgctgggcc tgttcagagt gacccccgag attccagccg gcctgccttctccaagaagc 1920gaaggcggag gcggatctgg cggcggagga tctagagagggacccgaact gtcccctgac 1980gatccagccg ggctgctgga tctgagacagggaatgttcg cccagctggt ggctcagaat 2040gtgctgctga ttgacggacctctgagctgg tactccgacc cagggctggc aggggtgtcc 2100ctgactgggggactgtccta caaagaagat acaaaagaac tggtggtggc taaagctggg2160gtgtactatg tgttttttca gctggaactg aggcgggtgg tggctggggagggctcagga 2220tctgtgtccc tggctctgca tctgcagcca ctgcgctctgctgctggcgc agctgcactg 2280gctctgactg tggacctgcc accagcctctagcgaggcca gaaacagcgc cttcgggttc 2340caaggacgcc tgctgcatctgagcgccgga cagcgcctgg gagtgcatct gcatactgaa 2400gccagagcccggcatgcttg gcagctgact cagggggcaa ctgtgctggg actgtttcgc2460gtgacacctg agatccctgc cggactgcca agccctagat cagaa25051111923DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideanti-TnC(18D4) Fc knob monomeric ligand(71-254) chain 111caggtgcaat tggtgcagtc tggggctgag gtgaagaagcctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgctataagctgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggagggatcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcagggtcaccatt actgcagaca aatccacgag cacagcctac 240atggagctgagcagcctgag atctgaggac accgccgtgt attactgtgc gaaaggtaac300ttctacggtg gtctggacta ctggggccaa gggaccaccg tgaccgtctcctcagctagc 360accaagggcc catcggtctt ccccctggca ccctcctccaagagcacctc tgggggcaca 420gcggccctgg gctgcctggt caaggactacttccccgaac cggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcggcgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540tactccctcagcagcgtggt gaccgtgccc tccagcagct tgggcaccca gacctacatc600tgcaacgtga atcacaagcc cagcaacacc aaggtggaca agaaagttgagcccaaatct 660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctgaagctgcagg gggaccgtca 720gtcttcctct tccccccaaa acccaaggacaccctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatggcaaggagtac 960aagtgcaagg tctccaacaa agccctcggc gcccccatcgagaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtacaccctgcccc cctgcagaga tgagctgacc 1080aagaaccagg tgtccctgtggtgtctggtc aagggcttct accccagcga tatcgccgtg 1140gagtgggagagcaacggcca gcctgagaac aactacaaga ccaccccccc tgtgctggac1200agcgacggca gcttcttcct gtactccaaa ctgaccgtgg acaagagccggtggcagcag 1260ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgcacaaccacta cacccagaag 1320tccctgagcc tgagccccgg cggaggcggcggaagcggag gaggaggatc cagagagggc 1380cctgagctga gccccgatgatcctgctgga ctgctggacc tgcggcaggg catgtttgct 1440cagctggtggcccagaacgt gctgctgatc gatggccccc tgtcctggta cagcgatcct1500ggactggctg gcgtgtcact gacaggcggc ctgagctaca aagaggacaccaaagaactg 1560gtggtggcca aggccggcgt gtactacgtg ttctttcagctggaactgcg gagagtggtg 1620gccggcgaag gatctggctc tgtgtctctggccctgcatc tgcagcctct gagaagcgct 1680gctggcgctg cagctctggcactgacagtg gatctgcctc ctgccagctc cgaggcccgg 1740aatagcgcatttgggtttca aggcaggctg ctgcacctgt ctgccggcca gaggctggga1800gtgcatctgc acacagaggc cagggctaga cacgcctggc agctgacacagggcgctaca 1860gtgctgggcc tgttcagagt gacccccgag attccagccggcctgccttc tccaagaagc 1920gaa 1923112835PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptideanti-TnC(18D4) Fc hole dimeric ligand (71-254) chain112Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser15 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser SerTyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu GluTrp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr AlaGln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser ThrSer Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu AspThr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Phe Tyr Gly Gly LeuAsp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser AlaSer Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser SerLys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu ValLys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val ProSer Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val AsnHis Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu ProLys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro AlaPro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe ProPro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro GluVal Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly LysGlu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Gly AlaPro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro ArgGlu Pro Gln Val Cys Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu LeuThr Lys Asn Gln Val Ser Leu Ser Cys 355 360 365Ala Val Lys Gly PheTyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly GlnPro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395400Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met HisGlu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser LeuSer Pro Gly Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly Ser ArgGlu Gly Pro Glu Leu Ser 450 455 460Pro Asp Asp Pro Ala Gly Leu LeuAsp Leu Arg Gln Gly Met Phe Ala465 470 475 480Gln Leu Val Ala GlnAsn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp 485 490 495Tyr Ser AspPro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser 500 505 510TyrLys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr 515 520525Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly530 535 540Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu ArgSer Ala545 550 555 560Ala Gly Ala Ala Ala Leu Ala Leu Thr Val AspLeu Pro Pro Ala Ser 565 570 575Ser Glu Ala Arg Asn Ser Ala Phe GlyPhe Gln Gly Arg Leu Leu His 580 585 590Leu Ser Ala Gly Gln Arg LeuGly Val His Leu His Thr Glu Ala Arg 595 600 605Ala Arg His Ala TrpGln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu 610 615 620Phe Arg ValThr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser625 630 635640Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu645 650 655Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg GlnGly Met 660 665 670Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu IleAsp Gly Pro Leu 675 680 685Ser Trp Tyr Ser Asp Pro Gly Leu Ala GlyVal Ser Leu Thr Gly Gly 690 695 700Leu Ser Tyr Lys Glu Asp Thr LysGlu Leu Val Val Ala Lys Ala Gly705 710 715 720Val Tyr Tyr Val PhePhe Gln Leu Glu Leu Arg Arg Val Val Ala Gly 725 730 735Glu Gly SerGly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg 740 745 750SerAla Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro 755 760765Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu770 775 780Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu HisThr Glu785 790 795 800Ala Arg Ala Arg His Ala Trp Gln Leu Thr GlnGly Ala Thr Val Leu 805 810 815Gly Leu Phe Arg Val Thr Pro Glu IlePro Ala Gly Leu Pro Ser Pro 820 825 830Arg Ser Glu835113641PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideanti-TnC(18D4) Fc knob monomeric ligand(71-254) chain 113Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val LysLys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly GlyThr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro GlyGln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly ThrAla Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr AlaAsp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn PheTyr Gly Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val ThrVal Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125LeuAla Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpAsn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe ProAla Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser ValVal Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr IleCys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp LysLys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys ProPro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230 235 240ValPhe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp TrpLeu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn LysAla Leu Gly Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala LysGly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro CysArg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 355 360 365LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu ThrVal Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Gly 435 440 445Gly Gly Gly Ser Gly GlyGly Gly Ser Arg Glu Gly Pro Glu Leu Ser 450 455 460Pro Asp Asp ProAla Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala465

470 475 480Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro LeuSer Trp 485 490 495Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu ThrGly Gly Leu Ser 500 505 510Tyr Lys Glu Asp Thr Lys Glu Leu Val ValAla Lys Ala Gly Val Tyr 515 520 525Tyr Val Phe Phe Gln Leu Glu LeuArg Arg Val Val Ala Gly Glu Gly 530 535 540Ser Gly Ser Val Ser LeuAla Leu His Leu Gln Pro Leu Arg Ser Ala545 550 555 560Ala Gly AlaAla Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser 565 570 575SerGlu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His 580 585590Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg595 600 605Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val LeuGly Leu 610 615 620Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu ProSer Pro Arg Ser625 630 635 640Glu1142130DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotideDimeric ligand (71-248) - CL* Fc knob chain114agagagggcc ctgagctgag ccccgatgat cctgctggac tgctggacctgcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcg atggccccctgtcctggtac 120agcgatcctg gactggctgg cgtgtcactg acaggcggcctgagctacaa agaggacacc 180aaagaactgg tggtggccaa ggccggcgtgtactacgtgt tctttcagct ggaactgcgg 240agagtggtgg ccggcgaaggatctggctct gtgtctctgg ccctgcatct gcagcctctg 300agatctgctgctggcgccgc tgctctggca ctgacagtgg atctgcctcc tgccagcagc360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgc tgcacctgtctgccggccag 420aggctgggag tgcatctgca cacagaggcc agggctagacacgcctggca gctgacacag 480ggcgctacag tgctgggcct gttcagagtgacccccgaga ttccagccgg actgggaggc 540ggcggatctg gcggcggaggatctagagaa ggacccgagc tgtcccctga cgatccagcc 600gggctgctggatctgagaca gggaatgttc gcccagctgg tggctcagaa tgtgctgctg660attgacggac ctctgagctg gtactccgac ccagggctgg caggggtgtccctgactggg 720ggactgtcct acaaagaaga tacaaaagaa ctggtggtggctaaagctgg ggtgtactat 780gtgttttttc agctggaact gaggcgggtggtggctgggg agggctcagg atctgtgtcc 840ctggctctgc atctgcagccactgcgctct gcagcagggg ctgcagcact ggccctgact 900gtggacctgcccccagcttc ttccgaggcc agaaacagcg ccttcgggtt ccaaggacgc960ctgctgcatc tgagcgccgg acagcgcctg ggagtgcatc tgcatactgaagccagagcc 1020cggcatgctt ggcagctgac tcagggggca actgtgctgggactgtttcg cgtgacacct 1080gagatccccg ctggactggg cggaggcggttccggagggg gaggatctcg tacggtggct 1140gcaccatctg tctttatcttcccacccagc gaccggaagc tgaagtctgg cacagccagc 1200gtcgtgtgcctgctgaataa cttctacccc cgcgaggcca aggtgcagtg gaaggtggac1260aatgccctgc agagcggcaa cagccaggaa agcgtgaccg agcaggacagcaaggactcc 1320acctacagcc tgagcagcac cctgaccctg agcaaggccgactacgagaa gcacaaggtg 1380tacgcctgcg aagtgaccca ccagggcctgtctagccccg tgaccaagag cttcaaccgg 1440ggcgagtgcg acaagacccacacctgtcct ccatgccctg cccctgaagc tgctggcggc 1500cctagcgtgttcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggacccct1560gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaagttcaattgg 1620tacgtggacg gcgtggaagt gcacaatgcc aagaccaagccgcgggagga gcagtacaac 1680agcacgtacc gtgtggtcag cgtcctcaccgtcctgcacc aggactggct gaatggcaag 1740gagtacaagt gcaaggtctccaacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1800aaagccaaagggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag1860ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcccagcgacatc 1920gccgtggagt gggagagcaa tgggcagccg gagaacaactacaagaccac gcctcccgtg 1980ctggactccg acggctcctt cttcctctacagcaagctca ccgtggacaa gagcaggtgg 2040cagcagggga acgtcttctcatgctccgtg atgcatgagg ctctgcacaa ccactacacg 2100cagaagagcctctccctgtc tccgggtaaa 2130115873DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic polynucleotideMonomeric ligand(71-248)-CH1* 115agagagggcc ctgagctgag ccccgatgat cctgctggactgctggacct gcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcgatggccccct gtcctggtac 120agcgatcctg gactggctgg cgtgtcactgacaggcggcc tgagctacaa agaggacacc 180aaagaactgg tggtggccaaggccggcgtg tactacgtgt tctttcagct ggaactgcgg 240agagtggtggccggcgaagg atctggctct gtgtctctgg ccctgcatct gcagcctctg300agatctgctg ctggcgccgc tgctctggca ctgacagtgg atctgcctcctgccagcagc 360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgctgcacctgtc tgccggccag 420aggctgggag tgcatctgca cacagaggccagggctagac acgcctggca gctgacacag 480ggcgctacag tgctgggcctgttcagagtg acccccgaga ttccagccgg actgggaggc 540ggaggttccggaggcggagg atctgctagc acaaagggcc ccagcgtgtt ccctctggcc600cctagcagca agagcacatc tggcggaaca gccgccctgg gctgcctggtggaagattac 660ttccccgagc ccgtgaccgt gtcctggaat tctggcgccctgacaagcgg cgtgcacacc 720tttccagccg tgctgcagag cagcggcctgtactctctga gcagcgtcgt gacagtgccc 780agcagctctc tgggcacccagacctacatc tgcaacgtga accacaagcc cagcaacacc 840aaggtggacgagaaggtgga acccaagtcc tgc 873116710PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideDimeric ligand (71-248) - CL* Fc knob chain 116Arg GluGly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 10 15LeuArg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 20 2530Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu LeuVal 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu GluLeu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val SerLeu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala AlaAla Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser Ser GluAla Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu Leu HisLeu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His Thr GluAla Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160Gly AlaThr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170175Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro180 185 190Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu ArgGln Gly 195 200 205Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu LeuIle Asp Gly Pro 210 215 220Leu Ser Trp Tyr Ser Asp Pro Gly Leu AlaGly Val Ser Leu Thr Gly225 230 235 240Gly Leu Ser Tyr Lys Glu AspThr Lys Glu Leu Val Val Ala Lys Ala 245 250 255Gly Val Tyr Tyr ValPhe Phe Gln Leu Glu Leu Arg Arg Val Val Ala 260 265 270Gly Glu GlySer Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu 275 280 285ArgSer Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro 290 295300Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln GlyArg305 310 315 320Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly ValHis Leu His Thr 325 330 335Glu Ala Arg Ala Arg His Ala Trp Gln LeuThr Gln Gly Ala Thr Val 340 345 350Leu Gly Leu Phe Arg Val Thr ProGlu Ile Pro Ala Gly Leu Gly Gly 355 360 365Gly Gly Ser Gly Gly GlyGly Ser Arg Thr Val Ala Ala Pro Ser Val 370 375 380Phe Ile Phe ProPro Ser Asp Arg Lys Leu Lys Ser Gly Thr Ala Ser385 390 395 400ValVal Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 405 410415Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val420 425 430Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser SerThr Leu 435 440 445Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys ValTyr Ala Cys Glu 450 455 460Val Thr His Gln Gly Leu Ser Ser Pro ValThr Lys Ser Phe Asn Arg465 470 475 480Gly Glu Cys Asp Lys Thr HisThr Cys Pro Pro Cys Pro Ala Pro Glu 485 490 495Ala Ala Gly Gly ProSer Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 500 505 510Thr Leu MetIle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 515 520 525ValSer His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 530 535540Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln TyrAsn545 550 555 560Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val LeuHis Gln Asp Trp 565 570 575Leu Asn Gly Lys Glu Tyr Lys Cys Lys ValSer Asn Lys Ala Leu Gly 580 585 590Ala Pro Ile Glu Lys Thr Ile SerLys Ala Lys Gly Gln Pro Arg Glu 595 600 605Pro Gln Val Tyr Thr LeuPro Pro Cys Arg Asp Glu Leu Thr Lys Asn 610 615 620Gln Val Ser LeuTrp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile625 630 635 640AlaVal Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 645 650655Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys660 665 670Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val PheSer Cys 675 680 685Ser Val Met His Glu Ala Leu His Asn His Tyr ThrGln Lys Ser Leu 690 695 700Ser Leu Ser Pro Gly Lys705710117291PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideMonomeric ligand (71-248)-CH1* 117Arg Glu GlyPro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 10 15Leu ArgGln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 20 25 30IleAsp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val 35 4045Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu LeuArg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser LeuAla Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala AlaLeu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser Ser Glu AlaArg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu Leu His LeuSer Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His Thr Glu AlaArg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160Gly Ala ThrVal Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170 175GlyLeu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys 180 185190Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly195 200 205Gly Thr Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe ProGlu Pro 210 215 220Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr SerGly Val His Thr225 230 235 240Phe Pro Ala Val Leu Gln Ser Ser GlyLeu Tyr Ser Leu Ser Ser Val 245 250 255Val Thr Val Pro Ser Ser SerLeu Gly Thr Gln Thr Tyr Ile Cys Asn 260 265 270Val Asn His Lys ProSer Asn Thr Lys Val Asp Glu Lys Val Glu Pro 275 280 285Lys Ser Cys2901182130DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideDimeric ligand (71-248) - CL Fc knob chain118agagagggcc ctgagctgag ccccgatgat cctgctggac tgctggacctgcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcg atggccccctgtcctggtac 120agcgatcctg gactggctgg cgtgtcactg acaggcggcctgagctacaa agaggacacc 180aaagaactgg tggtggccaa ggccggcgtgtactacgtgt tctttcagct ggaactgcgg 240agagtggtgg ccggcgaaggatctggctct gtgtctctgg ccctgcatct gcagcctctg 300agatctgctgctggcgccgc tgctctggca ctgacagtgg atctgcctcc tgccagcagc360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgc tgcacctgtctgccggccag 420aggctgggag tgcatctgca cacagaggcc agggctagacacgcctggca gctgacacag 480ggcgctacag tgctgggcct gttcagagtgacccccgaga ttccagccgg actgggaggc 540ggcggatctg gcggcggaggatctagagaa ggacccgagc tgtcccctga cgatccagcc 600gggctgctggatctgagaca gggaatgttc gcccagctgg tggctcagaa tgtgctgctg660attgacggac ctctgagctg gtactccgac ccagggctgg caggggtgtccctgactggg 720ggactgtcct acaaagaaga tacaaaagaa ctggtggtggctaaagctgg ggtgtactat 780gtgttttttc agctggaact gaggcgggtggtggctgggg agggctcagg atctgtgtcc 840ctggctctgc atctgcagccactgcgctct gcagcagggg ctgcagcact ggccctgact 900gtggacctgcccccagcttc ttccgaggcc agaaacagcg ccttcgggtt ccaaggacgc960ctgctgcatc tgagcgccgg acagcgcctg ggagtgcatc tgcatactgaagccagagcc 1020cggcatgctt ggcagctgac tcagggggca actgtgctgggactgtttcg cgtgacacct 1080gagatccccg ctggactggg cggaggcggttccggagggg gaggatctcg tacggtggcc 1140gctccctccg tgtttatctttcccccatcc gatgaacagc tgaaaagcgg caccgcctcc 1200gtcgtgtgtctgctgaacaa tttttaccct agggaagcta aagtgcagtg gaaagtggat1260aacgcactgc agtccggcaa ctcccaggaa tctgtgacag aacaggactccaaggacagc 1320acctactccc tgtcctccac cctgacactg tctaaggctgattatgagaa acacaaagtc 1380tacgcctgcg aagtcaccca tcagggcctgagctcgcccg tcacaaagag cttcaacagg 1440ggagagtgtg acaagacccacacctgtccc ccttgtcctg cccctgaagc tgctggcggc 1500ccttctgtgttcctgttccc cccaaagccc aaggacaccc tgatgatcag ccggaccccc1560gaagtgacct gcgtggtggt ggatgtgtcc cacgaggacc ctgaagtgaagttcaattgg 1620tacgtggacg gcgtggaagt gcacaatgcc aagaccaagccgcgggagga gcagtacaac 1680agcacgtacc gtgtggtcag cgtcctcaccgtcctgcacc aggactggct gaatggcaag 1740gagtacaagt gcaaggtctccaacaaagcc ctcggcgccc ccatcgagaa aaccatctcc 1800aaagccaaagggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag1860ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcccagcgacatc 1920gccgtggagt gggagagcaa tgggcagccg gagaacaactacaagaccac gcctcccgtg 1980ctggactccg acggctcctt cttcctctacagcaagctca ccgtggacaa gagcaggtgg 2040cagcagggga acgtcttctcatgctccgtg atgcatgagg ctctgcacaa ccactacacg 2100cagaagagcctctccctgtc tccgggtaaa 2130119873DNAArtificial SequenceDescriptionof Artificial Sequence Synthetic polynucleotideMonomeric ligand(71-248)-CH1 119agagagggcc ctgagctgag ccccgatgat cctgctggactgctggacct gcggcagggc 60atgtttgctc agctggtggc ccagaacgtg ctgctgatcgatggccccct gtcctggtac 120agcgatcctg gactggctgg cgtgtcactgacaggcggcc tgagctacaa agaggacacc 180aaagaactgg tggtggccaaggccggcgtg tactacgtgt tctttcagct ggaactgcgg 240agagtggtggccggcgaagg atctggctct gtgtctctgg ccctgcatct gcagcctctg300agatctgctg ctggcgccgc tgctctggca ctgacagtgg atctgcctcctgccagcagc 360gaggcccgga atagcgcatt tgggtttcaa ggcaggctgctgcacctgtc tgccggccag 420aggctgggag tgcatctgca cacagaggccagggctagac acgcctggca gctgacacag 480ggcgctacag tgctgggcctgttcagagtg acccccgaga ttccagccgg actgggaggc 540ggaggttccggaggcggagg atctgctagc accaaaggcc cttccgtgtt tcctctggct600cctagctcca agtccacctc tggaggcacc gctgctctcg gatgcctcgtgaaggattat 660tttcctgagc ctgtgacagt gtcctggaat agcggagcactgacctctgg agtgcatact 720ttccccgctg tgctgcagtc ctctggactgtacagcctga gcagcgtggt gacagtgccc 780agcagcagcc tgggcacccagacctacatc tgcaacgtga accacaagcc cagcaacacc 840aaggtggacaagaaggtgga acccaagtct tgt 873120710PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideDimeric ligand (71-248) - CL Fc knob chain 120Arg GluGly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 10 15LeuArg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 20 2530Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu LeuVal 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu GluLeu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val SerLeu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala AlaAla Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser Ser GluAla Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu Leu HisLeu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His Thr GluAla Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160Gly AlaThr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala

165 170 175Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg GluGly Pro 180 185 190Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu AspLeu Arg Gln Gly 195 200 205Met Phe Ala Gln Leu Val Ala Gln Asn ValLeu Leu Ile Asp Gly Pro 210 215 220Leu Ser Trp Tyr Ser Asp Pro GlyLeu Ala Gly Val Ser Leu Thr Gly225 230 235 240Gly Leu Ser Tyr LysGlu Asp Thr Lys Glu Leu Val Val Ala Lys Ala 245 250 255Gly Val TyrTyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala 260 265 270GlyGlu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu 275 280285Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro290 295 300Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe GlnGly Arg305 310 315 320Leu Leu His Leu Ser Ala Gly Gln Arg Leu GlyVal His Leu His Thr 325 330 335Glu Ala Arg Ala Arg His Ala Trp GlnLeu Thr Gln Gly Ala Thr Val 340 345 350Leu Gly Leu Phe Arg Val ThrPro Glu Ile Pro Ala Gly Leu Gly Gly 355 360 365Gly Gly Ser Gly GlyGly Gly Ser Arg Thr Val Ala Ala Pro Ser Val 370 375 380Phe Ile PhePro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser385 390 395400Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln405 410 415Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln GluSer Val 420 425 430Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser LeuSer Ser Thr Leu 435 440 445Thr Leu Ser Lys Ala Asp Tyr Glu Lys HisLys Val Tyr Ala Cys Glu 450 455 460Val Thr His Gln Gly Leu Ser SerPro Val Thr Lys Ser Phe Asn Arg465 470 475 480Gly Glu Cys Asp LysThr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 485 490 495Ala Ala GlyGly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 500 505 510ThrLeu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 515 520525Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly530 535 540Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu GlnTyr Asn545 550 555 560Ser Thr Tyr Arg Val Val Ser Val Leu Thr ValLeu His Gln Asp Trp 565 570 575Leu Asn Gly Lys Glu Tyr Lys Cys LysVal Ser Asn Lys Ala Leu Gly 580 585 590Ala Pro Ile Glu Lys Thr IleSer Lys Ala Lys Gly Gln Pro Arg Glu 595 600 605Pro Gln Val Tyr ThrLeu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn 610 615 620Gln Val SerLeu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile625 630 635640Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr645 650 655Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu TyrSer Lys 660 665 670Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly AsnVal Phe Ser Cys 675 680 685Ser Val Met His Glu Ala Leu His Asn HisTyr Thr Gln Lys Ser Leu 690 695 700Ser Leu Ser Pro Gly Lys705710121291PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideMonomeric ligand (71-248)-CH1 121Arg Glu GlyPro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 10 15Leu ArgGln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 20 25 30IleAsp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val 35 4045Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu LeuArg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser LeuAla Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala AlaLeu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser Ser Glu AlaArg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu Leu His LeuSer Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His Thr Glu AlaArg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160Gly Ala ThrVal Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170 175GlyLeu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ser Thr Lys 180 185190Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly195 200 205Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe ProGlu Pro 210 215 220Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr SerGly Val His Thr225 230 235 240Phe Pro Ala Val Leu Gln Ser Ser GlyLeu Tyr Ser Leu Ser Ser Val 245 250 255Val Thr Val Pro Ser Ser SerLeu Gly Thr Gln Thr Tyr Ile Cys Asn 260 265 270Val Asn His Lys ProSer Asn Thr Lys Val Asp Lys Lys Val Glu Pro 275 280 285Lys Ser Cys2901222469DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideanti-TnC(18D4) Fc hole dimeric ligand(71-248) chain 122caggtgcaat tggtgcagtc tggggctgag gtgaagaagcctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgctataagctgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggagggatcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcagggtcaccatt actgcagaca aatccacgag cacagcctac 240atggagctgagcagcctgag atctgaggac accgccgtgt attactgtgc gaaaggtaac300ttctacggtg gtctggacta ctggggccaa gggaccaccg tgaccgtctcctcagctagc 360accaagggcc cctccgtgtt ccccctggcc cccagcagcaagagcaccag cggcggcaca 420gccgctctgg gctgcctggt caaggactacttccccgagc ccgtgaccgt gtcctggaac 480agcggagccc tgacctccggcgtgcacacc ttccccgccg tgctgcagag ttctggcctg 540tatagcctgagcagcgtggt caccgtgcct tctagcagcc tgggcaccca gacctacatc600tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtggagcccaagagc 660tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctgaagctgcagg gggaccgtca 720gtcttcctct tccccccaaa acccaaggacaccctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatggcaaggagtac 960aagtgcaagg tctccaacaa agccctcggc gcccccatcgagaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtgcaccctgcccc catcccggga tgagctgacc 1080aagaaccagg tcagcctctcgtgcgcagtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggagagcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1200tccgacggct ccttcttcct cgtgagcaag ctcaccgtgg acaagagcaggtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgcacaaccacta cacgcagaag 1320agcctctccc tgtctccggg tggaggcggcggaagcggag gaggaggatc cagagagggc 1380cctgagctga gccctgatgatcctgccgga ctgctggacc tgcggcaggg aatgtttgcc 1440cagctggtggcccagaacgt gctgctgatc gatggccccc tgtcctggta cagcgatcct1500ggactggctg gcgtgtcact gacaggcggc ctgagctaca aagaggacaccaaagaactg 1560gtggtggcca aggccggcgt gtactacgtg ttctttcagctggaactgcg gagagtggtg 1620gccggcgaag gatctggctc tgtgtctctggccctgcatc tgcagcctct gagatctgct 1680gctggcgccg ctgctctggcactgacagtg gatctgcctc ctgccagcag cgaggcccgg 1740aatagcgcatttgggtttca aggcaggctg ctgcacctgt ctgccggcca gaggctggga1800gtgcatctgc acacagaggc cagggctaga cacgcctggc agctgacacagggcgctaca 1860gtgctgggcc tgttcagagt gacccccgag attccagcaggcctgggagg cggcggatct 1920ggcggcggag gatctagaga aggacccgagctgtcccccg acgatcccgc tgggctgctg 1980gatctgagac agggcatgttcgctcagctg gtggctcaga atgtgctgct gattgacgga 2040cctctgagctggtactccga cccagggctg gcaggggtgt ccctgactgg gggactgtcc2100tacaaagaag atacaaaaga actggtggtg gctaaagctg gggtgtactatgtgtttttt 2160cagctggaac tgaggcgggt ggtggctggg gagggctcaggatctgtgtc cctggctctg 2220catctgcagc cactgcgctc tgcagcaggggctgcagcac tggccctgac tgtggacctg 2280cccccagctt cttccgaggccagaaacagc gccttcgggt tccaaggacg cctgctgcat 2340ctgagcgccggacagcgcct gggagtgcat ctgcatactg aagccagagc ccggcatgct2400tggcagctga ctcagggggc aactgtgctg ggactgtttc gcgtgacacctgagatccca 2460gccgggctc 24691231905DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotideanti-TnC(18D4) Fc knob monomeric (71-248) ligand123caggtgcaat tggtgcagtc tggggctgag gtgaagaagc ctgggtcctcggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgcta taagctgggtgcgacaggcc 120cctggacaag ggctcgagtg gatgggaggg atcatccctatctttggtac agcaaactac 180gcacagaagt tccagggcag ggtcaccattactgcagaca aatccacgag cacagcctac 240atggagctga gcagcctgagatctgaggac accgccgtgt attactgtgc gaaaggtaac 300ttctacggtggtctggacta ctggggccaa gggaccaccg tgaccgtctc ctcagctagc360accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctctgggggcaca 420gcggccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacaccttcccggctg tcctacagtc ctcaggactc 540tactccctca gcagcgtggtgaccgtgccc tccagcagct tgggcaccca gacctacatc 600tgcaacgtgaatcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagggggaccgtca 720gtcttcctct tccccccaaa acccaaggac accctcatgatctcccggac ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaagaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataatgccaagaca aagccgcggg aggagcagta caacagcacg 900taccgtgtggtcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac960aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccatctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtac accctgcccccctgcagaga tgagctgacc 1080aagaaccagg tgtccctgtg gtgtctggtcaagggcttct accccagcga tatcgccgtg 1140gagtgggaga gcaacggccagcctgagaac aactacaaga ccaccccccc tgtgctggac 1200agcgacggcagcttcttcct gtactccaaa ctgaccgtgg acaagagccg gtggcagcag1260ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccactacacccagaag 1320tccctgagcc tgagccccgg cggaggcggc ggaagcggaggaggaggatc cagagagggc 1380cctgagctga gccctgatga tcctgccggactgctggacc tgcggcaggg aatgtttgcc 1440cagctggtgg cccagaacgtgctgctgatc gatggccccc tgtcctggta cagcgatcct 1500ggactggctggcgtgtcact gacaggcggc ctgagctaca aagaggacac caaagaactg1560gtggtggcca aggccggcgt gtactacgtg ttctttcagc tggaactgcggagagtggtg 1620gccggcgaag gatctggctc tgtgtctctg gccctgcatctgcagcctct gagatctgct 1680gctggcgccg ctgctctggc actgacagtggatctgcctc ctgccagcag cgaggcccgg 1740aatagcgcat ttgggtttcaaggcaggctg ctgcacctgt ctgccggcca gaggctggga 1800gtgcatctgcacacagaggc cagggctaga cacgcctggc agctgacaca gggcgctaca1860gtgctgggcc tgttcagagt gacccccgag attcctgccg ggctc1905124823PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideanti-TnC(18D4) Fc hole dimeric ligand (71-248)chain 124Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys ProGly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr PheSer Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln GlyLeu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala AsnTyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp LysSer Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Phe Tyr GlyGly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val SerSer Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala ProSer Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140CysLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuGln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn ValAsn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val GluPro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys ProAla Pro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu PhePro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala LeuGly Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro Gln Val Cys Thr Leu 340 345 350Pro Pro Ser Arg AspGlu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 355 360 365Ala Val LysGly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380AsnGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390395 400Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp LysSer 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu SerLeu Ser Pro Gly Gly 435 440 445Gly Gly Gly Ser Gly Gly Gly Gly SerArg Glu Gly Pro Glu Leu Ser 450 455 460Pro Asp Asp Pro Ala Gly LeuLeu Asp Leu Arg Gln Gly Met Phe Ala465 470 475 480Gln Leu Val AlaGln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp 485 490 495Tyr SerAsp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser 500 505510Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr515 520 525Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala GlyGlu Gly 530 535 540Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln ProLeu Arg Ser Ala545 550 555 560Ala Gly Ala Ala Ala Leu Ala Leu ThrVal Asp Leu Pro Pro Ala Ser 565 570 575Ser Glu Ala Arg Asn Ser AlaPhe Gly Phe Gln Gly Arg Leu Leu His 580 585 590Leu Ser Ala Gly GlnArg Leu Gly Val His Leu His Thr Glu Ala Arg 595 600 605Ala Arg HisAla Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu 610 615 620PheArg Val Thr Pro Glu Ile Pro Ala Gly Leu Gly Gly Gly Gly Ser625 630635 640Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro 645 650 655Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala 660 665 670Gln Asn Val Leu Leu Ile Asp Gly Pro Leu SerTrp Tyr Ser Asp Pro 675 680 685Gly Leu Ala Gly Val Ser Leu Thr GlyGly Leu Ser Tyr Lys Glu Asp 690 695 700Thr Lys Glu Leu Val Val AlaLys Ala Gly Val Tyr Tyr Val Phe Phe705 710 715 720Gln Leu Glu LeuArg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val 725 730 735Ser LeuAla Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala 740 745750Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg755 760 765Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu SerAla Gly 770 775 780Gln Arg Leu Gly Val His Leu His Thr Glu Ala ArgAla Arg His Ala785 790 795 800Trp Gln Leu Thr Gln Gly Ala Thr ValLeu Gly Leu Phe Arg Val Thr 805 810 815Pro Glu Ile Pro Ala Gly Leu820125635PRTArtificial SequenceDescription of

Artificial Sequence Synthetic polypeptideanti- TnC(18D4) Fc knobmonomeric (71-248) ligand 125Gln Val Gln Leu Val Gln Ser Gly AlaGlu Val Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys AlaSer Gly Gly Thr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg GlnAla Pro Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro IlePhe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val ThrIle Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu LeuSer Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala LysGly Asn Phe Tyr Gly Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val ThrVal Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu Gly Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205Asn Thr LysVal Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220HisThr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser225 230235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly ValGlu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320Lys Cys Lys ValSer Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 325 330 335Ile SerLys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345350Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu TrpGlu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr ProPro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe Leu Tyr SerLys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln Gly Asn ValPhe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His Asn His TyrThr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445Gly Gly GlySer Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser 450 455 460ProAsp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala465 470475 480Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu SerTrp 485 490 495Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr GlyGly Leu Ser 500 505 510Tyr Lys Glu Asp Thr Lys Glu Leu Val Val AlaLys Ala Gly Val Tyr 515 520 525Tyr Val Phe Phe Gln Leu Glu Leu ArgArg Val Val Ala Gly Glu Gly 530 535 540Ser Gly Ser Val Ser Leu AlaLeu His Leu Gln Pro Leu Arg Ser Ala545 550 555 560Ala Gly Ala AlaAla Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser 565 570 575Ser GluAla Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His 580 585590Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg595 600 605Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val LeuGly Leu 610 615 620Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu625630 6351261806DNAArtificial SequenceDescription of ArtificialSequence Synthetic polynucleotideFc hole dimeric ligand (71-248)chain 126gacaaaactc acacatgccc accgtgccca gcacctgaag ctgcagggggaccgtcagtc 60ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccctgaggtcaca 120tgcgtggtgg tggacgtgag ccacgaagac cctgaggtcaagttcaactg gtacgtggac 180ggcgtggagg tgcataatgc caagacaaagccgcgggagg agcagtacaa cagcacgtac 240cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaatggcaa ggagtacaag 300tgcaaggtctccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa360gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatgagctgaccaag 420aaccaggtca gcctctcgtg cgcagtcaaa ggcttctatcccagcgacat cgccgtggag 480tgggagagca atgggcagcc ggagaacaactacaagacca cgcctcccgt gctggactcc 540gacggctcct tcttcctcgtgagcaagctc accgtggaca agagcaggtg gcagcagggg 600aacgtcttctcatgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc660ctctccctgt ctccgggtgg aggcggcgga agcggaggag gaggatccagagagggccct 720gagctgagcc ctgatgatcc tgccggactg ctggacctgcggcagggaat gtttgcccag 780ctggtggccc agaacgtgct gctgatcgatggccccctgt cctggtacag cgatcctgga 840ctggctggcg tgtcactgacaggcggcctg agctacaaag aggacaccaa agaactggtg 900gtggccaaggccggcgtgta ctacgtgttc tttcagctgg aactgcggag agtggtggcc960ggcgaaggat ctggctctgt gtctctggcc ctgcatctgc agcctctgagatctgctgct 1020ggcgccgctg ctctggcact gacagtggat ctgcctcctgccagcagcga ggcccggaat 1080agcgcatttg ggtttcaagg caggctgctgcacctgtctg ccggccagag gctgggagtg 1140catctgcaca cagaggccagggctagacac gcctggcagc tgacacaggg cgctacagtg 1200ctgggcctgttcagagtgac ccccgagatt ccagcaggcc tgggaggcgg cggatctggc1260ggcggaggat ctagagaagg acccgagctg tcccccgacg atcccgctgggctgctggat 1320ctgagacagg gcatgttcgc tcagctggtg gctcagaatgtgctgctgat tgacggacct 1380ctgagctggt actccgaccc agggctggcaggggtgtccc tgactggggg actgtcctac 1440aaagaagata caaaagaactggtggtggct aaagctgggg tgtactatgt gttttttcag 1500ctggaactgaggcgggtggt ggctggggag ggctcaggat ctgtgtccct ggctctgcat1560ctgcagccac tgcgctctgc agcaggggct gcagcactgg ccctgactgtggacctgccc 1620ccagcttctt ccgaggccag aaacagcgcc ttcgggttccaaggacgcct gctgcatctg 1680agcgccggac agcgcctggg agtgcatctgcatactgaag ccagagcccg gcatgcttgg 1740cagctgactc agggggcaactgtgctggga ctgtttcgcg tgacacctga gatcccagcc 1800gggctc1806127602PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideFc hole dimeric ligand (71-248) chain 127AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1 5 1015Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerHis 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly ValGlu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr AsnSer Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn LysAla Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala LysGly Gln Pro Arg Glu Pro Gln Val 115 120 125Cys Thr Leu Pro Pro SerArg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Ser Cys AlaVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160TrpGlu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys SerVal Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu Ser Leu Ser 210 215 220Pro Gly Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Arg Glu Gly Pro225 230 235 240Glu Leu Ser Pro Asp Asp ProAla Gly Leu Leu Asp Leu Arg Gln Gly 245 250 255Met Phe Ala Gln LeuVal Ala Gln Asn Val Leu Leu Ile Asp Gly Pro 260 265 270Leu Ser TrpTyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly 275 280 285GlyLeu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala 290 295300Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val ValAla305 310 315 320Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu HisLeu Gln Pro Leu 325 330 335Arg Ser Ala Ala Gly Ala Ala Ala Leu AlaLeu Thr Val Asp Leu Pro 340 345 350Pro Ala Ser Ser Glu Ala Arg AsnSer Ala Phe Gly Phe Gln Gly Arg 355 360 365Leu Leu His Leu Ser AlaGly Gln Arg Leu Gly Val His Leu His Thr 370 375 380Glu Ala Arg AlaArg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val385 390 395 400LeuGly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Gly Gly 405 410415Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro420 425 430Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met PheAla Gln 435 440 445Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly ProLeu Ser Trp Tyr 450 455 460Ser Asp Pro Gly Leu Ala Gly Val Ser LeuThr Gly Gly Leu Ser Tyr465 470 475 480Lys Glu Asp Thr Lys Glu LeuVal Val Ala Lys Ala Gly Val Tyr Tyr 485 490 495Val Phe Phe Gln LeuGlu Leu Arg Arg Val Val Ala Gly Glu Gly Ser 500 505 510Gly Ser ValSer Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala 515 520 525GlyAla Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser 530 535540Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu HisLeu545 550 555 560Ser Ala Gly Gln Arg Leu Gly Val His Leu His ThrGlu Ala Arg Ala 565 570 575Arg His Ala Trp Gln Leu Thr Gln Gly AlaThr Val Leu Gly Leu Phe 580 585 590Arg Val Thr Pro Glu Ile Pro AlaGly Leu 595 6001281212DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotideFc hole monomericligand (71-248) chain 128gacaaaactc acacatgccc accgtgcccagcacctgaag ctgcaggggg accgtcagtc 60ttcctcttcc ccccaaaacc caaggacaccctcatgatct cccggacccc tgaggtcaca 120tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggac 180ggcgtggaggtgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac240cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaaggagtacaag 300tgcaaggtct ccaacaaagc cctcggcgcc cccatcgagaaaaccatctc caaagccaaa 360gggcagcccc gagaaccaca ggtgtgcaccctgcccccat cccgggatga gctgaccaag 420aaccaggtca gcctctcgtgcgcagtcaaa ggcttctatc ccagcgacat cgccgtggag 480tgggagagcaatgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc540gacggctcct tcttcctcgt gagcaagctc accgtggaca agagcaggtggcagcagggg 600aacgtcttct catgctccgt gatgcatgag gctctgcacaaccactacac gcagaagagc 660ctctccctgt ctccgggtag agagggccctgagctgagcc ctgatgatcc tgccggactg 720ctggacctgc ggcagggaatgtttgcccag ctggtggccc agaacgtgct gctgatcgat 780ggccccctgtcctggtacag cgatcctgga ctggctggcg tgtcactgac aggcggcctg840agctacaaag aggacaccaa agaactggtg gtggccaagg ccggcgtgtactacgtgttc 900tttcagctgg aactgcggag agtggtggcc ggcgaaggatctggctctgt gtctctggcc 960ctgcatctgc agcctctgag atctgctgctggcgccgctg ctctggcact gacagtggat 1020ctgcctcctg ccagcagcgaggcccggaat agcgcatttg ggtttcaagg caggctgctg 1080cacctgtctgccggccagag gctgggagtg catctgcaca cagaggccag ggctagacac1140gcctggcagc tgacacaggg cgctacagtg ctgggcctgt tcagagtgacccccgagatt 1200cctgccgggc tc 12121292439DNAArtificialSequenceDescription of Artificial Sequence Syntheticpolynucleotideanti-TnC(18D4) Fc knob dimeric ligand (71-248) chain129caggtgcaat tggtgcagtc tggggctgag gtgaagaagc ctgggtcctcggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgcta taagctgggtgcgacaggcc 120cctggacaag ggctcgagtg gatgggaggg atcatccctatctttggtac agcaaactac 180gcacagaagt tccagggcag ggtcaccattactgcagaca aatccacgag cacagcctac 240atggagctga gcagcctgagatctgaggac accgccgtgt attactgtgc gaaaggtaac 300ttctacggtggtctggacta ctggggccaa gggaccaccg tgaccgtctc ctcagctagc360accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctctgggggcaca 420gcggccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacaccttcccggctg tcctacagtc ctcaggactc 540tactccctca gcagcgtggtgaccgtgccc tccagcagct tgggcaccca gacctacatc 600tgcaacgtgaatcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagggggaccgtca 720gtcttcctct tccccccaaa acccaaggac accctcatgatctcccggac ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaagaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataatgccaagaca aagccgcggg aggagcagta caacagcacg 900taccgtgtggtcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac960aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccatctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtac accctgcccccctgcagaga tgagctgacc 1080aagaaccagg tgtccctgtg gtgtctggtcaagggcttct accccagcga tatcgccgtg 1140gagtgggaga gcaacggccagcctgagaac aactacaaga ccaccccccc tgtgctggac 1200agcgacggcagcttcttcct gtactccaaa ctgaccgtgg acaagagccg gtggcagcag1260ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc acaaccactacacccagaag 1320tccctgagcc tgagccccgg cagagagggc cctgagctgagccctgatga tcctgccgga 1380ctgctggacc tgcggcaggg aatgtttgcccagctggtgg cccagaacgt gctgctgatc 1440gatggccccc tgtcctggtacagcgatcct ggactggctg gcgtgtcact gacaggcggc 1500ctgagctacaaagaggacac caaagaactg gtggtggcca aggccggcgt gtactacgtg1560ttctttcagc tggaactgcg gagagtggtg gccggcgaag gatctggctctgtgtctctg 1620gccctgcatc tgcagcctct gagatctgct gctggcgccgctgctctggc actgacagtg 1680gatctgcctc ctgccagcag cgaggcccggaatagcgcat ttgggtttca aggcaggctg 1740ctgcacctgt ctgccggccagaggctggga gtgcatctgc acacagaggc cagggctaga 1800cacgcctggcagctgacaca gggcgctaca gtgctgggcc tgttcagagt gacccccgag1860attccagcag gcctgggagg cggcggatct ggcggcggag gatctagagaaggacccgag 1920ctgtcccccg acgatcccgc tgggctgctg gatctgagacagggcatgtt cgctcagctg 1980gtggctcaga atgtgctgct gattgacggacctctgagct ggtactccga cccagggctg 2040gcaggggtgt ccctgactgggggactgtcc tacaaagaag atacaaaaga actggtggtg 2100gctaaagctggggtgtacta tgtgtttttt cagctggaac tgaggcgggt ggtggctggg2160gagggctcag gatctgtgtc cctggctctg catctgcagc cactgcgctctgcagcaggg 2220gctgcagcac tggccctgac tgtggacctg cccccagcttcttccgaggc cagaaacagc 2280gccttcgggt tccaaggacg cctgctgcatctgagcgccg gacagcgcct gggagtgcat 2340ctgcatactg aagccagagcccggcatgct tggcagctga ctcagggggc aactgtgctg 2400ggactgtttcgcgtgacacc tgagatccca gccgggctc 2439130404PRTArtificialSequenceDescription of Artificial Sequence Synthetic polypeptideFchole monomeric ligand (71-248) chain 130Asp Lys Thr His Thr Cys ProPro Cys Pro Ala Pro Glu Ala Ala Gly1 5 10 15Gly Pro Ser Val Phe LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro GluVal Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn AlaLys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80ArgVal Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 9095Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu ProGln Val 115 120 125Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr LysAsn Gln Val Ser 130 135 140Leu Ser Cys Ala Val Lys Gly Phe Tyr ProSer Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser AspGly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 180 185 190Asp Lys SerArg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200

205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser210 215 220Pro Gly Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro AlaGly Leu225 230 235 240Leu Asp Leu Arg Gln Gly Met Phe Ala Gln LeuVal Ala Gln Asn Val 245 250 255Leu Leu Ile Asp Gly Pro Leu Ser TrpTyr Ser Asp Pro Gly Leu Ala 260 265 270Gly Val Ser Leu Thr Gly GlyLeu Ser Tyr Lys Glu Asp Thr Lys Glu 275 280 285Leu Val Val Ala LysAla Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu 290 295 300Leu Arg ArgVal Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala305 310 315320Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala325 330 335Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg AsnSer Ala 340 345 350Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser AlaGly Gln Arg Leu 355 360 365Gly Val His Leu His Thr Glu Ala Arg AlaArg His Ala Trp Gln Leu 370 375 380Thr Gln Gly Ala Thr Val Leu GlyLeu Phe Arg Val Thr Pro Glu Ile385 390 395 400Pro Ala GlyLeu131813PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideanti-TnC(18D4) Fc knob dimeric ligand (71-248)chain 131Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys ProGly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr PheSer Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln GlyLeu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala AsnTyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp LysSer Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg SerGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Phe Tyr GlyGly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110Thr Val Thr Val SerSer Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala ProSer Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140CysLeu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150155 160Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val LeuGln 165 170 175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr ValPro Ser Ser 180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn ValAsn His Lys Pro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val GluPro Lys Ser Cys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys ProAla Pro Glu Ala Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu PhePro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr ProGlu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265270Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu AsnGly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala LeuGly Ala Pro Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly GlnPro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Cys Arg AspGlu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys 355 360 365Leu Val LysGly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380AsnGly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp LysSer 405 410 415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val MetHis Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu SerLeu Ser Pro Gly Arg 435 440 445Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly Leu Leu Asp Leu 450 455 460Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val Leu Leu Ile465 470 475 480Asp Gly Pro LeuSer Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser 485 490 495Leu ThrGly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val 500 505510Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg515 520 525Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala LeuHis Leu 530 535 540Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala LeuAla Leu Thr Val545 550 555 560Asp Leu Pro Pro Ala Ser Ser Glu AlaArg Asn Ser Ala Phe Gly Phe 565 570 575Gln Gly Arg Leu Leu His LeuSer Ala Gly Gln Arg Leu Gly Val His 580 585 590Leu His Thr Glu AlaArg Ala Arg His Ala Trp Gln Leu Thr Gln Gly 595 600 605Ala Thr ValLeu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly 610 615 620LeuGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu625 630635 640Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln GlyMet 645 650 655Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu Ile AspGly Pro Leu 660 665 670Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly ValSer Leu Thr Gly Gly 675 680 685Leu Ser Tyr Lys Glu Asp Thr Lys GluLeu Val Val Ala Lys Ala Gly 690 695 700Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg Arg Val Val Ala Gly705 710 715 720Glu Gly Ser GlySer Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg 725 730 735Ser AlaAla Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro 740 745750Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu755 760 765Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu HisThr Glu 770 775 780Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln GlyAla Thr Val Leu785 790 795 800Gly Leu Phe Arg Val Thr Pro Glu IlePro Ala Gly Leu 805 8101321242DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotideFc knob monomericligand (71-248) chain 132gacaaaactc acacatgccc accgtgcccagcacctgaag ctgcaggggg accgtcagtc 60ttcctcttcc ccccaaaacc caaggacaccctcatgatct cccggacccc tgaggtcaca 120tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggac 180ggcgtggaggtgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac240cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaaggagtacaag 300tgcaaggtct ccaacaaagc cctcggcgcc cccatcgagaaaaccatctc caaagccaaa 360gggcagcccc gagaaccaca ggtgtacaccctgcccccct gcagagatga gctgaccaag 420aaccaggtgt ccctgtggtgtctggtcaag ggcttctacc ccagcgatat cgccgtggag 480tgggagagcaacggccagcc tgagaacaac tacaagacca ccccccctgt gctggacagc540gacggcagct tcttcctgta ctccaaactg accgtggaca agagccggtggcagcagggc 600aacgtgttca gctgcagcgt gatgcacgag gccctgcacaaccactacac ccagaagtcc 660ctgagcctga gccccggcgg aggcggcggaagcggaggag gaggatccag agagggccct 720gagctgagcc ctgatgatcctgccggactg ctggacctgc ggcagggaat gtttgcccag 780ctggtggcccagaacgtgct gctgatcgat ggccccctgt cctggtacag cgatcctgga840ctggctggcg tgtcactgac aggcggcctg agctacaaag aggacaccaaagaactggtg 900gtggccaagg ccggcgtgta ctacgtgttc tttcagctggaactgcggag agtggtggcc 960ggcgaaggat ctggctctgt gtctctggccctgcatctgc agcctctgag atctgctgct 1020ggcgccgctg ctctggcactgacagtggat ctgcctcctg ccagcagcga ggcccggaat 1080agcgcatttgggtttcaagg caggctgctg cacctgtctg ccggccagag gctgggagtg1140catctgcaca cagaggccag ggctagacac gcctggcagc tgacacagggcgctacagtg 1200ctgggcctgt tcagagtgac ccccgagatt cctgccgggc tc1242133414PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideFc knob monomeric ligand (71-248) chain 133AspLys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly1 5 1015Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val SerHis 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly ValGlu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr AsnSer Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His GlnAsp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn LysAla Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala LysGly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro CysArg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys LeuVal Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160TrpGlu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys SerVal Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys SerLeu Ser Leu Ser 210 215 220Pro Gly Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Arg Glu Gly Pro225 230 235 240Glu Leu Ser Pro Asp Asp ProAla Gly Leu Leu Asp Leu Arg Gln Gly 245 250 255Met Phe Ala Gln LeuVal Ala Gln Asn Val Leu Leu Ile Asp Gly Pro 260 265 270Leu Ser TrpTyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly 275 280 285GlyLeu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala 290 295300Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val ValAla305 310 315 320Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu HisLeu Gln Pro Leu 325 330 335Arg Ser Ala Ala Gly Ala Ala Ala Leu AlaLeu Thr Val Asp Leu Pro 340 345 350Pro Ala Ser Ser Glu Ala Arg AsnSer Ala Phe Gly Phe Gln Gly Arg 355 360 365Leu Leu His Leu Ser AlaGly Gln Arg Leu Gly Val His Leu His Thr 370 375 380Glu Ala Arg AlaArg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val385 390 395 400LeuGly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu 4054101341905DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideanti-TnC(18D4) Fc hole monomeric ligand(71-248) chain 134caggtgcaat tggtgcagtc tggggctgag gtgaagaagcctgggtcctc ggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgctataagctgggt gcgacaggcc 120cctggacaag ggctcgagtg gatgggagggatcatcccta tctttggtac agcaaactac 180gcacagaagt tccagggcagggtcaccatt actgcagaca aatccacgag cacagcctac 240atggagctgagcagcctgag atctgaggac accgccgtgt attactgtgc gaaaggtaac300ttctacggtg gtctggacta ctggggccaa gggaccaccg tgaccgtctcctcagctagc 360accaagggcc cctccgtgtt ccccctggcc cccagcagcaagagcaccag cggcggcaca 420gccgctctgg gctgcctggt caaggactacttccccgagc ccgtgaccgt gtcctggaac 480agcggagccc tgacctccggcgtgcacacc ttccccgccg tgctgcagag ttctggcctg 540tatagcctgagcagcgtggt caccgtgcct tctagcagcc tgggcaccca gacctacatc600tgcaacgtga accacaagcc cagcaacacc aaggtggaca agaaggtggagcccaagagc 660tgcgacaaaa ctcacacatg cccaccgtgc ccagcacctgaagctgcagg gggaccgtca 720gtcttcctct tccccccaaa acccaaggacaccctcatga tctcccggac ccctgaggtc 780acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtggaggtgcataa tgccaagaca aagccgcggg aggagcagta caacagcacg900taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatggcaaggagtac 960aagtgcaagg tctccaacaa agccctcggc gcccccatcgagaaaaccat ctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtgcaccctgcccc catcccggga tgagctgacc 1080aagaaccagg tcagcctctcgtgcgcagtc aaaggcttct atcccagcga catcgccgtg 1140gagtgggagagcaatgggca gccggagaac aactacaaga ccacgcctcc cgtgctggac1200tccgacggct ccttcttcct cgtgagcaag ctcaccgtgg acaagagcaggtggcagcag 1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgcacaaccacta cacgcagaag 1320agcctctccc tgtctccggg tggaggcggcggaagcggag gaggaggatc cagagagggc 1380cctgagctga gccctgatgatcctgccgga ctgctggacc tgcggcaggg aatgtttgcc 1440cagctggtggcccagaacgt gctgctgatc gatggccccc tgtcctggta cagcgatcct1500ggactggctg gcgtgtcact gacaggcggc ctgagctaca aagaggacaccaaagaactg 1560gtggtggcca aggccggcgt gtactacgtg ttctttcagctggaactgcg gagagtggtg 1620gccggcgaag gatctggctc tgtgtctctggccctgcatc tgcagcctct gagatctgct 1680gctggcgccg ctgctctggcactgacagtg gatctgcctc ctgccagcag cgaggcccgg 1740aatagcgcatttgggtttca aggcaggctg ctgcacctgt ctgccggcca gaggctggga1800gtgcatctgc acacagaggc cagggctaga cacgcctggc agctgacacagggcgctaca 1860gtgctgggcc tgttcagagt gacccccgag attcctgccg ggctc19051351806DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideFc knob dimeric ligand (71-248) chain135gacaaaactc acacatgccc accgtgccca gcacctgaag ctgcagggggaccgtcagtc 60ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccctgaggtcaca 120tgcgtggtgg tggacgtgag ccacgaagac cctgaggtcaagttcaactg gtacgtggac 180ggcgtggagg tgcataatgc caagacaaagccgcgggagg agcagtacaa cagcacgtac 240cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaatggcaa ggagtacaag 300tgcaaggtctccaacaaagc cctcggcgcc cccatcgaga aaaccatctc caaagccaaa360gggcagcccc gagaaccaca ggtgtacacc ctgcccccct gcagagatgagctgaccaag 420aaccaggtgt ccctgtggtg tctggtcaag ggcttctaccccagcgatat cgccgtggag 480tgggagagca acggccagcc tgagaacaactacaagacca ccccccctgt gctggacagc 540gacggcagct tcttcctgtactccaaactg accgtggaca agagccggtg gcagcagggc 600aacgtgttcagctgcagcgt gatgcacgag gccctgcaca accactacac ccagaagtcc660ctgagcctga gccccggcgg aggcggcgga agcggaggag gaggatccagagagggccct 720gagctgagcc ctgatgatcc tgccggactg ctggacctgcggcagggaat gtttgcccag 780ctggtggccc agaacgtgct gctgatcgatggccccctgt cctggtacag cgatcctgga 840ctggctggcg tgtcactgacaggcggcctg agctacaaag aggacaccaa agaactggtg 900gtggccaaggccggcgtgta ctacgtgttc tttcagctgg aactgcggag agtggtggcc960ggcgaaggat ctggctctgt gtctctggcc ctgcatctgc agcctctgagatctgctgct 1020ggcgccgctg ctctggcact gacagtggat ctgcctcctgccagcagcga ggcccggaat 1080agcgcatttg ggtttcaagg caggctgctgcacctgtctg ccggccagag gctgggagtg 1140catctgcaca cagaggccagggctagacac gcctggcagc tgacacaggg cgctacagtg 1200ctgggcctgttcagagtgac ccccgagatt ccagcaggcc tgggaggcgg cggatctggc1260ggcggaggat ctagagaagg acccgagctg tcccccgacg atcccgctgggctgctggat 1320ctgagacagg gcatgttcgc tcagctggtg gctcagaatgtgctgctgat tgacggacct 1380ctgagctggt actccgaccc agggctggcaggggtgtccc tgactggggg actgtcctac 1440aaagaagata caaaagaactggtggtggct aaagctgggg tgtactatgt gttttttcag 1500ctggaactgaggcgggtggt ggctggggag ggctcaggat ctgtgtccct ggctctgcat1560ctgcagccac tgcgctctgc agcaggggct gcagcactgg ccctgactgtggacctgccc 1620ccagcttctt ccgaggccag aaacagcgcc ttcgggttccaaggacgcct gctgcatctg 1680agcgccggac agcgcctggg agtgcatctgcatactgaag ccagagcccg gcatgcttgg 1740cagctgactc agggggcaactgtgctggga ctgtttcgcg tgacacctga gatcccagcc 1800gggctc1806136635PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideanti-TnC(18D4) Fc hole monomeric ligand(71-248) chain 136Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val LysLys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly GlyThr Phe Ser Ser Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro GlyGln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly ThrAla Asn Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr AlaAsp Lys Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser LeuArg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90

95Ala Lys Gly Asn Phe Tyr Gly Gly Leu Asp Tyr Trp Gly Gln Gly Thr100 105 110Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser ValPhe Pro 115 120 125Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly ThrAla Ala Leu Gly 130 135 140Cys Leu Val Lys Asp Tyr Phe Pro Glu ProVal Thr Val Ser Trp Asn145 150 155 160Ser Gly Ala Leu Thr Ser GlyVal His Thr Phe Pro Ala Val Leu Gln 165 170 175Ser Ser Gly Leu TyrSer Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190Ser Leu GlyThr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205AsnThr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215220His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly ProSer225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val AspVal Ser His Glu Asp Pro 260 265 270Glu Val Lys Phe Asn Trp Tyr ValAsp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg GluGlu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu ThrVal Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315 320LysCys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr 325 330335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu340 345 350Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser LeuSer Cys 355 360 365Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala ValGlu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr Lys ThrThr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe Phe LeuVal Ser Lys Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln Gln GlyAsn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu His AsnHis Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly 435 440 445GlyGly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu Leu Ser 450 455460Pro Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met PheAla465 470 475 480Gln Leu Val Ala Gln Asn Val Leu Leu Ile Asp GlyPro Leu Ser Trp 485 490 495Tyr Ser Asp Pro Gly Leu Ala Gly Val SerLeu Thr Gly Gly Leu Ser 500 505 510Tyr Lys Glu Asp Thr Lys Glu LeuVal Val Ala Lys Ala Gly Val Tyr 515 520 525Tyr Val Phe Phe Gln LeuGlu Leu Arg Arg Val Val Ala Gly Glu Gly 530 535 540Ser Gly Ser ValSer Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala545 550 555 560AlaGly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser 565 570575Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His580 585 590Leu Ser Ala Gly Gln Arg Leu Gly Val His Leu His Thr GluAla Arg 595 600 605Ala Arg His Ala Trp Gln Leu Thr Gln Gly Ala ThrVal Leu Gly Leu 610 615 620Phe Arg Val Thr Pro Glu Ile Pro Ala GlyLeu625 630 635137602PRTArtificial SequenceDescription of ArtificialSequence Synthetic polypeptideFc knob dimeric ligand (71-248) chain137Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly15 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr LeuMet 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp ValSer His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp GlyVal Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu HisGln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser AsnLys Ala Leu Gly Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys AlaLys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro ProCys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Trp CysLeu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys LeuThr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe SerCys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr GlnLys Ser Leu Ser Leu Ser 210 215 220Pro Gly Gly Gly Gly Gly Ser GlyGly Gly Gly Ser Arg Glu Gly Pro225 230 235 240Glu Leu Ser Pro AspAsp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly 245 250 255Met Phe AlaGln Leu Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro 260 265 270LeuSer Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly 275 280285Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala290 295 300Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg Arg ValVal Ala305 310 315 320Gly Glu Gly Ser Gly Ser Val Ser Leu Ala LeuHis Leu Gln Pro Leu 325 330 335Arg Ser Ala Ala Gly Ala Ala Ala LeuAla Leu Thr Val Asp Leu Pro 340 345 350Pro Ala Ser Ser Glu Ala ArgAsn Ser Ala Phe Gly Phe Gln Gly Arg 355 360 365Leu Leu His Leu SerAla Gly Gln Arg Leu Gly Val His Leu His Thr 370 375 380Glu Ala ArgAla Arg His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val385 390 395400Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Gly Gly405 410 415Gly Gly Ser Gly Gly Gly Gly Ser Arg Glu Gly Pro Glu LeuSer Pro 420 425 430Asp Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln GlyMet Phe Ala Gln 435 440 445Leu Val Ala Gln Asn Val Leu Leu Ile AspGly Pro Leu Ser Trp Tyr 450 455 460Ser Asp Pro Gly Leu Ala Gly ValSer Leu Thr Gly Gly Leu Ser Tyr465 470 475 480Lys Glu Asp Thr LysGlu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr 485 490 495Val Phe PheGln Leu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser 500 505 510GlySer Val Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala 515 520525Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser530 535 540Glu Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu LeuHis Leu545 550 555 560Ser Ala Gly Gln Arg Leu Gly Val His Leu HisThr Glu Ala Arg Ala 565 570 575Arg His Ala Trp Gln Leu Thr Gln GlyAla Thr Val Leu Gly Leu Phe 580 585 590Arg Val Thr Pro Glu Ile ProAla Gly Leu 595 6001381335DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotideDP47 Fc hole chain138gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtccctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggtccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggtagtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatctccagagaca attccaagaa cacgctgtat 240ctgcagatga acagcctgagagccgaggac acggccgtat attactgtgc gaaaggcagc 300ggatttgactactggggcca aggaaccctg gtcaccgtct cgagtgctag caccaagggc360ccatcggtct tccccctggc accctcctcc aagagcacct ctgggggcacagcggccctg 420ggctgcctgg tcaaggacta cttccccgaa ccggtgacggtgtcgtggaa ctcaggcgcc 480ctgaccagcg gcgtgcacac cttcccggctgtcctacagt cctcaggact ctactccctc 540agcagcgtgg tgaccgtgccctccagcagc ttgggcaccc agacctacat ctgcaacgtg 600aatcacaagcccagcaacac caaggtggac aagaaagttg agcccaaatc ttgtgacaaa660actcacacat gcccaccgtg cccagcacct gaagctgcag ggggaccgtcagtcttcctc 720ttccccccaa aacccaagga caccctcatg atctcccggacccctgaggt cacatgcgtg 780gtggtggacg tgagccacga agaccctgaggtcaagttca actggtacgt ggacggcgtg 840gaggtgcata atgccaagacaaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900gtcagcgtcctcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag960gtctccaaca aagccctcgg cgcccccatc gagaaaacca tctccaaagccaaagggcag 1020ccccgagaac cacaggtgtg caccctgccc ccatcccgggatgagctgac caagaaccag 1080gtcagcctct cgtgcgcagt caaaggcttctatcccagcg acatcgccgt ggagtgggag 1140agcaatgggc agccggagaacaactacaag accacgcctc ccgtgctgga ctccgacggc 1200tccttcttcctcgtgagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc1260ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaagagcctctcc 1320ctgtctccgg gtaaa 1335139645DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotideDP47 light chain 139gaaatcgtgt taacgcagtc tccaggcaccctgtctttgt ctccagggga aagagccacc 60ctctcttgca gggccagtca gagtgttagcagcagctact tagcctggta ccagcagaaa 120cctggccagg ctcccaggctcctcatctat ggagcatcca gcagggccac tggcatccca 180gacaggttcagtggcagtgg atccgggaca gacttcactc tcaccatcag cagactggag240cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgctgacgttcggc 300caggggacca aagtggaaat caaacgtacg gtggctgcaccatctgtctt catcttcccg 360ccatctgatg agcagttgaa atctggaactgcctctgttg tgtgcctgct gaataacttc 420tatcccagag aggccaaagtacagtggaag gtggataacg ccctccaatc gggtaactcc 480caggagagtgtcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg540acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagtcacccatcag 600ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt645140445PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideDP47 Fc hole chain 140Glu Val Gln Leu Leu GluSer Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala IleSer Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 7580Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu ValThr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe ProLeu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val ThrVal Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser LeuSer Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr GlnThr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200205Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys210 215 220Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser ValPhe Leu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met IleSer Arg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val SerHis Glu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp GlyVal Glu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu GlnTyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val LeuHis Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315320Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys325 330 335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu ProPro Ser 340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu SerCys Ala Val Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val GluTrp Glu Ser Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr ThrPro Pro Val Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu ValSer Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly AsnVal Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430HisTyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440445141215PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideDP47 light chain 141Glu Ile Val Leu Thr GlnSer Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr LeuSer Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30Tyr Leu Ala TrpTyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45Ile Tyr GlyAla Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60Gly SerGly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 7580Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro85 90 95Leu Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr ValAla 100 105 110Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu GlnLeu Lys Ser 115 120 125Gly Thr Ala Ser Val Val Cys Leu Leu Asn AsnPhe Tyr Pro Arg Glu 130 135 140Ala Lys Val Gln Trp Lys Val Asp AsnAla Leu Gln Ser Gly Asn Ser145 150 155 160Gln Glu Ser Val Thr GluGln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175Ser Ser Thr LeuThr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190Tyr AlaCys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200205Ser Phe Asn Arg Gly Glu Cys 210 2151422496DNAArtificialSequenceDescription of Artificial Sequence SyntheticpolynucleotideDP47 Fc hole chain fused to dimeric hu 4-1BBL(71-254) 142gaggtgcaat tgttggagtc tgggggaggc ttggtacagc ctggggggtccctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgcca tgagctgggtccgccaggct 120ccagggaagg ggctggagtg ggtctcagct attagtggtagtggtggtag cacatactac 180gcagactccg tgaagggccg gttcaccatctccagagaca attccaagaa cacgctgtat 240ctgcagatga acagcctgagagccgaggac acggccgtat attactgtgc gaaaggcagc 300ggatttgactactggggcca aggaaccctg gtcaccgtct cgagtgctag caccaagggc360ccctccgtgt tccccctggc ccccagcagc aagagcacca gcggcggcacagccgctctg 420ggctgcctgg tcaaggacta cttccccgag cccgtgaccgtgtcctggaa cagcggagcc 480ctgacctccg gcgtgcacac cttccccgccgtgctgcaga gttctggcct gtatagcctg 540agcagcgtgg tcaccgtgccttctagcagc ctgggcaccc agacctacat ctgcaacgtg 600aaccacaagcccagcaacac caaggtggac aagaaggtgg agcccaagag ctgcgacaaa660actcacacat gcccaccgtg cccagcacct gaagctgcag ggggaccgtcagtcttcctc 720ttccccccaa aacccaagga caccctcatg atctcccggacccctgaggt cacatgcgtg 780gtggtggacg tgagccacga agaccctgaggtcaagttca actggtacgt ggacggcgtg 840gaggtgcata atgccaagacaaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 900gtcagcgtcctcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag960gtctccaaca aagccctcgg cgcccccatc gagaaaacca tctccaaagccaaagggcag 1020ccccgagaac cacaggtgtg caccctgccc ccatcccgggatgagctgac caagaaccag 1080gtcagcctct cgtgcgcagt caaaggcttctatcccagcg acatcgccgt ggagtgggag 1140agcaatgggc agccggagaacaactacaag accacgcctc ccgtgctgga ctccgacggc 1200tccttcttcctcgtgagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc1260ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaagagcctctcc 1320ctgtctccgg gtggaggcgg cggaagcgga ggaggaggatccagagaggg ccctgagctg 1380agccccgatg atcctgctgg actgctggacctgcggcagg gcatgtttgc tcagctggtg

1440gcccagaacg tgctgctgat cgatggcccc ctgtcctggt acagcgatcctggactggct 1500ggcgtgtcac tgacaggcgg cctgagctac aaagaggacaccaaagaact ggtggtggcc 1560aaggccggcg tgtactacgt gttctttcagctggaactgc ggagagtggt ggccggcgaa 1620ggatctggct ctgtgtctctggccctgcat ctgcagcctc tgagaagcgc tgctggcgct 1680gcagctctggcactgacagt ggatctgcct cctgccagct ccgaggcccg gaatagcgca1740tttgggtttc aaggcaggct gctgcacctg tctgccggcc agaggctgggagtgcatctg 1800cacacagagg ccagggctag acacgcctgg cagctgacacagggcgctac agtgctgggc 1860ctgttcagag tgacccccga gattccagccggcctgcctt ctccaagaag cgaaggcgga 1920ggcggatctg gcggcggaggatctagagag ggacccgaac tgtcccctga cgatccagcc 1980gggctgctggatctgagaca gggaatgttc gcccagctgg tggctcagaa tgtgctgctg2040attgacggac ctctgagctg gtactccgac ccagggctgg caggggtgtccctgactggg 2100ggactgtcct acaaagaaga tacaaaagaa ctggtggtggctaaagctgg ggtgtactat 2160gtgttttttc agctggaact gaggcgggtggtggctgggg agggctcagg atctgtgtcc 2220ctggctctgc atctgcagccactgcgctct gctgctggcg cagctgcact ggctctgact 2280gtggacctgccaccagcctc tagcgaggcc agaaacagcg ccttcgggtt ccaaggacgc2340ctgctgcatc tgagcgccgg acagcgcctg ggagtgcatc tgcatactgaagccagagcc 2400cggcatgctt ggcagctgac tcagggggca actgtgctgggactgtttcg cgtgacacct 2460gagatccctg ccggactgcc aagccctaga tcagaa24961431914DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideDP47 Fc knob chain fused to monomeric hu4-1BBL (71-254) 143gaggtgcaat tgttggagtc tgggggaggc ttggtacagcctggggggtc cctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgccatgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagctattagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccggttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatgaacagcctgag agccgaggac acggccgtat attactgtgc gaaaggcagc300ggatttgact actggggcca aggaaccctg gtcaccgtct cgagtgctagcaccaagggc 360ccatcggtct tccccctggc accctcctcc aagagcacctctgggggcac agcggccctg 420ggctgcctgg tcaaggacta cttccccgaaccggtgacgg tgtcgtggaa ctcaggcgcc 480ctgaccagcg gcgtgcacaccttcccggct gtcctacagt cctcaggact ctactccctc 540agcagcgtggtgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg600aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatcttgtgacaaa 660actcacacat gcccaccgtg cccagcacct gaagctgcagggggaccgtc agtcttcctc 720ttccccccaa aacccaagga caccctcatgatctcccgga cccctgaggt cacatgcgtg 780gtggtggacg tgagccacgaagaccctgag gtcaagttca actggtacgt ggacggcgtg 840gaggtgcataatgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg900gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagtacaagtgcaag 960gtctccaaca aagccctcgg cgcccccatc gagaaaaccatctccaaagc caaagggcag 1020ccccgagaac cacaggtgta caccctgcccccctgcagag atgagctgac caagaaccag 1080gtgtccctgt ggtgtctggtcaagggcttc taccccagcg atatcgccgt ggagtgggag 1140agcaacggccagcctgagaa caactacaag accacccccc ctgtgctgga cagcgacggc1200agcttcttcc tgtactccaa actgaccgtg gacaagagcc ggtggcagcagggcaacgtg 1260ttcagctgca gcgtgatgca cgaggccctg cacaaccactacacccagaa gtccctgagc 1320ctgagccccg gcggaggcgg cggaagcggaggaggaggat ccagagaggg ccctgagctg 1380agccccgatg atcctgctggactgctggac ctgcggcagg gcatgtttgc tcagctggtg 1440gcccagaacgtgctgctgat cgatggcccc ctgtcctggt acagcgatcc tggactggct1500ggcgtgtcac tgacaggcgg cctgagctac aaagaggaca ccaaagaactggtggtggcc 1560aaggccggcg tgtactacgt gttctttcag ctggaactgcggagagtggt ggccggcgaa 1620ggatctggct ctgtgtctct ggccctgcatctgcagcctc tgagaagcgc tgctggcgct 1680gcagctctgg cactgacagtggatctgcct cctgccagct ccgaggcccg gaatagcgca 1740tttgggtttcaaggcaggct gctgcacctg tctgccggcc agaggctggg agtgcatctg1800cacacagagg ccagggctag acacgcctgg cagctgacac agggcgctacagtgctgggc 1860ctgttcagag tgacccccga gattccagcc ggcctgccttctccaagaag cgaa 1914144832PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptideDP47 Fc hole chain fusedto dimeric hu 4-1BBL (71-254) 144Glu Val Gln Leu Leu Glu Ser GlyGly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser GlySer Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu GlnMet Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95AlaLys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly CysLeu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser TrpAsn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val His Thr Phe ProAla Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser Leu Ser Ser ValVal Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln Thr Tyr IleCys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205Val Asp LysLys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220ProPro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu225 230235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr ProGlu 245 250 255Val Thr Cys Val Val Val Asp Val Ser His Glu Asp ProGlu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln Tyr Asn Ser ThrTyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu His Gln Asp TrpLeu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320Val Ser Asn LysAla Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335Ala LysGly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser 340 345350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser AsnGly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val LeuAsp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Val Ser Lys Leu ThrVal Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val Phe Ser CysSer Val Met His Glu Ala Leu His Asn 420 425 430His Tyr Thr Gln LysSer Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly 435 440 445Ser Gly GlyGly Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp 450 455 460ProAla Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val465 470475 480Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr SerAsp 485 490 495Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu SerTyr Lys Glu 500 505 510Asp Thr Lys Glu Leu Val Val Ala Lys Ala GlyVal Tyr Tyr Val Phe 515 520 525Phe Gln Leu Glu Leu Arg Arg Val ValAla Gly Glu Gly Ser Gly Ser 530 535 540Val Ser Leu Ala Leu His LeuGln Pro Leu Arg Ser Ala Ala Gly Ala545 550 555 560Ala Ala Leu AlaLeu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 565 570 575Arg AsnSer Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 580 585590Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His595 600 605Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu PheArg Val 610 615 620Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro ArgSer Glu Gly Gly625 630 635 640Gly Gly Ser Gly Gly Gly Gly Ser ArgGlu Gly Pro Glu Leu Ser Pro 645 650 655Asp Asp Pro Ala Gly Leu LeuAsp Leu Arg Gln Gly Met Phe Ala Gln 660 665 670Leu Val Ala Gln AsnVal Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr 675 680 685Ser Asp ProGly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr 690 695 700LysGlu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr705 710715 720Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala Gly Glu GlySer 725 730 735Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu ArgSer Ala Ala 740 745 750Gly Ala Ala Ala Leu Ala Leu Thr Val Asp LeuPro Pro Ala Ser Ser 755 760 765Glu Ala Arg Asn Ser Ala Phe Gly PheGln Gly Arg Leu Leu His Leu 770 775 780Ser Ala Gly Gln Arg Leu GlyVal His Leu His Thr Glu Ala Arg Ala785 790 795 800Arg His Ala TrpGln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe 805 810 815Arg ValThr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 820 825830145640PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideDP47 Fc knob chain fused to monomeric hu4-1BBL (71-254) 145Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ile Gly Ser Gly Ala SerThr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Gly Trp PheGly Gly Phe Asn Tyr Trp Gly Gln Gly Thr Leu 100 105 110Val Thr ValSer Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125AlaPro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135140Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer145 150 155 160Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro AlaVal Leu Gln Ser 165 170 175Ser Gly Leu Tyr Ser Leu Ser Ser Val ValThr Val Pro Ser Ser Ser 180 185 190Leu Gly Thr Gln Thr Tyr Ile CysAsn Val Asn His Lys Pro Ser Asn 195 200 205Thr Lys Val Asp Lys LysVal Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220Thr Cys Pro ProCys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val225 230 235 240PheLeu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250255Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu260 265 270Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His AsnAla Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys AlaLeu Gly Ala Pro Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys GlyGln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Cys ArgAsp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu 355 360 365ValLys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspSer385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr ValAsp Lys Ser Arg 405 410 415Trp Gln Gln Gly Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys SerLeu Ser Leu Ser Pro Gly Gly Gly 435 440 445Gly Gly Ser Gly Gly GlyGly Ser Arg Glu Gly Pro Glu Leu Ser Pro 450 455 460Asp Asp Pro AlaGly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln465 470 475 480LeuVal Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr 485 490495Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr500 505 510Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly ValTyr Tyr 515 520 525Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val AlaGly Glu Gly Ser 530 535 540Gly Ser Val Ser Leu Ala Leu His Leu GlnPro Leu Arg Ser Ala Ala545 550 555 560Gly Ala Ala Ala Leu Ala LeuThr Val Asp Leu Pro Pro Ala Ser Ser 565 570 575Glu Ala Arg Asn SerAla Phe Gly Phe Gln Gly Arg Leu Leu His Leu 580 585 590Ser Ala GlyGln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala 595 600 605ArgHis Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe 610 615620Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg SerGlu625 630 635 6401461335DNAArtificial SequenceDescription ofArtificial Sequence Synthetic polynucleotideDP47 heavy chain (huIgG1 PGLALA) 146gaggtgcaat tgttggagtc tgggggaggc ttggtacagcctggggggtc cctgagactc 60tcctgtgcag cctccggatt cacctttagc agttatgccatgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtctcagctattagtggta gtggtggtag cacatactac 180gcagactccg tgaagggccggttcaccatc tccagagaca attccaagaa cacgctgtat 240ctgcagatgaacagcctgag agccgaggac acggccgtat attactgtgc gaaaggcagc300ggatttgact actggggcca aggaaccctg gtcaccgtct cgagtgctagcaccaagggc 360ccatcggtct tccccctggc accctcctcc aagagcacctctgggggcac agcggccctg 420ggctgcctgg tcaaggacta cttccccgaaccggtgacgg tgtcgtggaa ctcaggcgcc 480ctgaccagcg gcgtgcacaccttcccggct gtcctacagt cctcaggact ctactccctc 540agcagcgtggtgaccgtgcc ctccagcagc ttgggcaccc agacctacat ctgcaacgtg600aatcacaagc ccagcaacac caaggtggac aagaaagttg agcccaaatcttgtgacaaa 660actcacacat gcccaccgtg cccagcacct gaagctgcagggggaccgtc agtcttcctc 720ttccccccaa aacccaagga caccctcatgatctcccgga cccctgaggt cacatgcgtg 780gtggtggacg tgagccacgaagaccctgag gtcaagttca actggtacgt ggacggcgtg 840gaggtgcataatgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg900gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagtacaagtgcaag 960gtctccaaca aagccctcgg cgcccccatc gagaaaaccatctccaaagc caaagggcag 1020ccccgagaac cacaggtgta caccctgcccccatcccggg atgagctgac caagaaccag 1080gtcagcctga cctgcctggtcaaaggcttc tatcccagcg acatcgccgt ggagtgggag 1140agcaatgggcagccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc1200tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagcaggggaacgtc 1260ttctcatgct ccgtgatgca tgaggctctg cacaaccactacacgcagaa gagcctctcc 1320ctgtctccgg gtaaa 1335147445PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideDP47 heavy chain (hu IgG1 PGLALA) 147Glu Val Gln Leu LeuGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met SerTrp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser AlaIle Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 7580Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Lys Gly Ser Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu ValThr 100 105 110Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe ProLeu Ala Pro 115 120 125Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala AlaLeu Gly Cys Leu Val 130 135 140Lys Asp Tyr Phe Pro Glu Pro Val ThrVal Ser Trp Asn Ser Gly Ala145 150 155 160Leu Thr Ser Gly Val HisThr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175Leu Tyr Ser LeuSer

Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190Thr Gln ThrTyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205ValAsp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215220Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val PheLeu225 230 235 240Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile SerArg Thr Pro Glu 245 250 255Val Thr Cys Val Val Val Asp Val Ser HisGlu Asp Pro Glu Val Lys 260 265 270Phe Asn Trp Tyr Val Asp Gly ValGlu Val His Asn Ala Lys Thr Lys 275 280 285Pro Arg Glu Glu Gln TyrAsn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300Thr Val Leu HisGln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys305 310 315 320ValSer Asn Lys Ala Leu Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330335Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser340 345 350Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys LeuVal Lys 355 360 365Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp GluSer Asn Gly Gln 370 375 380Pro Glu Asn Asn Tyr Lys Thr Thr Pro ProVal Leu Asp Ser Asp Gly385 390 395 400Ser Phe Phe Leu Tyr Ser LysLeu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415Gln Gly Asn Val PheSer Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430His Tyr ThrGln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 4404451481344DNAArtificial SequenceDescription of Artificial SequenceSynthetic polynucleotideanti-TnC(18D4) heavy chain (huIgG1 PGLALA)148caggtgcaat tggtgcagtc tggggctgag gtgaagaagc ctgggtcctcggtgaaggtc 60tcctgcaagg cctccggagg cacattcagc agctacgcta taagctgggtgcgacaggcc 120cctggacaag ggctcgagtg gatgggaggg atcatccctatctttggtac agcaaactac 180gcacagaagt tccagggcag ggtcaccattactgcagaca aatccacgag cacagcctac 240atggagctga gcagcctgagatctgaggac accgccgtgt attactgtgc gaaaggtaac 300ttctacggtggtctggacta ctggggccaa gggaccaccg tgaccgtctc ctcagctagc360accaagggcc catcggtctt ccccctggca ccctcctcca agagcacctctgggggcaca 420gcggccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac 480tcaggcgccc tgaccagcgg cgtgcacaccttcccggctg tcctacagtc ctcaggactc 540tactccctca gcagcgtggtgaccgtgccc tccagcagct tgggcaccca gacctacatc 600tgcaacgtgaatcacaagcc cagcaacacc aaggtggaca agaaagttga gcccaaatct660tgtgacaaaa ctcacacatg cccaccgtgc ccagcacctg aagctgcagggggaccgtca 720gtcttcctct tccccccaaa acccaaggac accctcatgatctcccggac ccctgaggtc 780acatgcgtgg tggtggacgt gagccacgaagaccctgagg tcaagttcaa ctggtacgtg 840gacggcgtgg aggtgcataatgccaagaca aagccgcggg aggagcagta caacagcacg 900taccgtgtggtcagcgtcct caccgtcctg caccaggact ggctgaatgg caaggagtac960aagtgcaagg tctccaacaa agccctcggc gcccccatcg agaaaaccatctccaaagcc 1020aaagggcagc cccgagaacc acaggtgtac accctgcccccatcccggga tgagctgacc 1080aagaaccagg tcagcctgac ctgcctggtcaaaggcttct atcccagcga catcgccgtg 1140gagtgggaga gcaatgggcagccggagaac aactacaaga ccacgcctcc cgtgctggac 1200tccgacggctccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag1260gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccactacacgcagaag 1320agcctctccc tgtctccggg taaa 1344149448PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptideanti-TnC(18D4) heavy chain (huIgG1 PGLALA) 149Gln ValGln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15SerVal Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 2530Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met35 40 45Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln LysPhe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser ThrAla Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr AlaVal Tyr Tyr Cys 85 90 95Ala Lys Gly Asn Phe Tyr Gly Gly Leu Asp TyrTrp Gly Gln Gly Thr 100 105 110Thr Val Thr Val Ser Ser Ala Ser ThrLys Gly Pro Ser Val Phe Pro 115 120 125Leu Ala Pro Ser Ser Lys SerThr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140Cys Leu Val Lys AspTyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn145 150 155 160Ser GlyAla Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170175Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser180 185 190Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His LysPro Ser 195 200 205Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys SerCys Asp Lys Thr 210 215 220His Thr Cys Pro Pro Cys Pro Ala Pro GluAla Ala Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro LysPro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val ThrCys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270Glu Val LysPhe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285LysThr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 290 295300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys GluTyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala ProIle Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg GluPro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Arg Asp Glu Leu ThrLys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe TyrPro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln ProGlu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400SerAsp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410415Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser ProGly Lys 435 440 44515010PRTArtificial SequenceDescription ofArtificial Sequence Synthetic peptidePeptide linker (G4S)2 150GlyGly Gly Gly Ser Gly Gly Gly Gly Ser1 5 1015110PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptidePeptidelinker (SG4)2 151Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly1 51015214PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker G4(SG4)2 152Gly Gly Gly Gly Ser GlyGly Gly Gly Ser Gly Gly Gly Gly1 5 1015310PRTArtificialSequenceDescription of Artificial Sequence Synthetic peptidePeptidelinker 153Gly Ser Pro Gly Ser Ser Ser Ser Gly Ser1 51015420PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker (G4S)4 154Gly Gly Gly Gly Ser GlyGly Gly Gly Ser Gly Gly Gly Gly Ser Gly1 5 10 15Gly Gly Gly Ser201558PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 155Gly Ser Gly Ser Gly Asn Gly Ser151568PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 156Gly Gly Ser Gly Ser Gly Ser Gly151576PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 157Gly Gly Ser Gly Ser Gly151584PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 158Gly Gly SerGly11598PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 159Gly Gly Ser Gly Asn Gly Ser Gly151608PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 160Gly Gly Asn Gly Ser Gly Ser Gly151616PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 161Gly Gly Asn Gly Ser Gly151625PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptidePeptide linker 162Gly Gly Gly Gly Ser1516319PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptideLeader Sequence 1 163Met Asp Trp Thr Trp Arg IleLeu Phe Leu Val Ala Ala Ala Thr Gly1 5 10 15Ala HisSer16457DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 1 (DNA1) 164atggactggacctggagaat cctcttcttg gtggcagcag ccacaggagc ccactcc5716557DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 1 (DNA 2) 165atggactggacctggaggat cctcttcttg gtggcagcag ccacaggagc ccactcc5716620PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptideLeader Sequence 2 166Met Asp Met Arg Val Pro AlaGln Leu Leu Gly Leu Leu Leu Leu Trp1 5 10 15Phe Pro Gly Ala2016766DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 2 (DNA) 167atggacatgagggtccccgc tcagctcctg ggcctcctgc tgctctggtt cccaggtgcc 60aggtgt6616819PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptideLeader Sequence 3 168Met Gly Trp Ser Cys Ile IleLeu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val HisSer16957DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 3 (DNA 1) 169atgggatggagctgtatcat cctcttcttg gtagcaacag ctaccggtgt gcattcg5717057DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 3 (DNA 2) 170atgggctggtcctgcatcat cctgtttctg gtggctaccg ccactggagt gcattcc5717157DNAArtificial SequenceDescription of Artificial SequenceSynthetic oligonucleotideLeader Sequence 3 (DNA 3) 171atgggctggtcctgcatcat cctgtttctg gtcgccacag ccaccggcgt gcactct 57172184PRThom*osapiens 172Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly LeuLeu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln AsnVal Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro GlyLeu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu AspThr Lys Glu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val PhePhe Gln Leu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly SerGly Ser Val Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser AlaAla Gly Ala Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro ProAla Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln GlyArg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140HisLeu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150155 160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile ProAla 165 170 175Gly Leu Pro Ser Pro Arg Ser Glu 180173170PRThom*osapiens 173Leu Asp Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala GlnAsn Val1 5 10 15Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp ProGly Leu Ala 20 25 30Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys GluAsp Thr Lys Glu 35 40 45Leu Val Val Ala Lys Ala Gly Val Tyr Tyr ValPhe Phe Gln Leu Glu 50 55 60Leu Arg Arg Val Val Ala Gly Glu Gly SerGly Ser Val Ser Leu Ala65 70 75 80Leu His Leu Gln Pro Leu Arg SerAla Ala Gly Ala Ala Ala Leu Ala 85 90 95Leu Thr Val Asp Leu Pro ProAla Ser Ser Glu Ala Arg Asn Ser Ala 100 105 110Phe Gly Phe Gln GlyArg Leu Leu His Leu Ser Ala Gly Gln Arg Leu 115 120 125Gly Val HisLeu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu 130 135 140ThrGln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile145 150155 160Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu 165 170174175PRThom*osapiens 174Asp Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe AlaGln Leu1 5 10 15Val Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu SerTrp Tyr Ser 20 25 30Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly GlyLeu Ser Tyr Lys 35 40 45Glu Asp Thr Lys Glu Leu Val Val Ala Lys AlaGly Val Tyr Tyr Val 50 55 60Phe Phe Gln Leu Glu Leu Arg Arg Val ValAla Gly Glu Gly Ser Gly65 70 75 80Ser Val Ser Leu Ala Leu His LeuGln Pro Leu Arg Ser Ala Ala Gly 85 90 95Ala Ala Ala Leu Ala Leu ThrVal Asp Leu Pro Pro Ala Ser Ser Glu 100 105 110Ala Arg Asn Ser AlaPhe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 115 120 125Ala Gly GlnArg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg 130 135 140HisAla Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg145 150155 160Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser Glu165 170 175175203PRThom*o sapiens 175Pro Trp Ala Val Ser Gly Ala ArgAla Ser Pro Gly Ser Ala Ala Ser1 5 10 15Pro Arg Leu Arg Glu Gly ProGlu Leu Ser Pro Asp Asp Pro Ala Gly 20 25 30Leu Leu Asp Leu Arg GlnGly Met Phe Ala Gln Leu Val Ala Gln Asn 35 40 45Val Leu Leu Ile AspGly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu 50 55 60Ala Gly Val SerLeu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys65 70 75 80Glu LeuVal Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu 85 90 95GluLeu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu 100 105110Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu115 120 125Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala ArgAsn Ser 130 135 140Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu SerAla Gly Gln Arg145 150 155 160Leu Gly Val His Leu His Thr Glu AlaArg Ala Arg His Ala Trp Gln 165 170 175Leu Thr Gln Gly Ala Thr ValLeu Gly Leu Phe Arg Val Thr Pro Glu 180 185 190Ile Pro Ala Gly LeuPro Ser Pro Arg Ser Glu 195 200176378PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidedimeric hu 4-1BBL (71-254) connected by (G4S)2 linker176Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp15 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val LeuLeu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu AlaGly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr LysGlu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala GlyAla Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala SerSer Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg LeuLeu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu HisThr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155

160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala165 170 175Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser GlyGly Gly 180 185 190Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly Leu 195 200 205Leu Asp Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val 210 215 220Leu Leu Ile Asp Gly Pro Leu SerTrp Tyr Ser Asp Pro Gly Leu Ala225 230 235 240Gly Val Ser Leu ThrGly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 245 250 255Leu Val ValAla Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu 260 265 270LeuArg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala 275 280285Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala290 295 300Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg AsnSer Ala305 310 315 320Phe Gly Phe Gln Gly Arg Leu Leu His Leu SerAla Gly Gln Arg Leu 325 330 335Gly Val His Leu His Thr Glu Ala ArgAla Arg His Ala Trp Gln Leu 340 345 350Thr Gln Gly Ala Thr Val LeuGly Leu Phe Arg Val Thr Pro Glu Ile 355 360 365Pro Ala Gly Leu ProSer Pro Arg Ser Glu 370 375177194PRTArtificial SequenceDescriptionof Artificial Sequence Synthetic polypeptidemonomeric hu 4-1BBL(71-254) plus (G4S)2 linker 177Arg Glu Gly Pro Glu Leu Ser Pro AspAsp Pro Ala Gly Leu Leu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser TrpTyr Ser Asp Pro Gly Leu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly LeuSer Tyr Lys Glu Asp Thr Lys Glu Leu Val 50 55 60Val Ala Lys Ala GlyVal Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg65 70 75 80Arg Val ValAla Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His 85 90 95Leu GlnPro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr 100 105110Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly115 120 125Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg LeuGly Val 130 135 140His Leu His Thr Glu Ala Arg Ala Arg His Ala TrpGln Leu Thr Gln145 150 155 160Gly Ala Thr Val Leu Gly Leu Phe ArgVal Thr Pro Glu Ile Pro Ala 165 170 175Gly Leu Pro Ser Pro Arg SerGlu Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190GlySer178718PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptidedimeric hu 4-1BBL (71-254)-CH1 Fc knob chain178Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp15 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val LeuLeu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu AlaGly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr LysGlu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala GlyAla Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala SerSer Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg LeuLeu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu HisThr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala165 170 175Gly Leu Pro Ser Pro Arg Ser Glu Gly Gly Gly Gly Ser GlyGly Gly 180 185 190Gly Ser Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly Leu 195 200 205Leu Asp Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val 210 215 220Leu Leu Ile Asp Gly Pro Leu SerTrp Tyr Ser Asp Pro Gly Leu Ala225 230 235 240Gly Val Ser Leu ThrGly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 245 250 255Leu Val ValAla Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu 260 265 270LeuArg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala 275 280285Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala290 295 300Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg AsnSer Ala305 310 315 320Phe Gly Phe Gln Gly Arg Leu Leu His Leu SerAla Gly Gln Arg Leu 325 330 335Gly Val His Leu His Thr Glu Ala ArgAla Arg His Ala Trp Gln Leu 340 345 350Thr Gln Gly Ala Thr Val LeuGly Leu Phe Arg Val Thr Pro Glu Ile 355 360 365Pro Ala Gly Leu ProSer Pro Arg Ser Glu Gly Gly Gly Gly Ser Gly 370 375 380Gly Gly GlySer Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala385 390 395400Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu405 410 415Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp AsnSer Gly 420 425 430Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala ValLeu Gln Ser Ser 435 440 445Gly Leu Tyr Ser Leu Ser Ser Val Val ThrVal Pro Ser Ser Ser Leu 450 455 460Gly Thr Gln Thr Tyr Ile Cys AsnVal Asn His Lys Pro Ser Asn Thr465 470 475 480Lys Val Asp Lys LysVal Glu Pro Lys Ser Cys Asp Lys Thr His Thr 485 490 495Cys Pro ProCys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 500 505 510LeuPhe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 515 520525Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val530 535 540Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr545 550 555 560Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr TyrArg Val Val Ser Val 565 570 575Leu Thr Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys 580 585 590Lys Val Ser Asn Lys Ala LeuGly Ala Pro Ile Glu Lys Thr Ile Ser 595 600 605Lys Ala Lys Gly GlnPro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 610 615 620Cys Arg AspGlu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val625 630 635640Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly645 650 655Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspSer Asp 660 665 670Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp 675 680 685Gln Gln Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala Leu His 690 695 700Asn His Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys705 710 715179301PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidemonomeric hu 4-1BBL (71-254)-CL 179Arg Glu Gly Pro GluLeu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 10 15Leu Arg Gln GlyMet Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 20 25 30Ile Asp GlyPro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val 35 40 45Ser LeuThr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu Leu Val 50 55 60ValAla Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg65 70 7580Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala LeuThr 100 105 110Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn SerAla Phe Gly 115 120 125Phe Gln Gly Arg Leu Leu His Leu Ser Ala GlyGln Arg Leu Gly Val 130 135 140His Leu His Thr Glu Ala Arg Ala ArgHis Ala Trp Gln Leu Thr Gln145 150 155 160Gly Ala Thr Val Leu GlyLeu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170 175Gly Leu Pro SerPro Arg Ser Glu Gly Gly Gly Gly Ser Gly Gly Gly 180 185 190Gly SerArg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser 195 200205Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn210 215 220Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val AspAsn Ala225 230 235 240Leu Gln Ser Gly Asn Ser Gln Glu Ser Val ThrGlu Gln Asp Ser Lys 245 250 255Asp Ser Thr Tyr Ser Leu Ser Ser ThrLeu Thr Leu Ser Lys Ala Asp 260 265 270Tyr Glu Lys His Lys Val TyrAla Cys Glu Val Thr His Gln Gly Leu 275 280 285Ser Ser Pro Val ThrLys Ser Phe Asn Arg Gly Glu Cys 290 295 300180205PRThom*o sapiens180Ala Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser Ala15 10 15Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro 20 25 30Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln LeuVal Ala 35 40 45Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp TyrSer Asp Pro 50 55 60Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu SerTyr Lys Glu Asp65 70 75 80Thr Lys Glu Leu Val Val Ala Lys Ala GlyVal Tyr Tyr Val Phe Phe 85 90 95Gln Leu Glu Leu Arg Arg Val Val AlaGly Glu Gly Ser Gly Ser Val 100 105 110Ser Leu Ala Leu His Leu GlnPro Leu Arg Ser Ala Ala Gly Ala Ala 115 120 125Ala Leu Ala Leu ThrVal Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg 130 135 140Asn Ser AlaPhe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly145 150 155160Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala165 170 175Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe ArgVal Thr 180 185 190Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg SerGlu 195 200 205181133PRThom*o sapiens 181Gln Val Ser His Arg Tyr ProArg Ile Gln Ser Ile Lys Val Gln Phe1 5 10 15Thr Glu Tyr Lys Lys GluLys Gly Phe Ile Leu Thr Ser Gln Lys Glu 20 25 30Asp Glu Ile Met LysVal Gln Asn Asn Ser Val Ile Ile Asn Cys Asp 35 40 45Gly Phe Tyr LeuIle Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asn 50 55 60Ile Ser LeuHis Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys65 70 75 80LysVal Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys 85 9095Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp100 105 110Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln AsnPro Gly 115 120 125Glu Phe Cys Val Leu 130182132PRThom*o sapiens182Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr15 10 15Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys GluAsp 20 25 30Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn CysAsp Gly 35 40 45Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln GluVal Asn Ile 50 55 60Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu PheGln Leu Lys Lys65 70 75 80Val Arg Ser Val Asn Ser Leu Met Val AlaSer Leu Thr Tyr Lys Asp 85 90 95Lys Val Tyr Leu Asn Val Thr Thr AspAsn Thr Ser Leu Asp Asp Phe 100 105 110His Val Asn Gly Gly Glu LeuIle Leu Ile His Gln Asn Pro Gly Glu 115 120 125Phe Cys Val Leu130183178PRThom*o sapiens 183Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly Leu Leu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala Gln LeuVal Ala Gln Asn Val Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser Trp TyrSer Asp Pro Gly Leu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly Leu SerTyr Lys Glu Asp Thr Lys Glu Leu Val 50 55 60Val Ala Lys Ala Gly ValTyr Tyr Val Phe Phe Gln Leu Glu Leu Arg65 70 75 80Arg Val Val AlaGly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His 85 90 95Leu Gln ProLeu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr 100 105 110ValAsp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly 115 120125Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val130 135 140His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln LeuThr Gln145 150 155 160Gly Ala Thr Val Leu Gly Leu Phe Arg Val ThrPro Glu Ile Pro Ala 165 170 175Gly Leu184366PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidedimeric hu 4-1BBL (71-248) connected by (G4S)2 linker184Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp15 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val LeuLeu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu AlaGly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr LysGlu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala GlyAla Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala SerSer Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg LeuLeu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu HisThr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala165 170 175Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg GluGly Pro 180 185 190Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu AspLeu Arg Gln Gly 195 200 205Met Phe Ala Gln Leu Val Ala Gln Asn ValLeu Leu Ile Asp Gly Pro 210 215 220Leu Ser Trp Tyr Ser Asp Pro GlyLeu Ala Gly Val Ser Leu Thr Gly225 230 235 240Gly Leu Ser Tyr LysGlu Asp Thr Lys Glu Leu Val Val Ala Lys Ala 245 250 255Gly Val TyrTyr Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val Ala 260 265 270GlyGlu Gly Ser Gly Ser Val Ser Leu Ala Leu His Leu Gln Pro Leu 275 280285Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro290 295 300Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly Phe GlnGly Arg305

310 315 320Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val His LeuHis Thr 325 330 335Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr GlnGly Ala Thr Val 340 345 350Leu Gly Leu Phe Arg Val Thr Pro Glu IlePro Ala Gly Leu 355 360 365185360PRTArtificial SequenceDescriptionof Artificial Sequence Synthetic polypeptidedimeric hu 4-1BBL(80-254) connected by (G4S)2 linker 185Asp Pro Ala Gly Leu Leu AspLeu Arg Gln Gly Met Phe Ala Gln Leu1 5 10 15Val Ala Gln Asn Val LeuLeu Ile Asp Gly Pro Leu Ser Trp Tyr Ser 20 25 30Asp Pro Gly Leu AlaGly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys 35 40 45Glu Asp Thr LysGlu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val 50 55 60Phe Phe GlnLeu Glu Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly65 70 75 80SerVal Ser Leu Ala Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 85 9095Ala Ala Ala Leu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu100 105 110Ala Arg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu HisLeu Ser 115 120 125Ala Gly Gln Arg Leu Gly Val His Leu His Thr GluAla Arg Ala Arg 130 135 140His Ala Trp Gln Leu Thr Gln Gly Ala ThrVal Leu Gly Leu Phe Arg145 150 155 160Val Thr Pro Glu Ile Pro AlaGly Leu Pro Ser Pro Arg Ser Glu Gly 165 170 175Gly Gly Gly Ser GlyGly Gly Gly Ser Asp Pro Ala Gly Leu Leu Asp 180 185 190Leu Arg GlnGly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu 195 200 205IleAsp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala Gly Val 210 215220Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu LeuVal225 230 235 240Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe GlnLeu Glu Leu Arg 245 250 255Arg Val Val Ala Gly Glu Gly Ser Gly SerVal Ser Leu Ala Leu His 260 265 270Leu Gln Pro Leu Arg Ser Ala AlaGly Ala Ala Ala Leu Ala Leu Thr 275 280 285Val Asp Leu Pro Pro AlaSer Ser Glu Ala Arg Asn Ser Ala Phe Gly 290 295 300Phe Gln Gly ArgLeu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val305 310 315 320HisLeu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln 325 330335Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala340 345 350Gly Leu Pro Ser Pro Arg Ser Glu 355360186416PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptidedimeric hu 4-1BBL (52-254) connected by (G4S)2linker 186Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser AlaAla Ser1 5 10 15Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp AspPro Ala Gly 20 25 30Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln LeuVal Ala Gln Asn 35 40 45Val Leu Leu Ile Asp Gly Pro Leu Ser Trp TyrSer Asp Pro Gly Leu 50 55 60Ala Gly Val Ser Leu Thr Gly Gly Leu SerTyr Lys Glu Asp Thr Lys65 70 75 80Glu Leu Val Val Ala Lys Ala GlyVal Tyr Tyr Val Phe Phe Gln Leu 85 90 95Glu Leu Arg Arg Val Val AlaGly Glu Gly Ser Gly Ser Val Ser Leu 100 105 110Ala Leu His Leu GlnPro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu 115 120 125Ala Leu ThrVal Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser 130 135 140AlaPhe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg145 150155 160Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His Ala TrpGln 165 170 175Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg ValThr Pro Glu 180 185 190Ile Pro Ala Gly Leu Pro Ser Pro Arg Ser GluGly Gly Gly Gly Ser 195 200 205Gly Gly Gly Gly Ser Pro Trp Ala ValSer Gly Ala Arg Ala Ser Pro 210 215 220Gly Ser Ala Ala Ser Pro ArgLeu Arg Glu Gly Pro Glu Leu Ser Pro225 230 235 240Asp Asp Pro AlaGly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln 245 250 255Leu ValAla Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp Tyr 260 265270Ser Asp Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr275 280 285Lys Glu Asp Thr Lys Glu Leu Val Val Ala Lys Ala Gly ValTyr Tyr 290 295 300Val Phe Phe Gln Leu Glu Leu Arg Arg Val Val AlaGly Glu Gly Ser305 310 315 320Gly Ser Val Ser Leu Ala Leu His LeuGln Pro Leu Arg Ser Ala Ala 325 330 335Gly Ala Ala Ala Leu Ala LeuThr Val Asp Leu Pro Pro Ala Ser Ser 340 345 350Glu Ala Arg Asn SerAla Phe Gly Phe Gln Gly Arg Leu Leu His Leu 355 360 365Ser Ala GlyGln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala 370 375 380ArgHis Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe385 390395 400Arg Val Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser Pro Arg SerGlu 405 410 415187189PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptidemonomeric hu 4-1BBL(71-254) plus (G4S)1 linker 187Arg Glu Gly Pro Glu Leu Ser Pro AspAsp Pro Ala Gly Leu Leu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala GlnLeu Val Ala Gln Asn Val Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser TrpTyr Ser Asp Pro Gly Leu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly LeuSer Tyr Lys Glu Asp Thr Lys Glu Leu Val 50 55 60Val Ala Lys Ala GlyVal Tyr Tyr Val Phe Phe Gln Leu Glu Leu Arg65 70 75 80Arg Val ValAla Gly Glu Gly Ser Gly Ser Val Ser Leu Ala Leu His 85 90 95Leu GlnPro Leu Arg Ser Ala Ala Gly Ala Ala Ala Leu Ala Leu Thr 100 105110Val Asp Leu Pro Pro Ala Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly115 120 125Phe Gln Gly Arg Leu Leu His Leu Ser Ala Gly Gln Arg LeuGly Val 130 135 140His Leu His Thr Glu Ala Arg Ala Arg His Ala TrpGln Leu Thr Gln145 150 155 160Gly Ala Thr Val Leu Gly Leu Phe ArgVal Thr Pro Glu Ile Pro Ala 165 170 175Gly Leu Pro Ser Pro Arg SerGlu Gly Gly Gly Gly Ser 180 185188188PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidemonomeric hu 4-1BBL (71-248) plus (G4S)2 linker 188ArgGlu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly Leu Leu Asp1 5 1015Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val Leu Leu20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala GlyVal 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys GluLeu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln LeuGlu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly Ser Gly Ser ValSer Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser Ala Ala Gly AlaAla Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro Pro Ala Ser SerGlu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln Gly Arg Leu LeuHis Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140His Leu His ThrGlu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150 155 160GlyAla Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile Pro Ala 165 170175Gly Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 180185189183PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptidemonomeric hu 4-1BBL (71-248) plus (G4S)1linker 189Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp Pro Ala Gly LeuLeu Asp1 5 10 15Leu Arg Gln Gly Met Phe Ala Gln Leu Val Ala Gln AsnVal Leu Leu 20 25 30Ile Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro GlyLeu Ala Gly Val 35 40 45Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu AspThr Lys Glu Leu Val 50 55 60Val Ala Lys Ala Gly Val Tyr Tyr Val PhePhe Gln Leu Glu Leu Arg65 70 75 80Arg Val Val Ala Gly Glu Gly SerGly Ser Val Ser Leu Ala Leu His 85 90 95Leu Gln Pro Leu Arg Ser AlaAla Gly Ala Ala Ala Leu Ala Leu Thr 100 105 110Val Asp Leu Pro ProAla Ser Ser Glu Ala Arg Asn Ser Ala Phe Gly 115 120 125Phe Gln GlyArg Leu Leu His Leu Ser Ala Gly Gln Arg Leu Gly Val 130 135 140HisLeu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln Leu Thr Gln145 150155 160Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu Ile ProAla 165 170 175Gly Leu Gly Gly Gly Gly Ser 180190276PRTArtificialSequenceDescription of Artificial Sequence Syntheticpolypeptidedimeric huOX40L (51-183) connected by (G4S)2 linker190Gln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe15 10 15Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln LysGlu 20 25 30Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile AsnCys Asp 35 40 45Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser GlnGlu Val Asn 50 55 60Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro LeuPhe Gln Leu Lys65 70 75 80Lys Val Arg Ser Val Asn Ser Leu Met ValAla Ser Leu Thr Tyr Lys 85 90 95Asp Lys Val Tyr Leu Asn Val Thr ThrAsp Asn Thr Ser Leu Asp Asp 100 105 110Phe His Val Asn Gly Gly GluLeu Ile Leu Ile His Gln Asn Pro Gly 115 120 125Glu Phe Cys Val LeuGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140Val Ser HisArg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr145 150 155160Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp165 170 175Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn CysAsp Gly 180 185 190Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser GlnGlu Val Asn Ile 195 200 205Ser Leu His Tyr Gln Lys Asp Glu Glu ProLeu Phe Gln Leu Lys Lys 210 215 220Val Arg Ser Val Asn Ser Leu MetVal Ala Ser Leu Thr Tyr Lys Asp225 230 235 240Lys Val Tyr Leu AsnVal Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe 245 250 255His Val AsnGly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu 260 265 270PheCys Val Leu 275191274PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptidedimeric huOX40L (52-183)connected by (G4S)2 linker 191Val Ser His Arg Tyr Pro Arg Ile GlnSer Ile Lys Val Gln Phe Thr1 5 10 15Glu Tyr Lys Lys Glu Lys Gly PheIle Leu Thr Ser Gln Lys Glu Asp 20 25 30Glu Ile Met Lys Val Gln AsnAsn Ser Val Ile Ile Asn Cys Asp Gly 35 40 45Phe Tyr Leu Ile Ser LeuLys Gly Tyr Phe Ser Gln Glu Val Asn Ile 50 55 60Ser Leu His Tyr GlnLys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys65 70 75 80Val Arg SerVal Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp 85 90 95Lys ValTyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe 100 105110His Val Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro Gly Glu115 120 125Phe Cys Val Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly SerVal Ser 130 135 140His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val GlnPhe Thr Glu Tyr145 150 155 160Lys Lys Glu Lys Gly Phe Ile Leu ThrSer Gln Lys Glu Asp Glu Ile 165 170 175Met Lys Val Gln Asn Asn SerVal Ile Ile Asn Cys Asp Gly Phe Tyr 180 185 190Leu Ile Ser Leu LysGly Tyr Phe Ser Gln Glu Val Asn Ile Ser Leu 195 200 205His Tyr GlnLys Asp Glu Glu Pro Leu Phe Gln Leu Lys Lys Val Arg 210 215 220SerVal Asn Ser Leu Met Val Ala Ser Leu Thr Tyr Lys Asp Lys Val225 230235 240Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser Leu Asp Asp Phe HisVal 245 250 255Asn Gly Gly Glu Leu Ile Leu Ile His Gln Asn Pro GlyGlu Phe Cys 260 265 270Val Leu192164PRThom*o sapiens 192Leu Asp LeuArg Gln Gly Met Phe Ala Gln Leu Val Ala Gln Asn Val1 5 10 15Leu LeuIle Asp Gly Pro Leu Ser Trp Tyr Ser Asp Pro Gly Leu Ala 20 25 30GlyVal Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu Asp Thr Lys Glu 35 4045Leu Val Val Ala Lys Ala Gly Val Tyr Tyr Val Phe Phe Gln Leu Glu50 55 60Leu Arg Arg Val Val Ala Gly Glu Gly Ser Gly Ser Val Ser LeuAla65 70 75 80Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala AlaAla Leu Ala 85 90 95Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu AlaArg Asn Ser Ala 100 105 110Phe Gly Phe Gln Gly Arg Leu Leu His LeuSer Ala Gly Gln Arg Leu 115 120 125Gly Val His Leu His Thr Glu AlaArg Ala Arg His Ala Trp Gln Leu 130 135 140Thr Gln Gly Ala Thr ValLeu Gly Leu Phe Arg Val Thr Pro Glu Ile145 150 155 160Pro Ala GlyLeu193169PRThom*o sapiens 193Asp Pro Ala Gly Leu Leu Asp Leu Arg GlnGly Met Phe Ala Gln Leu1 5 10 15Val Ala Gln Asn Val Leu Leu Ile AspGly Pro Leu Ser Trp Tyr Ser 20 25 30Asp Pro Gly Leu Ala Gly Val SerLeu Thr Gly Gly Leu Ser Tyr Lys 35 40 45Glu Asp Thr Lys Glu Leu ValVal Ala Lys Ala Gly Val Tyr Tyr Val 50 55 60Phe Phe Gln Leu Glu LeuArg Arg Val Val Ala Gly Glu Gly Ser Gly65 70 75 80Ser Val Ser LeuAla Leu His Leu Gln Pro Leu Arg Ser Ala Ala Gly 85 90 95Ala Ala AlaLeu Ala Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu 100 105 110AlaArg Asn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser 115 120125Ala Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg130 135 140His Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly LeuPhe Arg145 150 155 160Val Thr Pro Glu Ile Pro Ala Gly Leu165194197PRThom*o sapiens 194Pro Trp Ala Val Ser Gly Ala Arg Ala SerPro Gly Ser Ala Ala Ser1 5 10 15Pro Arg Leu Arg Glu Gly Pro Glu LeuSer Pro Asp Asp Pro Ala Gly 20 25 30Leu Leu Asp Leu Arg Gln Gly MetPhe Ala Gln Leu Val Ala Gln Asn 35 40 45Val Leu Leu Ile Asp Gly ProLeu Ser Trp Tyr Ser Asp Pro Gly

Leu 50 55 60Ala Gly Val Ser Leu Thr Gly Gly Leu Ser Tyr Lys Glu AspThr Lys65 70 75 80Glu Leu Val Val Ala Lys Ala Gly Val Tyr Tyr ValPhe Phe Gln Leu 85 90 95Glu Leu Arg Arg Val Val Ala Gly Glu Gly SerGly Ser Val Ser Leu 100 105 110Ala Leu His Leu Gln Pro Leu Arg SerAla Ala Gly Ala Ala Ala Leu 115 120 125Ala Leu Thr Val Asp Leu ProPro Ala Ser Ser Glu Ala Arg Asn Ser 130 135 140Ala Phe Gly Phe GlnGly Arg Leu Leu His Leu Ser Ala Gly Gln Arg145 150 155 160Leu GlyVal His Leu His Thr Glu Ala Arg Ala Arg His Ala Trp Gln 165 170175Leu Thr Gln Gly Ala Thr Val Leu Gly Leu Phe Arg Val Thr Pro Glu180 185 190Ile Pro Ala Gly Leu 195195620PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideDimeric hu OX40L (51-183) - CL* Fc knob chain 195Gln ValSer His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe1 5 10 15ThrGlu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu 20 2530Asp Glu Ile Met Lys Val Gln Asp Asn Ser Val Ile Ile Asn Cys Asp35 40 45Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu ValAsp 50 55 60Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe GlnLeu Lys65 70 75 80Lys Val Arg Ser Val Asn Ser Leu Met Val Ala SerLeu Thr Tyr Lys 85 90 95Asp Lys Val Tyr Leu Asn Val Thr Thr Asp AsnThr Ser Leu Asp Asp 100 105 110Phe His Val Asn Gly Gly Glu Leu IleLeu Ile His Gln Asn Pro Gly 115 120 125Glu Phe Cys Val Leu Gly GlyGly Gly Ser Gly Gly Gly Gly Ser Gln 130 135 140Val Ser His Arg TyrPro Arg Ile Gln Ser Ile Lys Val Gln Phe Thr145 150 155 160Glu TyrLys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu Asp 165 170175Glu Ile Met Lys Val Gln Asp Asn Ser Val Ile Ile Asn Cys Asp Gly180 185 190Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu ValAsp Ile 195 200 205Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu PheGln Leu Lys Lys 210 215 220Val Arg Ser Val Asn Ser Leu Met Val AlaSer Leu Thr Tyr Lys Asp225 230 235 240Lys Val Tyr Leu Asn Val ThrThr Asp Asn Thr Ser Leu Asp Asp Phe 245 250 255His Val Asn Gly GlyGlu Leu Ile Leu Ile His Gln Asn Pro Gly Glu 260 265 270Phe Cys ValLeu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Thr 275 280 285ValAla Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Arg Lys Leu 290 295300Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe TyrPro305 310 315 320Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn AlaLeu Gln Ser Gly 325 330 335Asn Ser Gln Glu Ser Val Thr Glu Gln AspSer Lys Asp Ser Thr Tyr 340 345 350Ser Leu Ser Ser Thr Leu Thr LeuSer Lys Ala Asp Tyr Glu Lys His 355 360 365Lys Val Tyr Ala Cys GluVal Thr His Gln Gly Leu Ser Ser Pro Val 370 375 380Thr Lys Ser PheAsn Arg Gly Glu Cys Asp Lys Thr His Thr Cys Pro385 390 395 400ProCys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe 405 410415Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val420 425 430Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu ValLys Phe 435 440 445Asn Trp Tyr Val Asp Gly Val Glu Val His Asn AlaLys Thr Lys Pro 450 455 460Arg Glu Glu Gln Tyr Asn Ser Thr Tyr ArgVal Val Ser Val Leu Thr465 470 475 480Val Leu His Gln Asp Trp LeuAsn Gly Lys Glu Tyr Lys Cys Lys Val 485 490 495Ser Asn Lys Ala LeuGly Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 500 505 510Lys Gly GlnPro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg 515 520 525AspGlu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 530 535540Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly GlnPro545 550 555 560Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu AspSer Asp Gly Ser 565 570 575Phe Phe Leu Tyr Ser Lys Leu Thr Val AspLys Ser Arg Trp Gln Gln 580 585 590Gly Asn Val Phe Ser Cys Ser ValMet His Glu Ala Leu His Asn His 595 600 605Tyr Thr Gln Lys Ser LeuSer Leu Ser Pro Gly Lys 610 615 620196246PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideMonomeric hu OX40L (51-183) - CH1* 196Gln Val Ser HisArg Tyr Pro Arg Ile Gln Ser Ile Lys Val Gln Phe1 5 10 15Thr Glu TyrLys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln Lys Glu 20 25 30Asp GluIle Met Lys Val Gln Asp Asn Ser Val Ile Ile Asn Cys Asp 35 40 45GlyPhe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser Gln Glu Val Asp 50 5560Ile Ser Leu His Tyr Gln Lys Asp Glu Glu Pro Leu Phe Gln Leu Lys6570 75 80Lys Val Arg Ser Val Asn Ser Leu Met Val Ala Ser Leu Thr TyrLys 85 90 95Asp Lys Val Tyr Leu Asn Val Thr Thr Asp Asn Thr Ser LeuAsp Asp 100 105 110Phe His Val Asn Gly Gly Glu Leu Ile Leu Ile HisGln Asn Pro Gly 115 120 125Glu Phe Cys Val Leu Gly Gly Gly Gly SerGly Gly Gly Gly Ser Ala 130 135 140Ser Thr Lys Gly Pro Ser Val PhePro Leu Ala Pro Ser Ser Lys Ser145 150 155 160Thr Ser Gly Gly ThrAla Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe 165 170 175Pro Glu ProVal Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 180 185 190ValHis Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 195 200205Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr210 215 220Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val AspGlu Lys225 230 235 240Val Glu Pro Lys Ser Cys 2451972201PRThom*osapiens 197Met Gly Ala Met Thr Gln Leu Leu Ala Gly Val Phe Leu AlaPhe Leu1 5 10 15Ala Leu Ala Thr Glu Gly Gly Val Leu Lys Lys Val IleArg His Lys 20 25 30Arg Gln Ser Gly Val Asn Ala Thr Leu Pro Glu GluAsn Gln Pro Val 35 40 45Val Phe Asn His Val Tyr Asn Ile Lys Leu ProVal Gly Ser Gln Cys 50 55 60Ser Val Asp Leu Glu Ser Ala Ser Gly GluLys Asp Leu Ala Pro Pro65 70 75 80Ser Glu Pro Ser Glu Ser Phe GlnGlu His Thr Val Asp Gly Glu Asn 85 90 95Gln Ile Val Phe Thr His ArgIle Asn Ile Pro Arg Arg Ala Cys Gly 100 105 110Cys Ala Ala Ala ProAsp Val Lys Glu Leu Leu Ser Arg Leu Glu Glu 115 120 125Leu Glu AsnLeu Val Ser Ser Leu Arg Glu Gln Cys Thr Ala Gly Ala 130 135 140GlyCys Cys Leu Gln Pro Ala Thr Gly Arg Leu Asp Thr Arg Pro Phe145 150155 160Cys Ser Gly Arg Gly Asn Phe Ser Thr Glu Gly Cys Gly Cys ValCys 165 170 175Glu Pro Gly Trp Lys Gly Pro Asn Cys Ser Glu Pro GluCys Pro Gly 180 185 190Asn Cys His Leu Arg Gly Arg Cys Ile Asp GlyGln Cys Ile Cys Asp 195 200 205Asp Gly Phe Thr Gly Glu Asp Cys SerGln Leu Ala Cys Pro Ser Asp 210 215 220Cys Asn Asp Gln Gly Lys CysVal Asn Gly Val Cys Ile Cys Phe Glu225 230 235 240Gly Tyr Ala GlyAla Asp Cys Ser Arg Glu Ile Cys Pro Val Pro Cys 245 250 255Ser GluGlu His Gly Thr Cys Val Asp Gly Leu Cys Val Cys His Asp 260 265270Gly Phe Ala Gly Asp Asp Cys Asn Lys Pro Leu Cys Leu Asn Asn Cys275 280 285Tyr Asn Arg Gly Arg Cys Val Glu Asn Glu Cys Val Cys AspGlu Gly 290 295 300Phe Thr Gly Glu Asp Cys Ser Glu Leu Ile Cys ProAsn Asp Cys Phe305 310 315 320Asp Arg Gly Arg Cys Ile Asn Gly ThrCys Tyr Cys Glu Glu Gly Phe 325 330 335Thr Gly Glu Asp Cys Gly LysPro Thr Cys Pro His Ala Cys His Thr 340 345 350Gln Gly Arg Cys GluGlu Gly Gln Cys Val Cys Asp Glu Gly Phe Ala 355 360 365Gly Val AspCys Ser Glu Lys Arg Cys Pro Ala Asp Cys His Asn Arg 370 375 380GlyArg Cys Val Asp Gly Arg Cys Glu Cys Asp Asp Gly Phe Thr Gly385 390395 400Ala Asp Cys Gly Glu Leu Lys Cys Pro Asn Gly Cys Ser Gly HisGly 405 410 415Arg Cys Val Asn Gly Gln Cys Val Cys Asp Glu Gly TyrThr Gly Glu 420 425 430Asp Cys Ser Gln Leu Arg Cys Pro Asn Asp CysHis Ser Arg Gly Arg 435 440 445Cys Val Glu Gly Lys Cys Val Cys GluGln Gly Phe Lys Gly Tyr Asp 450 455 460Cys Ser Asp Met Ser Cys ProAsn Asp Cys His Gln His Gly Arg Cys465 470 475 480Val Asn Gly MetCys Val Cys Asp Asp Gly Tyr Thr Gly Glu Asp Cys 485 490 495Arg AspArg Gln Cys Pro Arg Asp Cys Ser Asn Arg Gly Leu Cys Val 500 505510Asp Gly Gln Cys Val Cys Glu Asp Gly Phe Thr Gly Pro Asp Cys Ala515 520 525Glu Leu Ser Cys Pro Asn Asp Cys His Gly Gln Gly Arg CysVal Asn 530 535 540Gly Gln Cys Val Cys His Glu Gly Phe Met Gly LysAsp Cys Lys Glu545 550 555 560Gln Arg Cys Pro Ser Asp Cys His GlyGln Gly Arg Cys Val Asp Gly 565 570 575Gln Cys Ile Cys His Glu GlyPhe Thr Gly Leu Asp Cys Gly Gln His 580 585 590Ser Cys Pro Ser AspCys Asn Asn Leu Gly Gln Cys Val Ser Gly Arg 595 600 605Cys Ile CysAsn Glu Gly Tyr Ser Gly Glu Asp Cys Ser Glu Val Ser 610 615 620ProPro Lys Asp Leu Val Val Thr Glu Val Thr Glu Glu Thr Val Asn625 630635 640Leu Ala Trp Asp Asn Glu Met Arg Val Thr Glu Tyr Leu Val ValTyr 645 650 655Thr Pro Thr His Glu Gly Gly Leu Glu Met Gln Phe ArgVal Pro Gly 660 665 670Asp Gln Thr Ser Thr Ile Ile Gln Glu Leu GluPro Gly Val Glu Tyr 675 680 685Phe Ile Arg Val Phe Ala Ile Leu GluAsn Lys Lys Ser Ile Pro Val 690 695 700Ser Ala Arg Val Ala Thr TyrLeu Pro Ala Pro Glu Gly Leu Lys Phe705 710 715 720Lys Ser Ile LysGlu Thr Ser Val Glu Val Glu Trp Asp Pro Leu Asp 725 730 735Ile AlaPhe Glu Thr Trp Glu Ile Ile Phe Arg Asn Met Asn Lys Glu 740 745750Asp Glu Gly Glu Ile Thr Lys Ser Leu Arg Arg Pro Glu Thr Ser Tyr755 760 765Arg Gln Thr Gly Leu Ala Pro Gly Gln Glu Tyr Glu Ile SerLeu His 770 775 780Ile Val Lys Asn Asn Thr Arg Gly Pro Gly Leu LysArg Val Thr Thr785 790 795 800Thr Arg Leu Asp Ala Pro Ser Gln IleGlu Val Lys Asp Val Thr Asp 805 810 815Thr Thr Ala Leu Ile Thr TrpPhe Lys Pro Leu Ala Glu Ile Asp Gly 820 825 830Ile Glu Leu Thr TyrGly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr 835 840 845Ile Asp LeuThr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys 850 855 860ProAsp Thr Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met865 870875 880Ser Ser Asn Pro Ala Lys Glu Thr Phe Thr Thr Gly Leu Asp AlaPro 885 890 895Arg Asn Leu Arg Arg Val Ser Gln Thr Asp Asn Ser IleThr Leu Glu 900 905 910Trp Arg Asn Gly Lys Ala Ala Ile Asp Ser TyrArg Ile Lys Tyr Ala 915 920 925Pro Ile Ser Gly Gly Asp His Ala GluVal Asp Val Pro Lys Ser Gln 930 935 940Gln Ala Thr Thr Lys Thr ThrLeu Thr Gly Leu Arg Pro Gly Thr Glu945 950 955 960Tyr Gly Ile GlyVal Ser Ala Val Lys Glu Asp Lys Glu Ser Asn Pro 965 970 975Ala ThrIle Asn Ala Ala Thr Glu Leu Asp Thr Pro Lys Asp Leu Gln 980 985990Val Ser Glu Thr Ala Glu Thr Ser Leu Thr Leu Leu Trp Lys Thr Pro995 1000 1005Leu Ala Lys Phe Asp Arg Tyr Arg Leu Asn Tyr Ser LeuPro Thr 1010 1015 1020Gly Gln Trp Val Gly Val Gln Leu Pro Arg AsnThr Thr Ser Tyr 1025 1030 1035Val Leu Arg Gly Leu Glu Pro Gly GlnGlu Tyr Asn Val Leu Leu 1040 1045 1050Thr Ala Glu Lys Gly Arg HisLys Ser Lys Pro Ala Arg Val Lys 1055 1060 1065Ala Ser Thr Glu GlnAla Pro Glu Leu Glu Asn Leu Thr Val Thr 1070 1075 1080Glu Val GlyTrp Asp Gly Leu Arg Leu Asn Trp Thr Ala Ala Asp 1085 1090 1095GlnAla Tyr Glu His Phe Ile Ile Gln Val Gln Glu Ala Asn Lys 1100 11051110Val Glu Ala Ala Arg Asn Leu Thr Val Pro Gly Ser Leu Arg Ala1115 1120 1125Val Asp Ile Pro Gly Leu Lys Ala Ala Thr Pro Tyr ThrVal Ser 1130 1135 1140Ile Tyr Gly Val Ile Gln Gly Tyr Arg Thr ProVal Leu Ser Ala 1145 1150 1155Glu Ala Ser Thr Gly Glu Thr Pro AsnLeu Gly Glu Val Val Val 1160 1165 1170Ala Glu Val Gly Trp Asp AlaLeu Lys Leu Asn Trp Thr Ala Pro 1175 1180 1185Glu Gly Ala Tyr GluTyr Phe Phe Ile Gln Val Gln Glu Ala Asp 1190 1195 1200Thr Val GluAla Ala Gln Asn Leu Thr Val Pro Gly Gly Leu Arg 1205 1210 1215SerThr Asp Leu Pro Gly Leu Lys Ala Ala Thr His Tyr Thr Ile 1220 12251230Thr Ile Arg Gly Val Thr Gln Asp Phe Ser Thr Thr Pro Leu Ser1235 1240 1245Val Glu Val Leu Thr Glu Glu Val Pro Asp Met Gly AsnLeu Thr 1250 1255 1260Val Thr Glu Val Ser Trp Asp Ala Leu Arg LeuAsn Trp Thr Thr 1265 1270 1275Pro Asp Gly Thr Tyr Asp Gln Phe ThrIle Gln Val Gln Glu Ala 1280 1285 1290Asp Gln Val Glu Glu Ala HisAsn Leu Thr Val Pro Gly Ser Leu 1295 1300 1305Arg Ser Met Glu IlePro Gly Leu Arg Ala Gly Thr Pro Tyr Thr 1310 1315 1320Val Thr LeuHis Gly Glu Val Arg Gly His Ser Thr Arg Pro Leu 1325 1330 1335AlaVal Glu Val Val Thr Glu Asp Leu Pro Gln Leu Gly Asp Leu 1340 13451350Ala Val Ser Glu Val Gly Trp Asp Gly Leu Arg Leu Asn Trp Thr1355 1360 1365Ala Ala Asp Asn Ala Tyr Glu His Phe Val Ile Gln ValGln Glu 1370 1375 1380Val Asn Lys Val Glu Ala Ala Gln Asn Leu ThrLeu Pro Gly Ser 1385 1390 1395Leu Arg Ala Val Asp Ile Pro Gly LeuGlu Ala Ala Thr Pro Tyr 1400 1405 1410Arg Val Ser Ile Tyr Gly ValIle Arg Gly Tyr Arg Thr Pro Val 1415

1420 1425Leu Ser Ala Glu Ala Ser Thr Ala Lys Glu Pro Glu Ile GlyAsn 1430 1435 1440Leu Asn Val Ser Asp Ile Thr Pro Glu Ser Phe AsnLeu Ser Trp 1445 1450 1455Met Ala Thr Asp Gly Ile Phe Glu Thr PheThr Ile Glu Ile Ile 1460 1465 1470Asp Ser Asn Arg Leu Leu Glu ThrVal Glu Tyr Asn Ile Ser Gly 1475 1480 1485Ala Glu Arg Thr Ala HisIle Ser Gly Leu Pro Pro Ser Thr Asp 1490 1495 1500Phe Ile Val TyrLeu Ser Gly Leu Ala Pro Ser Ile Arg Thr Lys 1505 1510 1515Thr IleSer Ala Thr Ala Thr Thr Glu Ala Leu Pro Leu Leu Glu 1520 15251530Asn Leu Thr Ile Ser Asp Ile Asn Pro Tyr Gly Phe Thr Val Ser1535 1540 1545Trp Met Ala Ser Glu Asn Ala Phe Asp Ser Phe Leu ValThr Val 1550 1555 1560Val Asp Ser Gly Lys Leu Leu Asp Pro Gln GluPhe Thr Leu Ser 1565 1570 1575Gly Thr Gln Arg Lys Leu Glu Leu ArgGly Leu Ile Thr Gly Ile 1580 1585 1590Gly Tyr Glu Val Met Val SerGly Phe Thr Gln Gly His Gln Thr 1595 1600 1605Lys Pro Leu Arg AlaGlu Ile Val Thr Glu Ala Glu Pro Glu Val 1610 1615 1620Asp Asn LeuLeu Val Ser Asp Ala Thr Pro Asp Gly Phe Arg Leu 1625 1630 1635SerTrp Thr Ala Asp Glu Gly Val Phe Asp Asn Phe Val Leu Lys 1640 16451650Ile Arg Asp Thr Lys Lys Gln Ser Glu Pro Leu Glu Ile Thr Leu1655 1660 1665Leu Ala Pro Glu Arg Thr Arg Asp Ile Thr Gly Leu ArgGlu Ala 1670 1675 1680Thr Glu Tyr Glu Ile Glu Leu Tyr Gly Ile SerLys Gly Arg Arg 1685 1690 1695Ser Gln Thr Val Ser Ala Ile Ala ThrThr Ala Met Gly Ser Pro 1700 1705 1710Lys Glu Val Ile Phe Ser AspIle Thr Glu Asn Ser Ala Thr Val 1715 1720 1725Ser Trp Arg Ala ProThr Ala Gln Val Glu Ser Phe Arg Ile Thr 1730 1735 1740Tyr Val ProIle Thr Gly Gly Thr Pro Ser Met Val Thr Val Asp 1745 1750 1755GlyThr Lys Thr Gln Thr Arg Leu Val Lys Leu Ile Pro Gly Val 1760 17651770Glu Tyr Leu Val Ser Ile Ile Ala Met Lys Gly Phe Glu Glu Ser1775 1780 1785Glu Pro Val Ser Gly Ser Phe Thr Thr Ala Leu Asp GlyPro Ser 1790 1795 1800Gly Leu Val Thr Ala Asn Ile Thr Asp Ser GluAla Leu Ala Arg 1805 1810 1815Trp Gln Pro Ala Ile Ala Thr Val AspSer Tyr Val Ile Ser Tyr 1820 1825 1830Thr Gly Glu Lys Val Pro GluIle Thr Arg Thr Val Ser Gly Asn 1835 1840 1845Thr Val Glu Tyr AlaLeu Thr Asp Leu Glu Pro Ala Thr Glu Tyr 1850 1855 1860Thr Leu ArgIle Phe Ala Glu Lys Gly Pro Gln Lys Ser Ser Thr 1865 1870 1875IleThr Ala Lys Phe Thr Thr Asp Leu Asp Ser Pro Arg Asp Leu 1880 18851890Thr Ala Thr Glu Val Gln Ser Glu Thr Ala Leu Leu Thr Trp Arg1895 1900 1905Pro Pro Arg Ala Ser Val Thr Gly Tyr Leu Leu Val TyrGlu Ser 1910 1915 1920Val Asp Gly Thr Val Lys Glu Val Ile Val GlyPro Asp Thr Thr 1925 1930 1935Ser Tyr Ser Leu Ala Asp Leu Ser ProSer Thr His Tyr Thr Ala 1940 1945 1950Lys Ile Gln Ala Leu Asn GlyPro Leu Arg Ser Asn Met Ile Gln 1955 1960 1965Thr Ile Phe Thr ThrIle Gly Leu Leu Tyr Pro Phe Pro Lys Asp 1970 1975 1980Cys Ser GlnAla Met Leu Asn Gly Asp Thr Thr Ser Gly Leu Tyr 1985 1990 1995ThrIle Tyr Leu Asn Gly Asp Lys Ala Glu Ala Leu Glu Val Phe 2000 20052010Cys Asp Met Thr Ser Asp Gly Gly Gly Trp Ile Val Phe Leu Arg2015 2020 2025Arg Lys Asn Gly Arg Glu Asn Phe Tyr Gln Asn Trp LysAla Tyr 2030 2035 2040Ala Ala Gly Phe Gly Asp Arg Arg Glu Glu PheTrp Leu Gly Leu 2045 2050 2055Asp Asn Leu Asn Lys Ile Thr Ala GlnGly Gln Tyr Glu Leu Arg 2060 2065 2070Val Asp Leu Arg Asp His GlyGlu Thr Ala Phe Ala Val Tyr Asp 2075 2080 2085Lys Phe Ser Val GlyAsp Ala Lys Thr Arg Tyr Lys Leu Lys Val 2090 2095 2100Glu Gly TyrSer Gly Thr Ala Gly Asp Ser Met Ala Tyr His Asn 2105 2110 2115GlyArg Ser Phe Ser Thr Phe Asp Lys Asp Thr Asp Ser Ala Ile 2120 21252130Thr Asn Cys Ala Leu Ser Tyr Lys Gly Ala Phe Trp Tyr Arg Asn2135 2140 2145Cys His Arg Val Asn Leu Met Gly Arg Tyr Gly Asp AsnAsn His 2150 2155 2160Ser Gln Gly Val Asn Trp Phe His Trp Lys GlyHis Glu His Ser 2165 2170 2175Ile Gln Phe Ala Glu Met Lys Leu ArgPro Ser Asn Phe Arg Asn 2180 2185 2190Leu Glu Gly Arg Arg Lys ArgAla 2195 22001982322PRThom*o sapiens 198Met Gln Ser Gly Pro Arg ProPro Leu Pro Ala Pro Gly Leu Ala Leu1 5 10 15Ala Leu Thr Leu Thr MetLeu Ala Arg Leu Ala Ser Ala Ala Ser Phe 20 25 30Phe Gly Glu Asn HisLeu Glu Val Pro Val Ala Thr Ala Leu Thr Asp 35 40 45Ile Asp Leu GlnLeu Gln Phe Ser Thr Ser Gln Pro Glu Ala Leu Leu 50 55 60Leu Leu AlaAla Gly Pro Ala Asp His Leu Leu Leu Gln Leu Tyr Ser65 70 75 80GlyArg Leu Gln Val Arg Leu Val Leu Gly Gln Glu Glu Leu Arg Leu 85 9095Gln Thr Pro Ala Glu Thr Leu Leu Ser Asp Ser Ile Pro His Thr Val100 105 110Val Leu Thr Val Val Glu Gly Trp Ala Thr Leu Ser Val AspGly Phe 115 120 125Leu Asn Ala Ser Ser Ala Val Pro Gly Ala Pro LeuGlu Val Pro Tyr 130 135 140Gly Leu Phe Val Gly Gly Thr Gly Thr LeuGly Leu Pro Tyr Leu Arg145 150 155 160Gly Thr Ser Arg Pro Leu ArgGly Cys Leu His Ala Ala Thr Leu Asn 165 170 175Gly Arg Ser Leu LeuArg Pro Leu Thr Pro Asp Val His Glu Gly Cys 180 185 190Ala Glu GluPhe Ser Ala Ser Asp Asp Val Ala Leu Gly Phe Ser Gly 195 200 205ProHis Ser Leu Ala Ala Phe Pro Ala Trp Gly Thr Gln Asp Glu Gly 210 215220Thr Leu Glu Phe Thr Leu Thr Thr Gln Ser Arg Gln Ala Pro LeuAla225 230 235 240Phe Gln Ala Gly Gly Arg Arg Gly Asp Phe Ile TyrVal Asp Ile Phe 245 250 255Glu Gly His Leu Arg Ala Val Val Glu LysGly Gln Gly Thr Val Leu 260 265 270Leu His Asn Ser Val Pro Val AlaAsp Gly Gln Pro His Glu Val Ser 275 280 285Val His Ile Asn Ala HisArg Leu Glu Ile Ser Val Asp Gln Tyr Pro 290 295 300Thr His Thr SerAsn Arg Gly Val Leu Ser Tyr Leu Glu Pro Arg Gly305 310 315 320SerLeu Leu Leu Gly Gly Leu Asp Ala Glu Ala Ser Arg His Leu Gln 325 330335Glu His Arg Leu Gly Leu Thr Pro Glu Ala Thr Asn Ala Ser Leu Leu340 345 350Gly Cys Met Glu Asp Leu Ser Val Asn Gly Gln Arg Arg GlyLeu Arg 355 360 365Glu Ala Leu Leu Thr Arg Asn Met Ala Ala Gly CysArg Leu Glu Glu 370 375 380Glu Glu Tyr Glu Asp Asp Ala Tyr Gly HisTyr Glu Ala Phe Ser Thr385 390 395 400Leu Ala Pro Glu Ala Trp ProAla Met Glu Leu Pro Glu Pro Cys Val 405 410 415Pro Glu Pro Gly LeuPro Pro Val Phe Ala Asn Phe Thr Gln Leu Leu 420 425 430Thr Ile SerPro Leu Val Val Ala Glu Gly Gly Thr Ala Trp Leu Glu 435 440 445TrpArg His Val Gln Pro Thr Leu Asp Leu Met Glu Ala Glu Leu Arg 450 455460Lys Ser Gln Val Leu Phe Ser Val Thr Arg Gly Ala Arg His GlyGlu465 470 475 480Leu Glu Leu Asp Ile Pro Gly Ala Gln Ala Arg LysMet Phe Thr Leu 485 490 495Leu Asp Val Val Asn Arg Lys Ala Arg PheIle His Asp Gly Ser Glu 500 505 510Asp Thr Ser Asp Gln Leu Val LeuGlu Val Ser Val Thr Ala Arg Val 515 520 525Pro Met Pro Ser Cys LeuArg Arg Gly Gln Thr Tyr Leu Leu Pro Ile 530 535 540Gln Val Asn ProVal Asn Asp Pro Pro His Ile Ile Phe Pro His Gly545 550 555 560SerLeu Met Val Ile Leu Glu His Thr Gln Lys Pro Leu Gly Pro Glu 565 570575Val Phe Gln Ala Tyr Asp Pro Asp Ser Ala Cys Glu Gly Leu Thr Phe580 585 590Gln Val Leu Gly Thr Ser Ser Gly Leu Pro Val Glu Arg ArgAsp Gln 595 600 605Pro Gly Glu Pro Ala Thr Glu Phe Ser Cys Arg GluLeu Glu Ala Gly 610 615 620Ser Leu Val Tyr Val His Arg Gly Gly ProAla Gln Asp Leu Thr Phe625 630 635 640Arg Val Ser Asp Gly Leu GlnAla Ser Pro Pro Ala Thr Leu Lys Val 645 650 655Val Ala Ile Arg ProAla Ile Gln Ile His Arg Ser Thr Gly Leu Arg 660 665 670Leu Ala GlnGly Ser Ala Met Pro Ile Leu Pro Ala Asn Leu Ser Val 675 680 685GluThr Asn Ala Val Gly Gln Asp Val Ser Val Leu Phe Arg Val Thr 690 695700Gly Ala Leu Gln Phe Gly Glu Leu Gln Lys Gln Gly Ala Gly GlyVal705 710 715 720Glu Gly Ala Glu Trp Trp Ala Thr Gln Ala Phe HisGln Arg Asp Val 725 730 735Glu Gln Gly Arg Val Arg Tyr Leu Ser ThrAsp Pro Gln His His Ala 740 745 750Tyr Asp Thr Val Glu Asn Leu AlaLeu Glu Val Gln Val Gly Gln Glu 755 760 765Ile Leu Ser Asn Leu SerPhe Pro Val Thr Ile Gln Arg Ala Thr Val 770 775 780Trp Met Leu ArgLeu Glu Pro Leu His Thr Gln Asn Thr Gln Gln Glu785 790 795 800ThrLeu Thr Thr Ala His Leu Glu Ala Thr Leu Glu Glu Ala Gly Pro 805 810815Ser Pro Pro Thr Phe His Tyr Glu Val Val Gln Ala Pro Arg Lys Gly820 825 830Asn Leu Gln Leu Gln Gly Thr Arg Leu Ser Asp Gly Gln GlyPhe Thr 835 840 845Gln Asp Asp Ile Gln Ala Gly Arg Val Thr Tyr GlyAla Thr Ala Arg 850 855 860Ala Ser Glu Ala Val Glu Asp Thr Phe ArgPhe Arg Val Thr Ala Pro865 870 875 880Pro Tyr Phe Ser Pro Leu TyrThr Phe Pro Ile His Ile Gly Gly Asp 885 890 895Pro Asp Ala Pro ValLeu Thr Asn Val Leu Leu Val Val Pro Glu Gly 900 905 910Gly Glu GlyVal Leu Ser Ala Asp His Leu Phe Val Lys Ser Leu Asn 915 920 925SerAla Ser Tyr Leu Tyr Glu Val Met Glu Arg Pro Arg His Gly Arg 930 935940Leu Ala Trp Arg Gly Thr Gln Asp Lys Thr Thr Met Val Thr SerPhe945 950 955 960Thr Asn Glu Asp Leu Leu Arg Gly Arg Leu Val TyrGln His Asp Asp 965 970 975Ser Glu Thr Thr Glu Asp Asp Ile Pro PheVal Ala Thr Arg Gln Gly 980 985 990Glu Ser Ser Gly Asp Met Ala TrpGlu Glu Val Arg Gly Val Phe Arg 995 1000 1005Val Ala Ile Gln ProVal Asn Asp His Ala Pro Val Gln Thr Ile 1010 1015 1020Ser Arg IlePhe His Val Ala Arg Gly Gly Arg Arg Leu Leu Thr 1025 1030 1035ThrAsp Asp Val Ala Phe Ser Asp Ala Asp Ser Gly Phe Ala Asp 1040 10451050Ala Gln Leu Val Leu Thr Arg Lys Asp Leu Leu Phe Gly Ser Ile1055 1060 1065Val Ala Val Asp Glu Pro Thr Arg Pro Ile Tyr Arg PheThr Gln 1070 1075 1080Glu Asp Leu Arg Lys Arg Arg Val Leu Phe ValHis Ser Gly Ala 1085 1090 1095Asp Arg Gly Trp Ile Gln Leu Gln ValSer Asp Gly Gln His Gln 1100 1105 1110Ala Thr Ala Leu Leu Glu ValGln Ala Ser Glu Pro Tyr Leu Arg 1115 1120 1125Val Ala Asn Gly SerSer Leu Val Val Pro Gln Gly Gly Gln Gly 1130 1135 1140Thr Ile AspThr Ala Val Leu His Leu Asp Thr Asn Leu Asp Ile 1145 1150 1155ArgSer Gly Asp Glu Val His Tyr His Val Thr Ala Gly Pro Arg 1160 11651170Trp Gly Gln Leu Val Arg Ala Gly Gln Pro Ala Thr Ala Phe Ser1175 1180 1185Gln Gln Asp Leu Leu Asp Gly Ala Val Leu Tyr Ser HisAsn Gly 1190 1195 1200Ser Leu Ser Pro Arg Asp Thr Met Ala Phe SerVal Glu Ala Gly 1205 1210 1215Pro Val His Thr Asp Ala Thr Leu GlnVal Thr Ile Ala Leu Glu 1220 1225 1230Gly Pro Leu Ala Pro Leu LysLeu Val Arg His Lys Lys Ile Tyr 1235 1240 1245Val Phe Gln Gly GluAla Ala Glu Ile Arg Arg Asp Gln Leu Glu 1250 1255 1260Ala Ala GlnGlu Ala Val Pro Pro Ala Asp Ile Val Phe Ser Val 1265 1270 1275LysSer Pro Pro Ser Ala Gly Tyr Leu Val Met Val Ser Arg Gly 1280 12851290Ala Leu Ala Asp Glu Pro Pro Ser Leu Asp Pro Val Gln Ser Phe1295 1300 1305Ser Gln Glu Ala Val Asp Thr Gly Arg Val Leu Tyr LeuHis Ser 1310 1315 1320Arg Pro Glu Ala Trp Ser Asp Ala Phe Ser LeuAsp Val Ala Ser 1325 1330 1335Gly Leu Gly Ala Pro Leu Glu Gly ValLeu Val Glu Leu Glu Val 1340 1345 1350Leu Pro Ala Ala Ile Pro LeuGlu Ala Gln Asn Phe Ser Val Pro 1355 1360 1365Glu Gly Gly Ser LeuThr Leu Ala Pro Pro Leu Leu Arg Val Ser 1370 1375 1380Gly Pro TyrPhe Pro Thr Leu Leu Gly Leu Ser Leu Gln Val Leu 1385 1390 1395GluPro Pro Gln His Gly Ala Leu Gln Lys Glu Asp Gly Pro Gln 1400 14051410Ala Arg Thr Leu Ser Ala Phe Ser Trp Arg Met Val Glu Glu Gln1415 1420 1425Leu Ile Arg Tyr Val His Asp Gly Ser Glu Thr Leu ThrAsp Ser 1430 1435 1440Phe Val Leu Met Ala Asn Ala Ser Glu Met AspArg Gln Ser His 1445 1450 1455Pro Val Ala Phe Thr Val Thr Val LeuPro Val Asn Asp Gln Pro 1460 1465 1470Pro Ile Leu Thr Thr Asn ThrGly Leu Gln Met Trp Glu Gly Ala 1475 1480 1485Thr Ala Pro Ile ProAla Glu Ala Leu Arg Ser Thr Asp Gly Asp 1490 1495 1500Ser Gly SerGlu Asp Leu Val Tyr Thr Ile Glu Gln Pro Ser Asn 1505 1510 1515GlyArg Val Val Leu Arg Gly Ala Pro Gly Thr Glu Val Arg Ser 1520 15251530Phe Thr Gln Ala Gln Leu Asp Gly Gly Leu Val Leu Phe Ser His1535 1540 1545Arg Gly Thr Leu Asp Gly Gly Phe Arg Phe Arg Leu SerAsp Gly 1550 1555 1560Glu His Thr Ser Pro Gly His Phe Phe Arg ValThr Ala Gln Lys 1565 1570 1575Gln Val Leu Leu Ser Leu Lys Gly SerGln Thr Leu Thr Val Cys 1580 1585 1590Pro Gly Ser Val Gln Pro LeuSer Ser Gln Thr Leu Arg Ala Ser 1595 1600 1605Ser Ser Ala Gly ThrAsp Pro Gln Leu Leu Leu Tyr Arg Val Val 1610 1615 1620Arg Gly ProGln Leu Gly Arg Leu Phe His Ala Gln Gln Asp Ser 1625 1630 1635ThrGly Glu Ala Leu Val Asn Phe Thr Gln Ala Glu Val Tyr Ala 1640 16451650Gly Asn Ile Leu Tyr Glu His Glu Met Pro Pro Glu Pro Phe Trp1655 1660 1665Glu Ala His Asp

Thr Leu Glu Leu Gln Leu Ser Ser Pro Pro Ala 1670 1675 1680Arg AspVal Ala Ala Thr Leu Ala Val Ala Val Ser Phe Glu Ala 1685 16901695Ala Cys Pro Gln Arg Pro Ser His Leu Trp Lys Asn Lys Gly Leu1700 1705 1710Trp Val Pro Glu Gly Gln Arg Ala Arg Ile Thr Val AlaAla Leu 1715 1720 1725Asp Ala Ser Asn Leu Leu Ala Ser Val Pro SerPro Gln Arg Ser 1730 1735 1740Glu His Asp Val Leu Phe Gln Val ThrGln Phe Pro Ser Arg Gly 1745 1750 1755Gln Leu Leu Val Ser Glu GluPro Leu His Ala Gly Gln Pro His 1760 1765 1770Phe Leu Gln Ser GlnLeu Ala Ala Gly Gln Leu Val Tyr Ala His 1775 1780 1785Gly Gly GlyGly Thr Gln Gln Asp Gly Phe His Phe Arg Ala His 1790 1795 1800LeuGln Gly Pro Ala Gly Ala Ser Val Ala Gly Pro Gln Thr Ser 1805 18101815Glu Ala Phe Ala Ile Thr Val Arg Asp Val Asn Glu Arg Pro Pro1820 1825 1830Gln Pro Gln Ala Ser Val Pro Leu Arg Leu Thr Arg GlySer Arg 1835 1840 1845Ala Pro Ile Ser Arg Ala Gln Leu Ser Val ValAsp Pro Asp Ser 1850 1855 1860Ala Pro Gly Glu Ile Glu Tyr Glu ValGln Arg Ala Pro His Asn 1865 1870 1875Gly Phe Leu Ser Leu Val GlyGly Gly Leu Gly Pro Val Thr Arg 1880 1885 1890Phe Thr Gln Ala AspVal Asp Ser Gly Arg Leu Ala Phe Val Ala 1895 1900 1905Asn Gly SerSer Val Ala Gly Ile Phe Gln Leu Ser Met Ser Asp 1910 1915 1920GlyAla Ser Pro Pro Leu Pro Met Ser Leu Ala Val Asp Ile Leu 1925 19301935Pro Ser Ala Ile Glu Val Gln Leu Arg Ala Pro Leu Glu Val Pro1940 1945 1950Gln Ala Leu Gly Arg Ser Ser Leu Ser Gln Gln Gln LeuArg Val 1955 1960 1965Val Ser Asp Arg Glu Glu Pro Glu Ala Ala TyrArg Leu Ile Gln 1970 1975 1980Gly Pro Gln Tyr Gly His Leu Leu ValGly Gly Arg Pro Thr Ser 1985 1990 1995Ala Phe Ser Gln Phe Gln IleAsp Gln Gly Glu Val Val Phe Ala 2000 2005 2010Phe Thr Asn Phe SerSer Ser His Asp His Phe Arg Val Leu Ala 2015 2020 2025Leu Ala ArgGly Val Asn Ala Ser Ala Val Val Asn Val Thr Val 2030 2035 2040ArgAla Leu Leu His Val Trp Ala Gly Gly Pro Trp Pro Gln Gly 2045 20502055Ala Thr Leu Arg Leu Asp Pro Thr Val Leu Asp Ala Gly Glu Leu2060 2065 2070Ala Asn Arg Thr Gly Ser Val Pro Arg Phe Arg Leu LeuGlu Gly 2075 2080 2085Pro Arg His Gly Arg Val Val Arg Val Pro ArgAla Arg Thr Glu 2090 2095 2100Pro Gly Gly Ser Gln Leu Val Glu GlnPhe Thr Gln Gln Asp Leu 2105 2110 2115Glu Asp Gly Arg Leu Gly LeuGlu Val Gly Arg Pro Glu Gly Arg 2120 2125 2130Ala Pro Gly Pro AlaGly Asp Ser Leu Thr Leu Glu Leu Trp Ala 2135 2140 2145Gln Gly ValPro Pro Ala Val Ala Ser Leu Asp Phe Ala Thr Glu 2150 2155 2160ProTyr Asn Ala Ala Arg Pro Tyr Ser Val Ala Leu Leu Ser Val 2165 21702175Pro Glu Ala Ala Arg Thr Glu Ala Gly Lys Pro Glu Ser Ser Thr2180 2185 2190Pro Thr Gly Glu Pro Gly Pro Met Ala Ser Ser Pro GluPro Ala 2195 2200 2205Val Ala Lys Gly Gly Phe Leu Ser Phe Leu GluAla Asn Met Phe 2210 2215 2220Ser Val Ile Ile Pro Met Cys Leu ValLeu Leu Leu Leu Ala Leu 2225 2230 2235Ile Leu Pro Leu Leu Phe TyrLeu Arg Lys Arg Asn Lys Thr Gly 2240 2245 2250Lys His Asp Val GlnVal Leu Thr Ala Lys Pro Arg Asn Gly Leu 2255 2260 2265Ala Gly AspThr Glu Thr Phe Arg Lys Val Glu Pro Gly Gln Ala 2270 2275 2280IlePro Leu Thr Ala Val Pro Gly Gln Gly Pro Pro Pro Gly Gly 2285 22902295Gln Pro Asp Pro Glu Leu Leu Gln Phe Cys Arg Thr Pro Asn Pro2300 2305 2310Ala Leu Lys Asn Gly Gln Tyr Trp Val 231523201991210PRThom*o sapiens 199Met Arg Pro Ser Gly Thr Ala Gly AlaAla Leu Leu Ala Leu Leu Ala1 5 10 15Ala Leu Cys Pro Ala Ser Arg AlaLeu Glu Glu Lys Lys Val Cys Gln 20 25 30Gly Thr Ser Asn Lys Leu ThrGln Leu Gly Thr Phe Glu Asp His Phe 35 40 45Leu Ser Leu Gln Arg MetPhe Asn Asn Cys Glu Val Val Leu Gly Asn 50 55 60Leu Glu Ile Thr TyrVal Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys65 70 75 80Thr Ile GlnGlu Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val 85 90 95Glu ArgIle Pro Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr 100 105110Tyr Glu Asn Ser Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn115 120 125Lys Thr Gly Leu Lys Glu Leu Pro Met Arg Asn Leu Gln GluIle Leu 130 135 140His Gly Ala Val Arg Phe Ser Asn Asn Pro Ala LeuCys Asn Val Glu145 150 155 160Ser Ile Gln Trp Arg Asp Ile Val SerSer Asp Phe Leu Ser Asn Met 165 170 175Ser Met Asp Phe Gln Asn HisLeu Gly Ser Cys Gln Lys Cys Asp Pro 180 185 190Ser Cys Pro Asn GlySer Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln 195 200 205Lys Leu ThrLys Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg 210 215 220GlyLys Ser Pro Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys225 230235 240Thr Gly Pro Arg Glu Ser Asp Cys Leu Val Cys Arg Lys Phe ArgAsp 245 250 255Glu Ala Thr Cys Lys Asp Thr Cys Pro Pro Leu Met LeuTyr Asn Pro 260 265 270Thr Thr Tyr Gln Met Asp Val Asn Pro Glu GlyLys Tyr Ser Phe Gly 275 280 285Ala Thr Cys Val Lys Lys Cys Pro ArgAsn Tyr Val Val Thr Asp His 290 295 300Gly Ser Cys Val Arg Ala CysGly Ala Asp Ser Tyr Glu Met Glu Glu305 310 315 320Asp Gly Val ArgLys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val 325 330 335Cys AsnGly Ile Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn 340 345350Ala Thr Asn Ile Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp355 360 365Leu His Ile Leu Pro Val Ala Phe Arg Gly Asp Ser Phe ThrHis Thr 370 375 380Pro Pro Leu Asp Pro Gln Glu Leu Asp Ile Leu LysThr Val Lys Glu385 390 395 400Ile Thr Gly Phe Leu Leu Ile Gln AlaTrp Pro Glu Asn Arg Thr Asp 405 410 415Leu His Ala Phe Glu Asn LeuGlu Ile Ile Arg Gly Arg Thr Lys Gln 420 425 430His Gly Gln Phe SerLeu Ala Val Val Ser Leu Asn Ile Thr Ser Leu 435 440 445Gly Leu ArgSer Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser 450 455 460GlyAsn Lys Asn Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu465 470475 480Phe Gly Thr Ser Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg GlyGlu 485 490 495Asn Ser Cys Lys Ala Thr Gly Gln Val Cys His Ala LeuCys Ser Pro 500 505 510Glu Gly Cys Trp Gly Pro Glu Pro Arg Asp CysVal Ser Cys Arg Asn 515 520 525Val Ser Arg Gly Arg Glu Cys Val AspLys Cys Asn Leu Leu Glu Gly 530 535 540Glu Pro Arg Glu Phe Val GluAsn Ser Glu Cys Ile Gln Cys His Pro545 550 555 560Glu Cys Leu ProGln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro 565 570 575Asp AsnCys Ile Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val 580 585590Lys Thr Cys Pro Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp595 600 605Lys Tyr Ala Asp Ala Gly His Val Cys His Leu Cys His ProAsn Cys 610 615 620Thr Tyr Gly Cys Thr Gly Pro Gly Leu Glu Gly CysPro Thr Asn Gly625 630 635 640Pro Lys Ile Pro Ser Ile Ala Thr GlyMet Val Gly Ala Leu Leu Leu 645 650 655Leu Leu Val Val Ala Leu GlyIle Gly Leu Phe Met Arg Arg Arg His 660 665 670Ile Val Arg Lys ArgThr Leu Arg Arg Leu Leu Gln Glu Arg Glu Leu 675 680 685Val Glu ProLeu Thr Pro Ser Gly Glu Ala Pro Asn Gln Ala Leu Leu 690 695 700ArgIle Leu Lys Glu Thr Glu Phe Lys Lys Ile Lys Val Leu Gly Ser705 710715 720Gly Ala Phe Gly Thr Val Tyr Lys Gly Leu Trp Ile Pro Glu GlyGlu 725 730 735Lys Val Lys Ile Pro Val Ala Ile Lys Glu Leu Arg GluAla Thr Ser 740 745 750Pro Lys Ala Asn Lys Glu Ile Leu Asp Glu AlaTyr Val Met Ala Ser 755 760 765Val Asp Asn Pro His Val Cys Arg LeuLeu Gly Ile Cys Leu Thr Ser 770 775 780Thr Val Gln Leu Ile Thr GlnLeu Met Pro Phe Gly Cys Leu Leu Asp785 790 795 800Tyr Val Arg GluHis Lys Asp Asn Ile Gly Ser Gln Tyr Leu Leu Asn 805 810 815Trp CysVal Gln Ile Ala Lys Gly Met Asn Tyr Leu Glu Asp Arg Arg 820 825830Leu Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro835 840 845Gln His Val Lys Ile Thr Asp Phe Gly Leu Ala Lys Leu LeuGly Ala 850 855 860Glu Glu Lys Glu Tyr His Ala Glu Gly Gly Lys ValPro Ile Lys Trp865 870 875 880Met Ala Leu Glu Ser Ile Leu His ArgIle Tyr Thr His Gln Ser Asp 885 890 895Val Trp Ser Tyr Gly Val ThrVal Trp Glu Leu Met Thr Phe Gly Ser 900 905 910Lys Pro Tyr Asp GlyIle Pro Ala Ser Glu Ile Ser Ser Ile Leu Glu 915 920 925Lys Gly GluArg Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr 930 935 940MetIle Met Val Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys945 950955 960Phe Arg Glu Leu Ile Ile Glu Phe Ser Lys Met Ala Arg Asp ProGln 965 970 975Arg Tyr Leu Val Ile Gln Gly Asp Glu Arg Met His LeuPro Ser Pro 980 985 990Thr Asp Ser Asn Phe Tyr Arg Ala Leu Met AspGlu Glu Asp Met Asp 995 1000 1005Asp Val Val Asp Ala Asp Glu TyrLeu Ile Pro Gln Gln Gly Phe 1010 1015 1020Phe Ser Ser Pro Ser ThrSer Arg Thr Pro Leu Leu Ser Ser Leu 1025 1030 1035Ser Ala Thr SerAsn Asn Ser Thr Val Ala Cys Ile Asp Arg Asn 1040 1045 1050Gly LeuGln Ser Cys Pro Ile Lys Glu Asp Ser Phe Leu Gln Arg 1055 10601065Tyr Ser Ser Asp Pro Thr Gly Ala Leu Thr Glu Asp Ser Ile Asp1070 1075 1080Asp Thr Phe Leu Pro Val Pro Glu Tyr Ile Asn Gln SerVal Pro 1085 1090 1095Lys Arg Pro Ala Gly Ser Val Gln Asn Pro ValTyr His Asn Gln 1100 1105 1110Pro Leu Asn Pro Ala Pro Ser Arg AspPro His Tyr Gln Asp Pro 1115 1120 1125His Ser Thr Ala Val Gly AsnPro Glu Tyr Leu Asn Thr Val Gln 1130 1135 1140Pro Thr Cys Val AsnSer Thr Phe Asp Ser Pro Ala His Trp Ala 1145 1150 1155Gln Lys GlySer His Gln Ile Ser Leu Asp Asn Pro Asp Tyr Gln 1160 1165 1170GlnAsp Phe Phe Pro Lys Glu Ala Lys Pro Asn Gly Ile Phe Lys 1175 11801185Gly Ser Thr Ala Glu Asn Ala Glu Tyr Leu Arg Val Ala Pro Gln1190 1195 1200Ser Ser Glu Phe Ile Gly Ala 1205 1210200556PRThom*osapiens 200Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu ThrPro Met1 5 10 15Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val GluGlu Gly Asp 20 25 30Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser AspGly Pro Thr Gln 35 40 45Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu LysPro Phe Leu Lys Leu 50 55 60Ser Leu Gly Leu Pro Gly Leu Gly Ile HisMet Arg Pro Leu Ala Ile65 70 75 80Trp Leu Phe Ile Phe Asn Val SerGln Gln Met Gly Gly Phe Tyr Leu 85 90 95Cys Gln Pro Gly Pro Pro SerGlu Lys Ala Trp Gln Pro Gly Trp Thr 100 105 110Val Asn Val Glu GlySer Gly Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125Leu Gly GlyLeu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140SerSer Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala145 150155 160Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu ProPro 165 170 175Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu ThrMet Ala Pro 180 185 190Gly Ser Thr Leu Trp Leu Ser Cys Gly Val ProPro Asp Ser Val Ser 195 200 205Arg Gly Pro Leu Ser Trp Thr His ValHis Pro Lys Gly Pro Lys Ser 210 215 220Leu Leu Ser Leu Glu Leu LysAsp Asp Arg Pro Ala Arg Asp Met Trp225 230 235 240Val Met Glu ThrGly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255Gly LysTyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265270Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly275 280 285Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile PheCys Leu 290 295 300Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg AlaLeu Val Leu Arg305 310 315 320Arg Lys Arg Lys Arg Met Thr Asp ProThr Arg Arg Phe Phe Lys Val 325 330 335Thr Pro Pro Pro Gly Ser GlyPro Gln Asn Gln Tyr Gly Asn Val Leu 340 345 350Ser Leu Pro Thr ProThr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365Ala Gly LeuGly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380ValGln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly385 390395 400Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser GluGlu 405 410 415Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly GlnAsp Gln Leu 420 425 430Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro GluAsp Glu Pro Leu Gly 435 440 445Pro Glu Asp Glu Asp Ser Phe Ser AsnAla Glu Ser Tyr Glu Asn Glu 450 455 460Asp Glu Glu Leu Thr Gln ProVal Ala Arg Thr Met Asp Phe Leu Ser465 470 475 480Pro His Gly SerAla Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly 485 490 495Ser GlnSer Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln 500 505510Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala515 520 525Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp ProAla Trp 530 535 540Gly Gly Gly Gly Arg Met Gly Thr Trp Ser ThrArg545 550 555201297PRThom*o sapiens 201Met Thr Thr Pro Arg Asn SerVal Asn Gly Thr Phe Pro Ala Glu Pro1 5 10 15Met Lys Gly Pro Ile AlaMet Gln Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30Arg Met

Ser Ser Leu Val Gly Pro Thr Gln Ser Phe Phe Met Arg Glu 35 40 45SerLys Thr Leu Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 5560Ala Leu Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile6570 75 80Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr IleIle 85 90 95Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg LysCys Leu 100 105 110Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser LeuPhe Ala Ala Ile 115 120 125Ser Gly Met Ile Leu Ser Ile Met Asp IleLeu Asn Ile Lys Ile Ser 130 135 140His Phe Leu Lys Met Glu Ser LeuAsn Phe Ile Arg Ala His Thr Pro145 150 155 160Tyr Ile Asn Ile TyrAsn Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175Ser Pro SerThr Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190IleLeu Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200205Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys210 215 220Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu GlnThr Ile225 230 235 240Glu Ile Lys Glu Glu Val Val Gly Leu Thr GluThr Ser Ser Gln Pro 245 250 255Lys Asn Glu Glu Asp Ile Glu Ile IlePro Ile Gln Glu Glu Glu Glu 260 265 270Glu Glu Thr Glu Thr Asn PhePro Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285Ser Pro Ile Glu AsnAsp Ser Ser Pro 290 295202364PRThom*o sapiens 202Met Pro Leu Leu LeuLeu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala1 5 10 15Met Asp Pro AsnPhe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln 20 25 30Glu Gly LeuCys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro 35 40 45Tyr TyrAsp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly 50 55 60AlaIle Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln65 70 7580Glu Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro85 90 95Ser Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg ArgAsp 100 105 110Asn Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser ThrLys Tyr Ser 115 120 125Tyr Lys Ser Pro Gln Leu Ser Val His Val ThrAsp Leu Thr His Arg 130 135 140Pro Lys Ile Leu Ile Pro Gly Thr LeuGlu Pro Gly His Ser Lys Asn145 150 155 160Leu Thr Cys Ser Val SerTrp Ala Cys Glu Gln Gly Thr Pro Pro Ile 165 170 175Phe Ser Trp LeuSer Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr 180 185 190His SerSer Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr 195 200205Asn Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu210 215 220Arg Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn ProThr Thr225 230 235 240Gly Ile Phe Pro Gly Asp Gly Ser Gly Lys GlnGlu Thr Arg Ala Gly 245 250 255Val Val His Gly Ala Ile Gly Gly AlaGly Val Thr Ala Leu Leu Ala 260 265 270Leu Cys Leu Cys Leu Ile PhePhe Ile Val Lys Thr His Arg Arg Lys 275 280 285Ala Ala Arg Thr AlaVal Gly Arg Asn Asp Thr His Pro Thr Thr Gly 290 295 300Ser Ala SerPro Lys His Gln Lys Lys Ser Lys Leu His Gly Pro Thr305 310 315320Glu Thr Ser Ser Cys Ser Gly Ala Ala Pro Thr Val Glu Met Asp Glu325 330 335Glu Leu His Tyr Ala Ser Leu Asn Phe His Gly Met Asn ProSer Lys 340 345 350Asp Thr Ser Thr Glu Tyr Ser Glu Val Arg Thr Gln355 360203205PRThom*o sapiens 203Met Thr Pro Pro Glu Arg Leu Phe LeuPro Arg Val Cys Gly Thr Thr1 5 10 15Leu His Leu Leu Leu Leu Gly LeuLeu Leu Val Leu Leu Pro Gly Ala 20 25 30Gln Gly Leu Pro Gly Val GlyLeu Thr Pro Ser Ala Ala Gln Thr Ala 35 40 45Arg Gln His Pro Lys MetHis Leu Ala His Ser Thr Leu Lys Pro Ala 50 55 60Ala His Leu Ile GlyAsp Pro Ser Lys Gln Asn Ser Leu Leu Trp Arg65 70 75 80Ala Asn ThrAsp Arg Ala Phe Leu Gln Asp Gly Phe Ser Leu Ser Asn 85 90 95Asn SerLeu Leu Val Pro Thr Ser Gly Ile Tyr Phe Val Tyr Ser Gln 100 105110Val Val Phe Ser Gly Lys Ala Tyr Ser Pro Lys Ala Thr Ser Ser Pro115 120 125Leu Tyr Leu Ala His Glu Val Gln Leu Phe Ser Ser Gln TyrPro Phe 130 135 140His Val Pro Leu Leu Ser Ser Gln Lys Met Val TyrPro Gly Leu Gln145 150 155 160Glu Pro Trp Leu His Ser Met Tyr HisGly Ala Ala Phe Gln Leu Thr 165 170 175Gln Gly Asp Gln Leu Ser ThrHis Thr Asp Gly Ile Pro His Leu Val 180 185 190Leu Ser Pro Ser ThrVal Phe Phe Gly Ala Phe Ala Leu 195 200 205204233PRThom*o sapiens204Met Ser Thr Glu Ser Met Ile Arg Asp Val Glu Leu Ala Glu Glu Ala15 10 15Leu Pro Lys Lys Thr Gly Gly Pro Gln Gly Ser Arg Arg Cys LeuPhe 20 25 30Leu Ser Leu Phe Ser Phe Leu Ile Val Ala Gly Ala Thr ThrLeu Phe 35 40 45Cys Leu Leu His Phe Gly Val Ile Gly Pro Gln Arg GluGlu Phe Pro 50 55 60Arg Asp Leu Ser Leu Ile Ser Pro Leu Ala Gln AlaVal Arg Ser Ser65 70 75 80Ser Arg Thr Pro Ser Asp Lys Pro Val AlaHis Val Val Ala Asn Pro 85 90 95Gln Ala Glu Gly Gln Leu Gln Trp LeuAsn Arg Arg Ala Asn Ala Leu 100 105 110Leu Ala Asn Gly Val Glu LeuArg Asp Asn Gln Leu Val Val Pro Ser 115 120 125Glu Gly Leu Tyr LeuIle Tyr Ser Gln Val Leu Phe Lys Gly Gln Gly 130 135 140Cys Pro SerThr His Val Leu Leu Thr His Thr Ile Ser Arg Ile Ala145 150 155160Val Ser Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala Ile Lys Ser Pro165 170 175Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys Pro TrpTyr Glu 180 185 190Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu LysGly Asp Arg Leu 195 200 205Ser Ala Glu Ile Asn Arg Pro Asp Tyr LeuAsp Phe Ala Glu Ser Gly 210 215 220Gln Val Tyr Phe Gly Ile Ile AlaLeu225 230205244PRThom*o sapiens 205Met Gly Ala Leu Gly Leu Glu GlyArg Gly Gly Arg Leu Gln Gly Arg1 5 10 15Gly Ser Leu Leu Leu Ala ValAla Gly Ala Thr Ser Leu Val Thr Leu 20 25 30Leu Leu Ala Val Pro IleThr Val Leu Ala Val Leu Ala Leu Val Pro 35 40 45Gln Asp Gln Gly GlyLeu Val Thr Glu Thr Ala Asp Pro Gly Ala Gln 50 55 60Ala Gln Gln GlyLeu Gly Phe Gln Lys Leu Pro Glu Glu Glu Pro Glu65 70 75 80Thr AspLeu Ser Pro Gly Leu Pro Ala Ala His Leu Ile Gly Ala Pro 85 90 95LeuLys Gly Gln Gly Leu Gly Trp Glu Thr Thr Lys Glu Gln Ala Phe 100 105110Leu Thr Ser Gly Thr Gln Phe Ser Asp Ala Glu Gly Leu Ala Leu Pro115 120 125Gln Asp Gly Leu Tyr Tyr Leu Tyr Cys Leu Val Gly Tyr ArgGly Arg 130 135 140Ala Pro Pro Gly Gly Gly Asp Pro Gln Gly Arg SerVal Thr Leu Arg145 150 155 160Ser Ser Leu Tyr Arg Ala Gly Gly AlaTyr Gly Pro Gly Thr Pro Glu 165 170 175Leu Leu Leu Glu Gly Ala GluThr Val Thr Pro Val Leu Asp Pro Ala 180 185 190Arg Arg Gln Gly TyrGly Pro Leu Trp Tyr Thr Ser Val Gly Phe Gly 195 200 205Gly Leu ValGln Leu Arg Arg Gly Glu Arg Val Tyr Val Asn Ile Ser 210 215 220HisPro Asp Met Val Asp Phe Ala Arg Gly Lys Thr Phe Phe Gly Ala225 230235 240Val Met Val Gly206183PRThom*o sapiens 206Met Glu Arg Val GlnPro Leu Glu Glu Asn Val Gly Asn Ala Ala Arg1 5 10 15Pro Arg Phe GluArg Asn Lys Leu Leu Leu Val Ala Ser Val Ile Gln 20 25 30Gly Leu GlyLeu Leu Leu Cys Phe Thr Tyr Ile Cys Leu His Phe Ser 35 40 45Ala LeuGln Val Ser His Arg Tyr Pro Arg Ile Gln Ser Ile Lys Val 50 55 60GlnPhe Thr Glu Tyr Lys Lys Glu Lys Gly Phe Ile Leu Thr Ser Gln65 70 7580Lys Glu Asp Glu Ile Met Lys Val Gln Asn Asn Ser Val Ile Ile Asn85 90 95Cys Asp Gly Phe Tyr Leu Ile Ser Leu Lys Gly Tyr Phe Ser GlnGlu 100 105 110Val Asn Ile Ser Leu His Tyr Gln Lys Asp Glu Glu ProLeu Phe Gln 115 120 125Leu Lys Lys Val Arg Ser Val Asn Ser Leu MetVal Ala Ser Leu Thr 130 135 140Tyr Lys Asp Lys Val Tyr Leu Asn ValThr Thr Asp Asn Thr Ser Leu145 150 155 160Asp Asp Phe His Val AsnGly Gly Glu Leu Ile Leu Ile His Gln Asn 165 170 175Pro Gly Glu PheCys Val Leu 180207261PRThom*o sapiens 207Met Ile Glu Thr Tyr Asn GlnThr Ser Pro Arg Ser Ala Ala Thr Gly1 5 10 15Leu Pro Ile Ser Met LysIle Phe Met Tyr Leu Leu Thr Val Phe Leu 20 25 30Ile Thr Gln Met IleGly Ser Ala Leu Phe Ala Val Tyr Leu His Arg 35 40 45Arg Leu Asp LysIle Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val 50 55 60Phe Met LysThr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser65 70 75 80LeuLeu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys 85 9095Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu100 105 110Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His ValIle Ser 115 120 125Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln TrpAla Glu Lys Gly 130 135 140Tyr Tyr Thr Met Ser Asn Asn Leu Val ThrLeu Glu Asn Gly Lys Gln145 150 155 160Leu Thr Val Lys Arg Gln GlyLeu Tyr Tyr Ile Tyr Ala Gln Val Thr 165 170 175Phe Cys Ser Asn ArgGlu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser 180 185 190Leu Cys LeuLys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala 195 200 205AlaAsn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His 210 215220Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe ValAsn225 230 235 240Val Thr Asp Pro Ser Gln Val Ser His Gly Thr GlyPhe Thr Ser Phe 245 250 255Gly Leu Leu Lys Leu 260208281PRThom*osapiens 208Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr TrpVal Asp1 5 10 15Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr ValLeu Pro Cys 20 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg ArgPro Pro Pro Pro 35 40 45Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro ProPro Pro Pro Leu Pro 50 55 60Pro Leu Pro Leu Pro Pro Leu Lys Lys ArgGly Asn His Ser Thr Gly65 70 75 80Leu Cys Leu Leu Val Met Phe PheMet Val Leu Val Ala Leu Val Gly 85 90 95Leu Gly Leu Gly Met Phe GlnLeu Phe His Leu Gln Lys Glu Leu Ala 100 105 110Glu Leu Arg Glu SerThr Ser Gln Met His Thr Ala Ser Ser Leu Glu 115 120 125Lys Gln IleGly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg 130 135 140LysVal Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu145 150155 160Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val LysTyr 165 170 175Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu TyrPhe Val Tyr 180 185 190Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys AsnAsn Leu Pro Leu Ser 195 200 205His Lys Val Tyr Met Arg Asn Ser LysTyr Pro Gln Asp Leu Val Met 210 215 220Met Glu Gly Lys Met Met SerTyr Cys Thr Thr Gly Gln Met Trp Ala225 230 235 240Arg Ser Ser TyrLeu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His 245 250 255Leu TyrVal Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser 260 265270Gln Thr Phe Phe Gly Leu Tyr Lys Leu 275 280209193PRThom*o sapiens209Met Pro Glu Glu Gly Ser Gly Cys Ser Val Arg Arg Arg Pro Tyr Gly15 10 15Cys Val Leu Arg Ala Ala Leu Val Pro Leu Val Ala Gly Leu ValIle 20 25 30Cys Leu Val Val Cys Ile Gln Arg Phe Ala Gln Ala Gln GlnGln Leu 35 40 45Pro Leu Glu Ser Leu Gly Trp Asp Val Ala Glu Leu GlnLeu Asn His 50 55 60Thr Gly Pro Gln Gln Asp Pro Arg Leu Tyr Trp GlnGly Gly Pro Ala65 70 75 80Leu Gly Arg Ser Phe Leu His Gly Pro GluLeu Asp Lys Gly Gln Leu 85 90 95Arg Ile His Arg Asp Gly Ile Tyr MetVal His Ile Gln Val Thr Leu 100 105 110Ala Ile Cys Ser Ser Thr ThrAla Ser Arg His His Pro Thr Thr Leu 115 120 125Ala Val Gly Ile CysSer Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg 130 135 140Leu Ser PheHis Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro145 150 155160Leu Ala Arg Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu165 170 175Pro Ser Arg Asn Thr Asp Glu Thr Phe Phe Gly Val Gln TrpVal Arg 180 185 190Pro210234PRThom*o sapiens 210Met Asp Pro Gly LeuGln Gln Ala Leu Asn Gly Met Ala Pro Pro Gly1 5 10 15Asp Thr Ala MetHis Val Pro Ala Gly Ser Val Ala Ser His Leu Gly 20 25 30Thr Thr SerArg Ser Tyr Phe Tyr Leu Thr Thr Ala Thr Leu Ala Leu 35 40 45Cys LeuVal Phe Thr Val Ala Thr Ile Met Val Leu Val Val Gln Arg 50 55 60ThrAsp Ser Ile Pro Asn Ser Pro Asp Asn Val Pro Leu Lys Gly Gly65 70 7580Asn Cys Ser Glu Asp Leu Leu Cys Ile Leu Lys Arg Ala Pro Phe Lys85 90 95Lys Ser Trp Ala Tyr Leu Gln Val Ala Lys His Leu Asn Lys ThrLys 100 105 110Leu Ser Trp Asn Lys Asp Gly Ile Leu His Gly Val ArgTyr Gln Asp 115 120 125Gly Asn Leu Val Ile Gln Phe Pro Gly Leu TyrPhe Ile Ile Cys Gln 130 135 140Leu Gln Phe Leu Val Gln Cys Pro AsnAsn Ser Val Asp Leu Lys Leu145 150 155 160Glu Leu Leu Ile Asn LysHis Ile Lys Lys Gln Ala Leu Val Thr Val 165 170 175Cys Glu Ser GlyMet Gln Thr Lys His Val Tyr Gln Asn Leu Ser Gln 180 185 190Phe LeuLeu Asp Tyr Leu Gln Val Asn Thr Thr Ile Ser Val Asn Val 195 200205Asp Thr Phe Gln Tyr Ile Asp Thr Ser Thr Phe Pro Leu Glu Asn Val210 215 220Leu Ser Ile Phe Leu

Tyr Ser Asn Ser Asp225 230211254PRThom*o sapiens 211Met Glu Tyr AlaSer Asp Ala Ser Leu Asp Pro Glu Ala Pro Trp Pro1 5 10 15Pro Ala ProArg Ala Arg Ala Cys Arg Val Leu Pro Trp Ala Leu Val 20 25 30Ala GlyLeu Leu Leu Leu Leu Leu Leu Ala Ala Ala Cys Ala Val Phe 35 40 45LeuAla Cys Pro Trp Ala Val Ser Gly Ala Arg Ala Ser Pro Gly Ser 50 5560Ala Ala Ser Pro Arg Leu Arg Glu Gly Pro Glu Leu Ser Pro Asp Asp6570 75 80Pro Ala Gly Leu Leu Asp Leu Arg Gln Gly Met Phe Ala Gln LeuVal 85 90 95Ala Gln Asn Val Leu Leu Ile Asp Gly Pro Leu Ser Trp TyrSer Asp 100 105 110Pro Gly Leu Ala Gly Val Ser Leu Thr Gly Gly LeuSer Tyr Lys Glu 115 120 125Asp Thr Lys Glu Leu Val Val Ala Lys AlaGly Val Tyr Tyr Val Phe 130 135 140Phe Gln Leu Glu Leu Arg Arg ValVal Ala Gly Glu Gly Ser Gly Ser145 150 155 160Val Ser Leu Ala LeuHis Leu Gln Pro Leu Arg Ser Ala Ala Gly Ala 165 170 175Ala Ala LeuAla Leu Thr Val Asp Leu Pro Pro Ala Ser Ser Glu Ala 180 185 190ArgAsn Ser Ala Phe Gly Phe Gln Gly Arg Leu Leu His Leu Ser Ala 195 200205Gly Gln Arg Leu Gly Val His Leu His Thr Glu Ala Arg Ala Arg His210 215 220Ala Trp Gln Leu Thr Gln Gly Ala Thr Val Leu Gly Leu PheArg Val225 230 235 240Thr Pro Glu Ile Pro Ala Gly Leu Pro Ser ProArg Ser Glu 245 250212281PRThom*o sapiens 212Met Ala Met Met Glu ValGln Gly Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val IlePhe Thr Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr ValTyr Phe Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser LysSer Gly Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp AspPro Asn Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 7580Lys Trp Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser85 90 95Glu Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile SerPro 100 105 110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala HisIle Thr Gly 115 120 125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser ProAsn Ser Lys Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn SerTrp Glu Ser Ser Arg Ser Gly145 150 155 160His Ser Phe Leu Ser AsnLeu His Leu Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys GlyPhe Tyr Tyr Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln GluGlu Ile Lys Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200205Tyr Ile Tyr Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys210 215 220Ser Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr GlyLeu Tyr225 230 235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu LysGlu Asn Asp Arg Ile 245 250 255Phe Val Ser Val Thr Asn Glu His LeuIle Asp Met Asp His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe LeuVal Gly 275 280213317PRThom*o sapiens 213Met Arg Arg Ala Ser Arg AspTyr Thr Lys Tyr Leu Arg Gly Ser Glu1 5 10 15Glu Met Gly Gly Gly ProGly Ala Pro His Glu Gly Pro Leu His Ala 20 25 30Pro Pro Pro Pro AlaPro His Gln Pro Pro Ala Ala Ser Arg Ser Met 35 40 45Phe Val Ala LeuLeu Gly Leu Gly Leu Gly Gln Val Val Cys Ser Val 50 55 60Ala Leu PhePhe Tyr Phe Arg Ala Gln Met Asp Pro Asn Arg Ile Ser65 70 75 80GluAsp Gly Thr His Cys Ile Tyr Arg Ile Leu Arg Leu His Glu Asn 85 9095Ala Asp Phe Gln Asp Thr Thr Leu Glu Ser Gln Asp Thr Lys Leu Ile100 105 110Pro Asp Ser Cys Arg Arg Ile Lys Gln Ala Phe Gln Gly AlaVal Gln 115 120 125Lys Glu Leu Gln His Ile Val Gly Ser Gln His IleArg Ala Glu Lys 130 135 140Ala Met Val Asp Gly Ser Trp Leu Asp LeuAla Lys Arg Ser Lys Leu145 150 155 160Glu Ala Gln Pro Phe Ala HisLeu Thr Ile Asn Ala Thr Asp Ile Pro 165 170 175Ser Gly Ser His LysVal Ser Leu Ser Ser Trp Tyr His Asp Arg Gly 180 185 190Trp Ala LysIle Ser Asn Met Thr Phe Ser Asn Gly Lys Leu Ile Val 195 200 205AsnGln Asp Gly Phe Tyr Tyr Leu Tyr Ala Asn Ile Cys Phe Arg His 210 215220His Glu Thr Ser Gly Asp Leu Ala Thr Glu Tyr Leu Gln Leu MetVal225 230 235 240Tyr Val Thr Lys Thr Ser Ile Lys Ile Pro Ser SerHis Thr Leu Met 245 250 255Lys Gly Gly Ser Thr Lys Tyr Trp Ser GlyAsn Ser Glu Phe His Phe 260 265 270Tyr Ser Ile Asn Val Gly Gly PhePhe Lys Leu Arg Ser Gly Glu Glu 275 280 285Ile Ser Ile Glu Val SerAsn Pro Ser Leu Leu Asp Pro Asp Gln Asp 290 295 300Ala Thr Tyr PheGly Ala Phe Lys Val Arg Asp Ile Asp305 310 315214249PRThom*o sapiens214Met Ala Ala Arg Arg Ser Gln Arg Arg Arg Gly Arg Arg Gly Glu Pro15 10 15Gly Thr Ala Leu Leu Val Pro Leu Ala Leu Gly Leu Gly Leu AlaLeu 20 25 30Ala Cys Leu Gly Leu Leu Leu Ala Val Val Ser Leu Gly SerArg Ala 35 40 45Ser Leu Ser Ala Gln Glu Pro Ala Gln Glu Glu Leu ValAla Glu Glu 50 55 60Asp Gln Asp Pro Ser Glu Leu Asn Pro Gln Thr GluGlu Ser Gln Asp65 70 75 80Pro Ala Pro Phe Leu Asn Arg Leu Val ArgPro Arg Arg Ser Ala Pro 85 90 95Lys Gly Arg Lys Thr Arg Ala Arg ArgAla Ile Ala Ala His Tyr Glu 100 105 110Val His Pro Arg Pro Gly GlnAsp Gly Ala Gln Ala Gly Val Asp Gly 115 120 125Thr Val Ser Gly TrpGlu Glu Ala Arg Ile Asn Ser Ser Ser Pro Leu 130 135 140Arg Tyr AsnArg Gln Ile Gly Glu Phe Ile Val Thr Arg Ala Gly Leu145 150 155160Tyr Tyr Leu Tyr Cys Gln Val His Phe Asp Glu Gly Lys Ala Val Tyr165 170 175Leu Lys Leu Asp Leu Leu Val Asp Gly Val Leu Ala Leu ArgCys Leu 180 185 190Glu Glu Phe Ser Ala Thr Ala Ala Ser Ser Leu GlyPro Gln Leu Arg 195 200 205Leu Cys Gln Val Ser Gly Leu Leu Ala LeuArg Pro Gly Ser Ser Leu 210 215 220Arg Ile Arg Thr Leu Pro Trp AlaHis Leu Lys Ala Ala Pro Phe Leu225 230 235 240Thr Tyr Phe Gly LeuPhe Gln Val His 245215250PRThom*o sapiens 215Met Pro Ala Ser Ser ProPhe Leu Leu Ala Pro Lys Gly Pro Pro Gly1 5 10 15Asn Met Gly Gly ProVal Arg Glu Pro Ala Leu Ser Val Ala Leu Trp 20 25 30Leu Ser Trp GlyAla Ala Leu Gly Ala Val Ala Cys Ala Met Ala Leu 35 40 45Leu Thr GlnGln Thr Glu Leu Gln Ser Leu Arg Arg Glu Val Ser Arg 50 55 60Leu GlnGly Thr Gly Gly Pro Ser Gln Asn Gly Glu Gly Tyr Pro Trp65 70 7580Gln Ser Leu Pro Glu Gln Ser Ser Asp Ala Leu Glu Ala Trp Glu Asn85 90 95Gly Glu Arg Ser Arg Lys Arg Arg Ala Val Leu Thr Gln Lys GlnLys 100 105 110Lys Gln His Ser Val Leu His Leu Val Pro Ile Asn AlaThr Ser Lys 115 120 125Asp Asp Ser Asp Val Thr Glu Val Met Trp GlnPro Ala Leu Arg Arg 130 135 140Gly Arg Gly Leu Gln Ala Gln Gly TyrGly Val Arg Ile Gln Asp Ala145 150 155 160Gly Val Tyr Leu Leu TyrSer Gln Val Leu Phe Gln Asp Val Thr Phe 165 170 175Thr Met Gly GlnVal Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr 180 185 190Leu PheArg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr 195 200205Asn Ser Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile210 215 220Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys Leu Asn LeuSer Pro225 230 235 240His Gly Thr Phe Leu Gly Phe Val Lys Leu 245250216285PRThom*o sapiens 216Met Asp Asp Ser Thr Glu Arg Glu Gln SerArg Leu Thr Ser Cys Leu1 5 10 15Lys Lys Arg Glu Glu Met Lys Leu LysGlu Cys Val Ser Ile Leu Pro 20 25 30Arg Lys Glu Ser Pro Ser Val ArgSer Ser Lys Asp Gly Lys Leu Leu 35 40 45Ala Ala Thr Leu Leu Leu AlaLeu Leu Ser Cys Cys Leu Thr Val Val 50 55 60Ser Phe Tyr Gln Val AlaAla Leu Gln Gly Asp Leu Ala Ser Leu Arg65 70 75 80Ala Glu Leu GlnGly His His Ala Glu Lys Leu Pro Ala Gly Ala Gly 85 90 95Ala Pro LysAla Gly Leu Glu Glu Ala Pro Ala Val Thr Ala Gly Leu 100 105 110LysIle Phe Glu Pro Pro Ala Pro Gly Glu Gly Asn Ser Ser Gln Asn 115 120125Ser Arg Asn Lys Arg Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln130 135 140Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr IleGln Lys145 150 155 160Gly Ser Tyr Thr Phe Val Pro Trp Leu Leu SerPhe Lys Arg Gly Ser 165 170 175Ala Leu Glu Glu Lys Glu Asn Lys IleLeu Val Lys Glu Thr Gly Tyr 180 185 190Phe Phe Ile Tyr Gly Gln ValLeu Tyr Thr Asp Lys Thr Tyr Ala Met 195 200 205Gly His Leu Ile GlnArg Lys Lys Val His Val Phe Gly Asp Glu Leu 210 215 220Ser Leu ValThr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu225 230 235240Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly245 250 255Asp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln IleSer Leu 260 265 270Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys LeuLeu 275 280 285217240PRThom*o sapiens 217Met Glu Glu Ser Val Val ArgPro Ser Val Phe Val Val Asp Gly Gln1 5 10 15Thr Asp Ile Pro Phe ThrArg Leu Gly Arg Ser His Arg Arg Gln Ser 20 25 30Cys Ser Val Ala ArgVal Gly Leu Gly Leu Leu Leu Leu Leu Met Gly 35 40 45Ala Gly Leu AlaVal Gln Gly Trp Phe Leu Leu Gln Leu His Trp Arg 50 55 60Leu Gly GluMet Val Thr Arg Leu Pro Asp Gly Pro Ala Gly Ser Trp65 70 75 80GluGln Leu Ile Gln Glu Arg Arg Ser His Glu Val Asn Pro Ala Ala 85 9095His Leu Thr Gly Ala Asn Ser Ser Leu Thr Gly Ser Gly Gly Pro Leu100 105 110Leu Trp Glu Thr Gln Leu Gly Leu Ala Phe Leu Arg Gly LeuSer Tyr 115 120 125His Asp Gly Ala Leu Val Val Thr Lys Ala Gly TyrTyr Tyr Ile Tyr 130 135 140Ser Lys Val Gln Leu Gly Gly Val Gly CysPro Leu Gly Leu Ala Ser145 150 155 160Thr Ile Thr His Gly Leu TyrLys Arg Thr Pro Arg Tyr Pro Glu Glu 165 170 175Leu Glu Leu Leu ValSer Gln Gln Ser Pro Cys Gly Arg Ala Thr Ser 180 185 190Ser Ser ArgVal Trp Trp Asp Ser Ser Phe Leu Gly Gly Val Val His 195 200 205LeuGlu Ala Gly Glu Lys Val Val Val Arg Val Leu Asp Glu Arg Leu 210 215220Val Arg Leu Arg Asp Gly Thr Arg Ser Tyr Phe Gly Ala Phe MetVal225 230 235 240218251PRThom*o sapiens 218Met Ala Glu Asp Leu GlyLeu Ser Phe Gly Glu Thr Ala Ser Val Glu1 5 10 15Met Leu Pro Glu HisGly Ser Cys Arg Pro Lys Ala Arg Ser Ser Ser 20 25 30Ala Arg Trp AlaLeu Thr Cys Cys Leu Val Leu Leu Pro Phe Leu Ala 35 40 45Gly Leu ThrThr Tyr Leu Leu Val Ser Gln Leu Arg Ala Gln Gly Glu 50 55 60Ala CysVal Gln Phe Gln Ala Leu Lys Gly Gln Glu Phe Ala Pro Ser65 70 7580His Gln Gln Val Tyr Ala Pro Leu Arg Ala Asp Gly Asp Lys Pro Arg85 90 95Ala His Leu Thr Val Val Arg Gln Thr Pro Thr Gln His Phe LysAsn 100 105 110Gln Phe Pro Ala Leu His Trp Glu His Glu Leu Gly LeuAla Phe Thr 115 120 125Lys Asn Arg Met Asn Tyr Thr Asn Lys Phe LeuLeu Ile Pro Glu Ser 130 135 140Gly Asp Tyr Phe Ile Tyr Ser Gln ValThr Phe Arg Gly Met Thr Ser145 150 155 160Glu Cys Ser Glu Ile ArgGln Ala Gly Arg Pro Asn Lys Pro Asp Ser 165 170 175Ile Thr Val ValIle Thr Lys Val Thr Asp Ser Tyr Pro Glu Pro Thr 180 185 190Gln LeuLeu Met Gly Thr Lys Ser Val Cys Glu Val Gly Ser Asn Trp 195 200205Phe Gln Pro Ile Tyr Leu Gly Ala Met Phe Ser Leu Gln Glu Gly Asp210 215 220Lys Leu Met Val Asn Val Ser Asp Ile Ser Leu Val Asp TyrThr Lys225 230 235 240Glu Asp Lys Thr Phe Phe Gly Ala Phe Leu Leu245 250219199PRThom*o sapiens 219Met Thr Leu His Pro Ser Pro Ile ThrCys Glu Phe Leu Phe Ser Thr1 5 10 15Ala Leu Ile Ser Pro Lys Met CysLeu Ser His Leu Glu Asn Met Pro 20 25 30Leu Ser His Ser Arg Thr GlnGly Ala Gln Arg Ser Ser Trp Lys Leu 35 40 45Trp Leu Phe Cys Ser IleVal Met Leu Leu Phe Leu Cys Ser Phe Ser 50 55 60Trp Leu Ile Phe IlePhe Leu Gln Leu Glu Thr Ala Lys Glu Pro Cys65 70 75 80Met Ala LysPhe Gly Pro Leu Pro Ser Lys Trp Gln Met Ala Ser Ser 85 90 95Glu ProPro Cys Val Asn Lys Val Ser Asp Trp Lys Leu Glu Ile Leu 100 105110Gln Asn Gly Leu Tyr Leu Ile Tyr Gly Gln Val Ala Pro Asn Ala Asn115 120 125Tyr Asn Asp Val Ala Pro Phe Glu Val Arg Leu Tyr Lys AsnLys Asp 130 135 140Met Ile Gln Thr Leu Thr Asn Lys Ser Lys Ile GlnAsn Val Gly Gly145 150 155 160Thr Tyr Glu Leu His Val Gly Asp ThrIle Asp Leu Ile Phe Asn Ser 165 170 175Glu His Gln Val Leu Lys AsnAsn Thr Tyr Trp Gly Ile Ile Leu Leu 180 185 190Ala Asn Pro Gln PheIle Ser 195220391PRThom*o sapiens 220Met Gly Tyr Pro Glu Val Glu ArgArg Glu Leu Leu Pro Ala Ala Ala1 5 10 15Pro Arg Glu Arg Gly Ser GlnGly Cys Gly Cys Gly Gly Ala Pro Ala 20 25 30Arg Ala Gly Glu Gly AsnSer Cys Leu Leu Phe Leu Gly Phe Phe Gly 35 40 45Leu Ser Leu Ala LeuHis Leu Leu Thr Leu Cys Cys Tyr Leu Glu Leu 50 55 60Arg Ser Glu LeuArg Arg Glu Arg Gly Ala Glu Ser Arg Leu Gly Gly65 70 75 80Ser GlyThr Pro Gly Thr Ser Gly Thr Leu Ser Ser Leu Gly Gly Leu 85 90 95AspPro Asp Ser Pro Ile Thr Ser His Leu Gly Gln Pro Ser Pro Lys 100 105110Gln Gln Pro Leu Glu Pro Gly Glu Ala Ala Leu His Ser Asp SerGln

115 120 125Asp Gly His Gln Met Ala Leu Leu Asn Phe Phe Phe Pro AspGlu Lys 130 135 140Pro Tyr Ser Glu Glu Glu Ser Arg Arg Val Arg ArgAsn Lys Arg Ser145 150 155 160Lys Ser Asn Glu Gly Ala Asp Gly ProVal Lys Asn Lys Lys Lys Gly 165 170 175Lys Lys Ala Gly Pro Pro GlyPro Asn Gly Pro Pro Gly Pro Pro Gly 180 185 190Pro Pro Gly Pro GlnGly Pro Pro Gly Ile Pro Gly Ile Pro Gly Ile 195 200 205Pro Gly ThrThr Val Met Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 210 215 220ProGln Gly Pro Pro Gly Leu Gln Gly Pro Ser Gly Ala Ala Asp Lys225 230235 240Ala Gly Thr Arg Glu Asn Gln Pro Ala Val Val His Leu Gln GlyGln 245 250 255Gly Ser Ala Ile Gln Val Lys Asn Asp Leu Ser Gly GlyVal Leu Asn 260 265 270Asp Trp Ser Arg Ile Thr Met Asn Pro Lys ValPhe Lys Leu His Pro 275 280 285Arg Ser Gly Glu Leu Glu Val Leu ValAsp Gly Thr Tyr Phe Ile Tyr 290 295 300Ser Gln Val Glu Val Tyr TyrIle Asn Phe Thr Asp Phe Ala Ser Tyr305 310 315 320Glu Val Val ValAsp Glu Lys Pro Phe Leu Gln Cys Thr Arg Ser Ile 325 330 335Glu ThrGly Lys Thr Asn Tyr Asn Thr Cys Tyr Thr Ala Gly Val Cys 340 345350Leu Leu Lys Ala Arg Gln Lys Ile Ala Val Lys Met Val His Ala Asp355 360 365Ile Ser Ile Asn Met Ser Lys His Thr Thr Phe Phe Gly AlaIle Arg 370 375 380Leu Gly Glu Ala Pro Ala Ser3853902216PRTArtificial SequenceDescription of Artificial SequenceSynthetic 6xHis tag 221His His His His His His1 522250PRTArtificialSequenceDescription of Artificial Sequence SyntheticpolypeptideMISC_FEATURE(1)..(50)This sequence may encompass 1-10"Gly Gly Gly Gly Ser" repeating unitsSee specification as filed fordetailed description of substitutions and preferred embodiments222Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly15 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly 20 25 30Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser GlyGly Gly 35 40 45Gly Ser 5022350PRTArtificial SequenceDescription ofArtificial Sequence Synthetic polypeptideMISC_FEATURE(1)..(50)Thissequence may encompass 1-10 "Ser Gly Gly Gly Gly" repeatingunitsSee specification as filed for detailed description ofsubstitutions and preferred embodiments 223Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 20 25 30Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45Gly Gly5022454PRTArtificial SequenceDescription of Artificial SequenceSynthetic polypeptideMISC_FEATURE(5)..(54)This region may encompass1-10 "Ser Gly Gly Gly Gly" repeating unitsSee specification asfiled for detailed description of substitutions and preferredembodiments 224Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly1 5 10 15Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly 20 25 30Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Gly Gly Gly 35 40 45Gly Ser Gly Gly Gly Gly5022515PRTArtificial SequenceDescription of Artificial SequenceSynthetic peptide 225Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys IleGlu Trp His Glu1 5 10 15

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