WO2025083273A1

WO2025083273A1 - Binding molecules

Info

Publication number: WO2025083273A1
Application number: PCT/EP2024/079608
Authority: WO
Inventors: Aymen Al-Shamkhani; Mark Austin; Sarah Buchan; Denice Tsz Yau CHAN; Maria Groves; Stuart HAYNES; Lesley JENKINSON; Kirsty Jane MARTIN; Hannah METCALFE; Steffen Preissler; Louise SLATER; Jessica WHITEHOUSE
Original assignee: Cancer Research Technology Limited; Medimmune Limited
Priority date: 2023-10-18
Filing date: 2024-10-18
Publication date: 2025-04-24
Also published as: GB202315963D0

Abstract

An antigen binding protein, such as an antibody or antigen-binding fragment thereof, capable of binding specifically to an epitope formed by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2).

Description

Binding Molecules Technical Field The invention relates to antigen-binding proteins, such as monoclonal antibodies and antigen- binding fragments thereof, that bind to human Death receptor 3 (DR3) also known as TNFRSF25 (tumour necrosis factor receptor superfamily 25), preferably antigen-binding proteins that bind specifically to human DR3 and agonise the activity of the DR3 receptor; the invention relates to uses of such antigen-binding proteins as medicaments and for treatment, as monotherapy or in combination with standard of care (SOC) or other therapies. Background to the Invention The advent of immunotherapy with checkpoint inhibitors (CPI) has revolutionised the treatment of some cancers, but the response rates in most cancers remain low and there is a desire to develop new therapies. CPI such as anti-PD-1/PD-L1, anti-CTLA-4 and anti-LAG3 prevent inhibitory signals and thereby alleviate immunosuppression allowing tumour-specific T cells to target cancer cells. Immunostimulatory antibodies (ISA) target immune activatory receptors on T cells and deliver co-stimulatory signals that combine with signals delivered through antigen recognition by the T cell receptor (TCR). A combination of TCR and co- stimulatory receptor signalling is required for productive T cell responses. Signalling by co-stimulatory receptors is normally controlled tightly through regulated expression of membrane-bound costimulatory ligands on antigen presenting cells. Microbial derived molecules such as lipopolysaccharide, CpG or double stranded RNA instigate the expression of co-stimulatory ligands by activating innate receptors e.g., Toll-like receptors. Conversely, the absence of these stimulatory molecules leads to blunted T cell responses against tumour antigens. ISA are able to ‘short circuit’ this process by directly targeting and activating co-stimulatory receptors on T cells, thus augmenting signals emanating from engagement of tumour antigens by the TCR. However, clinical development of ISA has lagged behind those of CPI largely because the antibodies generated fail to fully mimic the immunostimulatory effects of the natural costimulatory ligands. Here, we describe methods for the generation of potent ISA targeting the costimulatory receptor DR3. Death receptor 3 (DR3), also known as TNFRSF25, and its corresponding ligand, tumour necrosis factor-like ligand 1A (TL1A), belong to the tumour necrosis factor receptor superfamily (TNFRSF) and tumour necrosis factor superfamily (TNFSF), respectively. DR3 is largely expressed by T cells and activation of DR3 by its ligand, TL1A, stimulates NFκB/JNK signalling and amplifies protective T cell responses, making it a potential target for cancer 1

241018 CMAL003WO1 DCA immunotherapy. Soluble TL1A is limited as a therapeutic agent because it can be neutralized by decoy receptor 3 (DcR3) which is elevated in some tumours. WO0135995 (Tittle, et al.) described anti-DR3 antibodies that bind CRD1 that were generated by immunising using DR3 amino acids 1 to 32 and boosting with DR3 amino acids 1 to 13, the anti-DR3 antibodies are proposed for the treatment of autoimmune disease. WO0064465 (Human Genome Sciences, Inc.) described anti-DR3 antibodies proposed for use in the treatment of ulcerative colitis and Crohn’s disease. "DR3/TNFRSF25 antibody" DR3/TNFRSF25 Antibody (1H2) (H00008718-M07): Novus Biologicals described murine IgG2a anti-DR3 antibody, 1H2, that binds CRD1, which was generated by immunisation using amino acids 28 - 124 of DR3. WO2011106707 (Human Genome Sciences. Inc.) described an anti-DR3 antibody, 11H08, that inhibits TL1A binding to DR3 and acts as an agonist of TL1A activity. Monomeric anti- DR3 Fab fragments were generated that did not show agonistic activity. WO2012117067 (Novo Nordisk AS) described antagonistic anti-DR3 antibodies, 072, 073, 076 and Fab thereof that block the TL1A:DR3 interaction; the Fab with monovalent specificity for DR3 did not cluster receptors and did not have agonistic activity. WO2015152430 (Kyowa Hakko Kirin Co., Ltd) described anti-death receptor 3 (DR3) antagonistic IgG antibodies and antibody fragments thereof, wherein the antibodies and the antibody fragments thereof display a decreased agonistic activity or no agonistic activity for DR3 through their binding. WO2016081455 & US20120014950 (Pelican Therapeutics, Inc) described agonistic TNFRSF25 (DR3) specific antibodies, including monoclonal antibody PTX-25, and antigen binding fragments thereof, for use to stimulate proliferation of human T cells, for use in the treatment of human cancer patients. Antibodies described therein bind to an epitope formed by amino acids 64-69 of human DR3. Neutralizing and agonistic antibodies, as well as ligand-based approaches targeting the DR3/TL1A pathway, may be used to treat diseases, including inflammatory and immune- mediated diseases as well as cancer. Accordingly, there is a desire to identify antibodies 2

241018 CMAL003WO1 DCA capable of modulating the DR3/TL1A pathway, and in particular antibodies capable of agonising the DR3/TL1A pathway to provide a therapeutic approach for patients with cancer. Statements of invention The invention provides: 1. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, capable of binding specifically to an epitope formed by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). 2. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof of clause 1, capable of binding specifically to an epitope formed by residues of the amino acid sequence 37-45 of human DR3 (SEQ ID NO: 1) and thereby agonising a DR3 pathway, wherein optionally agonism may be assessed in a T cell proliferation assay. 3. An antigen-binding protein of clause 1 or clause 2, such as an antibody or antigen-binding fragment thereof, wherein the antigen binding protein is a murine, chimeric, humanised, or human antibody or antigen-binding fragment thereof. 4. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, of any preceding clause, comprising an antigen-binding site comprising framework sequences (FW1 to FW4) and CDRs (HCDR1, HCRD2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively) selected from: (a) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 (Clone B9); or (b) SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 (Clone F10), wherein the sequences are defined according to Kabat nomenclature. 5. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, of any preceding clause, wherein the antigen-binding site comprises the VH and / or VL domain sequence of, or a VH and / or VL domain sequence with at least 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identity to, a clone selected from: (a) humanised Clone B9 of VHg1/VLg11 (H1L11) of VH SEQ ID NO: 19 and VL SEQ ID NO: 20, respectively; 3

241018 CMAL003WO1 DCA (b) murine Clone B9 of VH SEQ ID NO: 15 and VL SEQ ID NO: 16, respectively; (c) murine Clone F10 of VH SEQ ID NO: 17 and VL SEQ ID NO: 18, respectively; and (d) a humanised version of Clone B9 of Figure 19 and Table 1, wherein the sequences are defined according to Kabat nomenclature. 6. An antigen-binding protein, such as a humanised or murine antibody or antigen-binding fragment thereof, of any preceding clause, wherein the antibody comprises the VH and / or VL domain of: (a) humanised Clone B9 VHg1/VLg11 (H1L11) of VH SEQ ID NO: 19 and VL SEQ ID NO: 20, respectively; (b) murine Clone B9 of VH SEQ ID NO: 15 and VL SEQ ID NO: 16, respectively; (c) murine Clone F10 of VH SEQ ID NO: 17 and VL SEQ ID NO: 18, respectively; or (d) a humanised version of Clone B9 shown in Figure 19 and Table 1, wherein the sequences are defined according to Kabat nomenclature. 7. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof capable of competing with an antibody according to any one of clauses 1 to 6 for binding to an epitope formed by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) when assessed in a competition assay. 8. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses comprising a human Fc selected from hIgG1, hIgG1-SELF, hIgG2, hIgG4, IgG1V11, IgG1 N297A, IgG1 N297Q, IgG1 N297A LALA-PG, IgG1 N297Q LALA-PG and IgG1 LALA-PG. 9. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, characterised in that the antigen binding protein is monovalent, bivalent, trivalent, or tetravalent for binding human DR3 (SEQ ID NO: 2). 10. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a multivalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). 11. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a bivalent, monospecific 4

241018 CMAL003WO1 DCA antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). 12. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc capable of binding to FcγR (for FcR mediated cross-linking), e.g., (e.g., B9-hIgG1 (B9-H1)). 13. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc that is silent (not capable of binding to FcγR), such as IgG1 N297A. 14. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a trivalent or tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). 15. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) or IgG1 Fc is absent. 16. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) and / or Fc is absent. 5

241018 CMAL003WO1 DCA 17. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 N297A Fc in which Fc function is silent (does not bind FcγR), e.g., “Fc-silent” TET-B9-N297A. 18. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc (capable of binding FcγR); e.g., tetravalent (TET) B9- H1 (TET-B9-H1). 19. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc. 20. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding clauses, wherein the antigen-binding protein comprises a Fab:IgG tetravalent construct. 21. A multivalent antigen-binding protein of any one of clauses 1 to 20 for use to agonise DR3 without cross-linking by Fc receptors. 22. A multivalent antigen-binding protein of any one of clauses 1 to 21 for use to agonise DR3 wherein the multivalent antigen-binding protein lacks effector function. 23. A hybridoma comprising an antigen-binding protein, such as an antibody or antigen- binding fragment thereof, according to any one of the preceding clauses. 24. A “vaccine” composition comprising an isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof, according to any one of clauses 1 to 22 and an excipient. 6

241018 CMAL003WO1 DCA 25. An isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof, according to any one of clauses 1 to 22. 26. An isolated recombinant DNA sequence of clause 25 which is a vector. 27. An isolated recombinant DNA sequence of clause 26 which is an expression vector. 28. An isolated recombinant DNA sequence of clause 26 or 27 encoding an antibody or antigen-binding fragment thereof, according to any one of clauses 1 to 22 under control of a promoter. 29. A host cell comprising a DNA or RNA sequence according to any one of clauses 25 to 28. 30. A host cell of clause 29 capable of expressing an isolated antibody or antigen-binding fragment thereof, of any one of clauses 1 to 22. 31. A method of making an isolated antibody or antigen-binding fragment thereof, of any one of clauses 1 to 22 comprising culturing a host cell according to clause 29 or 30 in conditions suitable for expression of the isolated antibody or antigen-binding fragment thereof. 32. A composition comprising: (a) an isolated antibody or antigen-binding fragment thereof, according to any one of clauses 1 to 22 and an excipient, preferably a pharmaceutically-acceptable excipient, or (b) an isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof according to any one of clauses 1 to 22 and an excipient, preferably a pharmaceutically-acceptable excipient. 33. An antibody or antigen-binding fragment thereof of any one of clauses 1 to 22, or composition of clause 24 or 32 for use as a medicament or for use in diagnosis. 34. An antibody or antigen-binding fragment thereof any one of clauses 1 to 22, or a composition of clause 24 or 32, for use in the prophylactic or therapeutic treatment of a cancer, for example wherein the cancer is selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin’s lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSC) and 7

241018 CMAL003WO1 DCA skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied. 35. An antibody or antigen-binding fragment thereof any one of clauses 1 to 22, or a composition of clause 24 or 32, for use in the prophylactic or therapeutic treatment of cancer in combination with one or more 2^nd therapy optionally selected from an anti-PD-1 mAb, vaccine, chemotherapy, kinase inhibitor, tumour targeting mAb (e.g., anti-CD20, anti-HER2, anti-VEGFR), antibody-drug conjugate, bispecific T cell engager, checkpoint inhibitors targeting immune receptors (e.g., PD-1, PD-L1, CTLA-4, LAG3), mAb targeting other co- stimulatory receptors (e.g., CD27, GITR, OX40, 4-1BB, ICOS, CD28), STING agonists and agents targeting myeloid suppressor cells. 36. An antibody or antigen-binding fragment thereof of any one of clauses 1 to 22, or a composition of clause 24 or 32, for use to stimulate the proliferation and differentiation of T cells into effector, cytotoxic and memory T cells. 37. An antibody or antigen-binding fragment thereof of any one of clauses 1 to 22, or a composition of clause 32, for use to identify human DR3 protein (SEQ ID NO: 2) comprising an epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2), 38. An antibody or antigen-binding fragment thereof of any one of clauses 1 to 22, or a composition of clause 32, for use in a diagnostic test for a cancer, for example wherein the cancer is selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin’s lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied. 39. A diagnostic kit comprising an antibody or antigen-binding fragment thereof of any one of clauses 1 to 22, or a composition of clause 32, and a reagent capable of detecting an immunological (antigen-antibody) complex which contains said antibody or antigen-binding fragment thereof, wherein optionally said antibody or antigen-binding fragment is immobilized on a solid support (e.g., microplate well), and / or wherein optionally said immunological complex which contains said antibody or antigen-binding fragment is detectable by ELISA or an alternative immunoassay method or by lateral flow. 8

241018 CMAL003WO1 DCA 40. A diagnostic kit according to clause 39, further comprising one or more control standards and / or specimen diluent and / or washing buffer. Detailed description The invention relates to antibodies, such as murine and humanised antibodies and antigen- binding fragments thereof capable of binding specifically to an isolated recombinant peptide comprising an epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) (UniProt Q93038; Ensembl gene ID, human: ENSG00000215788; NCBI gene ID, human: 8718). The invention further relates to antibodies, such as murine and humanised antibodies and antigen binding fragments thereof that comprise at least one CDR-based antigen-binding site, specific for an epitope comprised within residues 37 to 45 of human DR3. Murine and humanised antibodies and antigen binding fragments thereof of the invention bind specifically to human DR3 species that include epitopes formed by residues 37 – 45 (, SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). An antibody or antigen-binding fragment thereof of the invention may be produced by recombinant means. A “recombinant antibody” is an antibody which has been produced by a recombinantly engineered host cell. An antibody or antigen-binding fragment thereof in accordance with the invention is optionally isolated or purified. The term “antibody” or “antibody molecule” describes an immunoglobulin whether natural or partly or wholly synthetically produced. An antigen-binding protein of the invention may be an antibody, preferably a monoclonal antibody, and may be human or non-human, chimeric or humanised. The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies are the immunoglobulin isotypes, such as immunoglobulin G, and their isotypic subclasses, such as IgG1, IgG2, IgG3 and IgG4, as well as fragments thereof. The four human subclasses (IgG1, IgG2, IgG3 and IgG4) each contain a different heavy chain; but they are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system. IgG1 and IgG4 contain two inter-chain disulphide bonds in the hinge region, IgG2 has 4 and IgG3 has 11 inter-chain disulphide bonds. The terms “antibody” and “antibody molecule”, as used herein, includes antibody fragments, such as Fab fragment and single chain variable fragment (scFv), provided that said fragments 9

241018 CMAL003WO1 DCA comprise a CDR-based antigen binding site for an epitope comprising residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv) and domain antibodies (sdAbs, such as VH, VHH or VL). Unless the context requires otherwise, the terms “antigen-binding protein”, “antibody” or “antibody molecule”, as used herein, is thus equivalent to “antibody or antigen-binding fragment thereof”. Antibodies are immunoglobulins, which have the same basic structure consisting of two heavy and two light chains forming two Fab arms containing identical domains that are attached by a flexible hinge region to the stem of the antibody, the Fc domain, giving the classical ‘Y’ shape. The Fab domains consist of two variable and two constant domains, with a variable heavy (VH) and constant heavy 1 (CH1) domain on the heavy chain and a variable light (VL) and constant light (CL) domain on the light chain. The two variable domains (VH and VL) form the variable fragment (Fv), which provides the CDR-based antigen specificity of the antibody, with the constant domains (CH1 and VL) acting as a structural framework. Each variable domain contains three hypervariable loops, known as complementarity determining regions (CDRs). On each of the VH and VL the three CDRs (CDR1, CDR2, and CDR3) are flanked by four less-variable framework (FR) regions (FR1, FW2, FW3 and FW4) to give a structure FW1- CDR1-FW2-CDR2-FW3-CDR3-FW4. The CDRs provide a specific antigen recognition site on the surface of the antibody. Both Kabat and ImMunoGeneTics (IMGT) numbering nomenclature may be used herein. Generally, unless otherwise indicated (explicitly or by context) amino acid residues are numbered herein according to the Kabat numbering scheme (Kabat et al., 1991, J Immunol 147(5): 1709-19). For those instances when the IMGT numbering scheme is used, amino acid residues are numbered herein according to the ImMunoGeneTics (IMGT) numbering scheme described in Lefranc et al., 2005, Dev Comp Immunol 29(3): 185-203. Antibody structure was analysed using publicly available abYsis.org web-based antibody research system which includes pre-analysed sequence data from the European Molecular Biology Laboratory European Nucleotide Archive (EMBL-ENA) and Kabat as well as structure data from the Protein Data Bank (Swindell et al., 2017, J Mol Biol 429(3):356-364). Quote: “A defining characteristic of abYsis is that the sequences are automatically numbered with a series of popular schemes such as Kabat and Chothia and then annotated with key information such as complementarity-determining regions and potential post-translational modifications” (Swindell et al., 2017, J Mol Biol 429(3):356-364). 10 241018 CMAL003WO1 DCA It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing the CDRs into a different immunoglobulin framework or grafting variable regions onto a different immunoglobulin constant region. Introduction of the CDRs of one immunoglobulin into another immunoglobulin is described for example in EP-A-184187, GB2188638A or EP-A-239400. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced. Antibody humanisation involves the transfer, or “grafting”, of critical non-human amino acids onto a human antibody framework. Primarily this includes the grafting of amino acids in the complementarity-determining regions (CDRs), but potentially also other framework amino acids critical for the VH:VL interface and for orientation of the CDRs. Humanisation seeks to introduce human content to reduce the risk of immunogenicity, while retaining the original binding activity of the non-human parental antibody. The term "humanised antibody" is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences; optionally additional framework region modifications can be made within the human framework sequences. The term "humanised antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences and optimised (for example by affinity maturation). Optimisation may involve modification or one more amino acid residues in one or more of the CDRs and / or in one or more framework sequence to modulate or improve a biological property of the humanised antibody, e.g., to increase affinity, or to modulate the on rate and/ or off rate for binding of the antibody to its target epitope. As antibodies can be modified in several ways, the term “antigen-binding protein” or "antibody" should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, an aptamer, affimer or bicyclic peptide, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A- 0120694 and EP-A-0125023. An example of an antibody fragment comprising both CDR sequences and CH3 domain is a minibody, which comprises a scFv joined to a CH3 domain (Hu et al. (1996) Cancer Res 56(13): 3055-61). 11 241018 CMAL003WO1 DCA A domain (single-domain) antibody is a peptide, usually about 110 amino acids long, comprising one variable domain (VH) of a heavy-chain antibody, or of an IgG. A single-domain antibody (sdAb), (e.g., nanobody™ VHH, or human VH domain antibody), is an antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody (comprising two heavy and two light chains), it is an antigen-binding protein able to bind selectively to a specific antigen. Domain antibodies have a molecular weight of only 12–15 kDa and are thus much smaller than antibodies composed of two heavy protein chains and two light chains (150–160 kDa), and domain antibodies are even smaller than Fab fragments (~50 kDa, one light chain and half a heavy chain) and single-chain variable fragments (~25 kDa, two variable domains, one from a light and one from a heavy chain). Single-domain antibodies have been engineered from heavy-chain antibodies found in camelids; these are termed VHH fragments. Cartilaginous fish also have heavy-chain antibodies (IgNAR, 'immunoglobulin new antigen receptor'), from which single-domain antibodies called VNAR fragments can be obtained. A domain (single domain) antibody may be a VH or VL. A domain antibody may be a VH or VL of human or murine origin. Although most single-domain antibodies are heavy chain variable domains, light chain single-domain antibodies (VL) have also been shown to bind specifically to target epitopes. Protein scaffolds have relatively defined three-dimensional structures and typically contain one or more regions which are amenable to specific or random amino acid sequence variation, to produce antigen-binding regions within the scaffold that are capable of binding to an antigen. An antibody or antigen-binding fragment of the invention binds to an epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). Binding in this context may refer to specific binding. The term "specific" may refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner(s), here an epitope within residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). The term “specific” is also applicable where the antibody is specific for particular epitopes, such as an epitope comprised within residues that are carried by a number of antigens in which case the antibody molecule will be able to bind to the various antigens carrying the epitope. Preferably antibodies and antigen-binding fragments thereof of the invention bind to an epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and agonise a DR3 pathway. 12 241018 CMAL003WO1 DCA In particularly preferred aspects of the invention murine and humanised antibodies and antigen-binding fragments thereof of the invention bind human DR3 at an epitope formed and defined by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). In preferred embodiments, an antibody or an antigen-binding fragment thereof of the invention may comprise the set of six CDRs (HCDR1 (SYAMS – SEQ ID NO: 3), HCDR2 (TISDGDSYSYFPDSVKD – SEQ ID NO: 4), HCDR3 (DRIYGSVQYYAMDY – SEQ ID NO: 5), LCDR1 (RASESVEFSGTSLMQ – SEQ ID NO: 6), LCDR2 (AASNVES – SEQ ID NO: 7), and LCDR3 (QQSRKLPYT – SEQ ID NO: 8)) of Clone B9. In preferred embodiments, an antibody or an antigen-binding fragment thereof of the invention may comprise the set of six CDRs (HCDR1 (AYAMS – SEQ ID NO: 9), HCDR2 (TISDGDPYTYYPDNVKG – SEQ ID NO: 10), HCDR3 (ERNDYDQYYTMDY – SEQ ID NO: 11), LCDR1 (RASKSVSTSGYNYLH – SEQ ID NO: 12), LCDR2 (LASNLES – SEQ ID NO: 13), and LCDR3 (QHSRELPWT – SEQ ID NO: 14)) of Clone F10. An antibody or an antigen-binding fragment thereof of the invention may comprise the VH (SEQ ID NO: 15) and / or VL (SEQ ID NO: 16) sequence of antibody clone B9. An antibody or an antigen-binding fragment thereof of the invention may comprise the VH sequence (SEQ ID NO: 17) and / or VL (SEQ ID NO: 18) sequence of antibody clone F10. An antibody or an antigen-binding fragment thereof of the invention may comprise the VH and / or VL sequence of a humanised variant of antibody clone B9. An antibody or an antigen-binding fragment thereof of the invention may comprise the VH and / or VL sequence of a humanised variant of antibody clone B9, e.g., an antibody or an antigen- binding fragment thereof of the invention may comprise the VH (SEQ ID NO: 19) and / or VL (SEQ ID NO: 20) sequence of VHg1/VLg11 (H1L11) (a humanised variant of antibody clone B9). An antibody or an antigen-binding fragment thereof of the invention may comprise the VH and / or VL sequence of a humanised variant of antibody clone B9 shown in Figure 19 and Table 1. 13 241018 CMAL003WO1 DCA An antibody or an antigen-binding fragment thereof of the invention may comprise the VH sequence and / or VL sequence of a humanised variant of antibody clone F10. An antibody or an antigen-binding fragment thereof of the invention may comprise a humanised variant of the VH and VL sequence of B9 or a humanised variant of the VH and VL sequence of F10. An antibody or an antigen-binding fragment thereof of the invention may comprise a humanised variant of the VH and VL sequence of B9 as shown in Figure 19 and Table 1. An antibody or an antigen-binding fragment thereof of the invention may comprise the VH (SEQ ID NO: 19) and VL (SEQ ID NO: 20) sequence of VHg1/VLg11 (H1L11) (a humanised variant of antibody clone B9). An antibody or an antigen-binding fragment thereof of the invention may comprise the VH and VL sequence of a humanised variant of antibody clone B9 as shown in Table 1. SEQ ID NO: Clone Amino acid sequence SEQ ID NO: 21 VHg10_VLg11.seq VH PRT SEQ ID NO: 22 VHg10_VLg11.seq VL PRT SEQ ID NO: 23 VHg10_VLg12.seq VH PRT SEQ ID NO: 24 VHg10_VLg12.seq VL PRT SEQ ID NO: 25 VHg10_VLg4.seq VH PRT SEQ ID NO: 26 VHg10_VLg4.seq VL PRT SEQ ID NO: 27 VHg10_VLg5.seq VH PRT SEQ ID NO: 28 VHg10_VLg5.seq VL PRT SEQ ID NO: 29 VHg10_VLg6.seq VH PRT SEQ ID NO: 30 VHg10_VLg6.seq VL PRT SEQ ID NO: 31 VHg1_VLg11.seq VH PRT SEQ ID NO: 32 VHg1_VLg11.seq VL PRT SEQ ID NO: 33 VHg1_VLg12.seq VH PRT SEQ ID NO: 34 VHg1_VLg12.seq VL PRT SEQ ID NO: 35 VHg1_VLg2.seq VH PRT SEQ ID NO: 36 VHg1_VLg2.seq VL PRT SEQ ID NO: 37 VHg1_VLg4.seq VH PRT SEQ ID NO: 38 VHg1_VLg4.seq VL PRT SEQ ID NO: 39 VHg2_VLg10.seq VH PRT SEQ ID NO: 40 VHg2_VLg10.seq VL PRT SEQ ID NO: 41 VHg2_VLg11.seq VH PRT SEQ ID NO: 42 VHg2_VLg11.seq VL PRT 14 241018 CMAL003WO1 DCA SEQ ID NO: 43 VHg2_VLg12.seq VH PRT SEQ ID NO: 44 VHg2_VLg12.seq VL PRT SEQ ID NO: 45 VHg2_VLg13.seq VH PRT SEQ ID NO: 46 VHg2_VLg13.seq VL PRT SEQ ID NO: 47 VHg2_VLg2.seq VH PRT SEQ ID NO: 48 VHg2_VLg2.seq VL PRT SEQ ID NO: 49 VHg2_VLg4.seq VH PRT SEQ ID NO: 50 VHg2_VLg4.seq VL PRT SEQ ID NO: 51 VHg3_VLg11.seq VH PRT SEQ ID NO: 52 VHg3_VLg11.seq VL PRT SEQ ID NO: 53 VHg3_VLg12.seq VH PRT SEQ ID NO: 54 VHg3_VLg12.seq VL PRT SEQ ID NO: 55 VHg3_VLg13.seq VH PRT SEQ ID NO: 56 VHg3_VLg13.seq VL PRT SEQ ID NO: 57 VHg3_VLg4.seq VH PRT SEQ ID NO: 58 VHg3_VLg4.seq VL PRT SEQ ID NO: 59 VHg5_VLg11.seq VH PRT SEQ ID NO: 60 VHg5_VLg11.seq VL PRT SEQ ID NO: 61 VHg5_VLg12.seq VH PRT SEQ ID NO: 62 VHg5_VLg12.seq VL PRT SEQ ID NO: 63 VHg5_VLg13.seq VH PRT SEQ ID NO: 64 VHg5_VLg13.seq VL PRT SEQ ID NO: 65 VHg5_VLg2.seq VH PRT SEQ ID NO: 66 VHg5_VLg2.seq VL PRT SEQ ID NO: 67 VHg6_VLg1.seq VH PRT SEQ ID NO: 68 VHg6_VLg1.seq VL PRT SEQ ID NO: 69 VHg6_VLg11.seq VH PRT SEQ ID NO: 70 VHg6_VLg11.seq VL PRT SEQ ID NO: 71 VHg6_VLg12.seq VH PRT SEQ ID NO: 72 VHg6_VLg12.seq VL PRT SEQ ID NO: 73 VHg6_VLg13.seq VH PRT SEQ ID NO: 74 VHg6_VLg13.seq VL PRT SEQ ID NO: 75 VHg6_VLg2.seq VH PRT SEQ ID NO: 76 VHg6_VLg2.seq VL PRT SEQ ID NO: 77 VHg6_VLg4.seq VH PRT SEQ ID NO: 78 VHg6_VLg4.seq VL PRT SEQ ID NO: 79 VHg9_VLg11.seq VH PRT SEQ ID NO: 80 VHg9_VLg11.seq VL PRT Table 1. VH and VL sequences of humanised variants of antibody clone B9. 15 241018 CMAL003WO1 DCA An antibody or an antigen-binding fragment thereof of the invention may comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 further amino acid modifications in the VH and / or VL sequences, provided that functional properties of the antibody are retained. A modification may be an amino acid substitution, deletion or insertion. Preferably, the modification is a substitution. Amino acids may be referred to by their one letter or three letter codes, or by their full name. The one and three letter codes, as well as the full names, of each of the twenty standard amino acids are set out below. Amino acid One letter code Three letter code alanine A Ala arginine R Arg asparagine N Asn aspartic acid D Asp cysteine C Cys glutamic acid E Glu glutamine Q Gln glycine G Gly histidine H His isoleucine I Ile leucine L Leu lysine K Lys methionine M Met phenylalanine F Phe proline P Pro serine S Ser threonine T Thr tryptophan W Trp tyrosine Y Tyr valine V Val Table 2. Amino acids, one and three-letter codes. In preferred embodiments in which one or more amino acids are substituted with another amino acid, the substitutions may be conservative substitutions, for example according to the 16 241018 CMAL003WO1 DCA following table. In some embodiments, amino acids in the same category in the middle column are substituted for one another, i.e., a non-polar amino acid is substituted with another non- polar amino acid, for example. In some embodiments, amino acids in the same line in the rightmost column are substituted for one another. ALIPHATIC Non-polar G A P I L V Polar - C S T M uncharged N Q Polar - charged D E K R AROMATIC H F W Y Table 3. Amino acids In some embodiments, substitution(s) may be functionally conservative. That is, in some embodiments the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., binding affinity) of the antibody molecule comprising the substitution as compared to the equivalent unsubstituted antibody molecule. In a preferred embodiment, an antibody or an antigen-binding fragment thereof of the invention may comprise a VH and / or VL domain sequence with one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), preferably 20 alterations or fewer, 15 alterations or fewer, 10 alterations or fewer, 5 alterations or fewer, 4 alterations or fewer, 3 alterations or fewer, 2 alterations or fewer, or 1 alteration compared with the VH and / or VL sequences of the invention set forth herein. In preferred embodiments, a humanised antibody or an antigen-binding fragment thereof of the invention may comprise a humanised VH domain sequence of B9 set forth in SEQ ID NO: 19, e.g., a humanised VH domain with an amino acid sequence which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of B9 set forth in SEQ ID NO: 19. 17 241018 CMAL003WO1 DCA In preferred embodiments, a humanised antibody or an antigen-binding fragment thereof of the invention may comprise a humanised VL domain amino acid sequence of B9 set forth in SEQ ID NO: 20 e.g., a humanised VL domain with an amino acid sequence which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of B9 set forth in SEQ ID NO: 20. In preferred embodiments, a humanised antibody or an antigen-binding fragment thereof of the invention may comprise a humanised VH domain sequence of F10, e.g., a humanised VH domain with an amino acid sequence which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VH sequence of Clone F10. In preferred embodiments, a humanised antibody or an antigen-binding fragment thereof of the invention may comprise a humanised VL domain amino acid sequence of F10 e.g., a humanised VL domain with an amino acid sequence which has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the VL sequence of clone F10. Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g., BLAST (which uses the method of Altschul et al. (1990) J. MoI. Biol.215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol.147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm may be used (Nucl. Acids Res. (1997) 253389-3402). Sequence identity may be defined using the Bioedit, ClustalW algorithm. 18 241018 CMAL003WO1 DCA Alignments were performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms (Edgar (2004a) Nucleic Acids Res 32:1792-7; Edgar (2004b) BMC Bioinformatics 5:113.). The antibody may comprise a CH2 domain. The CH2 domain is preferably located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody is preferably the CH2 domain of human IgG1, IgG2, IgG3, or IgG4, more preferably the CH2 domain of human IgG1. The sequences of human IgG domains are known in the art. The antibody may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. Preferably, the hinge region, or part thereof, is a human IgG1, IgG2, IgG3 or IgG4 hinge region, or part thereof. More preferably, the hinge region, or part thereof, is an IgG1 hinge region, or part thereof. The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, most preferably a human IgG1 CH3 domain. An antibody of the invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. The sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art. An antibody of the invention may comprise a human IgG constant region, e.g., a human IgG1 constant region. A key mechanism of action for therapeutic antibodies in oncology is the targeted killing of tumour cells through recruitment of the immune system, which is achieved through interaction of the Fc domain with the complement component C1q or Fcγ receptors. C1q is a large multi-subunit protein, which can initiate CDC on binding to the Fc region of antibodies. Binding of C1q is the first step in the complement cascade, which induces a series of protein hydrolysis events, resulting in the formation of the membrane attack complex on the surface of the target cell. While C1q binds to the external surface of the Fc heavy chains, the affinity is impacted by the Fc glycan structure and in particular, galactose is known to increase C1q binding and therefore C1q activity. The structure of the hinge region can also inhibit binding by C1q and Fcγ receptors. Human IgG2 and IgG4 have more rigid and inaccessible hinge regions, which inhibit binding, resulting in lower Fc-mediated effector functionality in IgG2 and IgG4 molecules, compared to IgG1. 19 241018 CMAL003WO1 DCA Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM) and FcδR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classed as a high affinity receptor (nanomolar range KD) while FcγRII and FcγRIII are low to intermediate affinity (micromolar range KD). In antibody dependent cellular cytotoxicity (ADCC), FcγRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function varies for IgG subtypes. Although this is dependent on the allotype and specific FcγR in simple terms ADCC effector function is high for human IgG1 and IgG3, and low for IgG2 and IgG4. See below for IgG subtype variation in effector functions, ranked in decreasing potency. Effector Function Species IgG Subtype Potency ADCC Human IgG1≥IgG3>>IgG4>IgG2 Mouse IgG2b>IgG2a>IgG1>>IgG3 C1q Binding Human IgG3>IgG1>>IgG2>IgG4 Mouse IgG2a≥IgG2b>IgG3>IgG1 Table 4. Effector function and IgG subtype potency FcγRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions. Antibodies of the invention may have an Fc with effector function, with enhanced effector function or with reduced effector function. 20 241018 CMAL003WO1 DCA The potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such as ADCC, antibody-dependent cell-mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC). A number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D/A330L/I332E (“3M”), F243L or G236A. Alternatively, enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, FcγRs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity. Afucosylated (non-fucosylated) antibodies, exhibit greatly enhanced ADCC activity through increased binding to FcγRIIIa. An antibody of the invention may comprise a human IgG1 Fc (e.g., B9-hIgG1 (B9-H1)). An antibody of the invention may comprise an IgG1 V11 Fc (G237D/P238D/H268D/P271G/A330R) with enhanced or tailored affinity for hFcyRIIB (inhibitory). An antibody of the invention may comprise an IgG1 SELF Fc (S267E/L328F) conferring enhanced affinity for hFcyRIIB (inhibitory) & hFcyRIIA (activation). Activation of ADCC, ADCP and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred. Due to their lack of effector functions, IgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, IgG4 molecules can exchange half- molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of IgG4 antibodies with a reduced propensity for Fab-arm exchange. Fc engineering approaches have been used to determine the key interaction sites for the IgG1 Fc domain with Fcγ receptors and C1q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1q to a region covering the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fcγ receptors, such as FcγRI, FcγRlla, FcγRllb, FcγRIII and/or to 21 241018 CMAL003WO1 DCA complement. CH2 domains of human lgG domains normally bind to Fcγ receptors and complement, decreased binding to Fcγ receptors is expected to decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and decreased binding to complement is expected to decrease the complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations to decrease or abrogate binding of the CH2 domain to one or more Fcγ receptors and/or complement are known in the art. An antibody molecule of the invention may comprise an Fc with modifications K322A/L234A/L235A or L234F/L235E/P331S (“TM”), which almost completely abolish FcγR and C1q binding. An antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be decreased by mutation of Pro329 (position according to EU numbering), e.g., to either P329A or P329G. The antibody molecule of the invention may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA- PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering). Additionally or alternatively an antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297 (position 84.4 by IMGT numbering). Modification of glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction may confer a loss of binding to FcRs; a loss of binding to FcRs has been observed in N297 point mutations. An antibody molecule of the invention may comprise an Fc with an N297A, N297G or N297Q mutation. An antibody molecule of the invention with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor, or following the expression of Fc domains in bacteria. An antibody of the invention may comprise a human IgG Fc without effector function (“Fc silent”), for example, IgG1 N297A or IgG1 N297Q, e.g., “Fc-silent” B9-N297A or B9-N297Q. IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q/M428L variant, conferred an approximately 2-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y/S254T/T256E variant (“YTE”), gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy. 22 241018 CMAL003WO1 DCA Immunoglobulins are known to have a modular architecture comprising discrete domains, which can be combined in a multitude of different ways to create multivalent binding molecules, e.g., bivalent, trivalent, tetravalent or pentavalent binding molecules. Multivalent formats may be monospecific or multispecific. Multispecific binding molecules may be bispecific, trispecific, tetraspecific or pentaspecific antibody formats. Exemplary multivalent and multispecific antibody formats are described in Spiess et al. (2015) Mol Immunol 67: 95- 106 and Kontermann (2012) Mabs 4(2): 182-97, for example; antibodies of the invention may be employed in such formats. The invention contemplates the use of antigen-binding fragments of anti-human DR3 (anti- hDR3) antibodies of the invention described herein. Antibody fragments, which recognize specific epitopes, can be generated by known techniques. The antibody fragments are antigen binding portions of an antibody, such as F(ab′)2, Fab′, Fab, Fv, scFv and the like. Other antibody fragments include, but are not limited to: the F(ab)′2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab′ fragments, which can be generated by reducing disulphide bridges of the F(ab)′2 fragments. Alternatively, Fab′ expression libraries can be constructed to allow rapid and easy identification of monoclonal Fab′ fragments with the desired specificity. The present invention encompasses antigen- binding proteins that comprise antibodies and antigen-binding fragments thereof in accordance with the invention. A single chain Fv molecule (scFv) comprises a VL domain and a VH domain. The VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L). A scFv molecule is denoted as either VL-L-VH if the VL domain is the N-terminal part of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal part of the scFv molecule. Methods for making scFv molecules and designing suitable peptide linkers are known in the art. An antibody fragment can be prepared by proteolytic hydrolysis of the full-length antibody or by expression in a host of the DNA coding for the fragment. An antibody fragment can be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. An antibody fragment can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulphide linkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, an enzymatic cleavage using papain produces two monovalent Fab fragments and an Fc 23 241018 CMAL003WO1 DCA fragment directly. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). A CDR is a segment of the variable region of an antibody that is complementary in structure to the epitope to which the antibody binds and is more variable than the rest of the variable region. Accordingly, a CDR is sometimes referred to as hypervariable region. A variable region comprises three CDRs. CDR peptides can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, provided that the fragments bind to the antigen that is recognized by the intact antibody. Antibody fusion proteins and fragments thereof can be prepared by a variety of conventional procedures, ranging from glutaraldehyde linkage to more specific linkages between functional groups. The antibodies and/or antibody fragments are preferably covalently bound to one another, directly or through a linker moiety, through one or more functional groups on the antibody or fragment, e.g., amine, carboxyl, phenyl, thiol, or hydroxyl groups. Various conventional linkers in addition to glutaraldehyde can be used, e.g., diisiocyanates, diiosothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimide- hydroxysuccinimide esters, and the like. A simple method to produce murine, chimeric, humanised and human DR3 antibody fusion proteins is to mix the antibodies or fragments in the presence of glutaraldehyde to form an antibody fusion protein. The initial Schiff base linkages can be stabilized, e.g., by borohydride reduction to secondary amines. A diiosothiocyanate or carbodiimide can be used in place of glutaraldehyde as a non-site-specific linker. A multivalent, monospecific antibody of the invention may be an antibody fusion protein comprising at least two murine, chimeric, humanised or human anti-hDR3 mAbs, or antigen- binding fragments thereof, wherein at least two of the mAbs or antigen-binding fragments bind to the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). A multivalent, bispecific antibody of the invention may be an antibody fusion protein comprising at least two murine, chimeric, humanised or human anti-DR3 mAbs, or antigen-binding fragments thereof, wherein at least one of the mAbs or antigen-binding fragment thereof binds to the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) 24 241018 CMAL003WO1 DCA and a second mAb or antigen-binding fragment thereof binds to a different epitope of human DR3 or an epitope of a different antigen. For example, a bispecific DR3 antibody fusion protein may comprise an anti-hDR3 mAb or fragment thereof and second antibody or fragment thereof and that binds to a different antigen. Such a bispecific DR3 antibody fusion protein can be prepared, for example, by obtaining an F(ab′)2 fragments. The interchain disulphide bridges of the antibody F(ab′)2 fragment are gently reduced with cysteine, taking care to avoid light-heavy chain linkage, to form Fab′-SH fragments. The SH group(s) is (are) activated with an excess of bis-maleimide linker (1,1′- (methylenedi-4,1-phenylene)bis-maleimide). The anti-hDR3 mAb is converted to Fab′-SH and then reacted with the activated second Fab′-SH fragment to obtain a bispecific anti-hDR3 antibody fusion protein. A poly-specific NTI-DR3 antibody fusion protein can be obtained by adding DR3 antigen binding moieties to a bispecific chimeric, humanised or human DR3 antibody fusion protein. For example, a bispecific antibody fusion protein can be reacted with 2-iminothiolane to introduce one or more sulphydryl groups for use in coupling the bispecific fusion protein to a third DR3 antigen mAb or fragment, using the bis-maleimide activation procedure described above. These techniques for producing antibody composites are well known to those of skill in the art. Bispecific antibodies can be made by a variety of conventional methods, e.g., disulphide cleavage and reformation of mixtures of whole IgG or, preferably F(ab′)2 fragments, fusions of more than one hybridoma to form polyomas that produce antibodies having more than one specificity, and by genetic engineering. Bispecific antibody fusion proteins have been prepared by oxidative cleavage of Fab′ fragments resulting from reductive cleavage of different antibodies. This is advantageously carried out by mixing two different F(ab′)2 fragments produced by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of Fab′ fragments, followed by oxidative reformation of the disulphide linkages to produce a mixture of F(ab′)2 fragments including bispecific antibody fusion proteins containing a Fab′ portion specific to each of the original epitopes. General techniques for the preparation of antibody fusion proteins are known to those of skill in the art. More selective linkage can be achieved by using a heterobifunctional linker such as maleimidehydroxysuccinimide ester. Reaction of the ester with an antibody or fragment will derivatise amine groups on the antibody or fragment, and the derivative can then be reacted with, e.g., an antibody Fab fragment having free sulfhydryl groups (or, a larger fragment or 25 241018 CMAL003WO1 DCA intact antibody with sulfhydryl groups appended thereto by, e.g., Traut's Reagent). Such a linker is less likely to crosslink groups in the same antibody and improves the selectivity of the linkage. It is advantageous to link the antibodies or fragments at sites remote from the antigen binding sites. This can be accomplished by, e.g., linkage to cleaved interchain sulphydryl groups, as noted above. Another method involves reacting an antibody having an oxidized carbohydrate portion with another antibody, which has at least one free amine function. This results in an initial Schiff base (mime) linkage, which is preferably stabilized by reduction to a secondary amine, e.g., by borohydride reduction, to form the final composite. ScFvs with linkers greater than 12 amino acid residues in length (for example, 15- or 18- residue linkers) allow interacting between the VH and VL domains on the same chain and generally form a mixture of monomers, dimers (termed diabodies) and small amounts of higher mass multimers. ScFvs with linkers of 5 or less amino acid residues, however, prohibit intramolecular pairing of the VH and VL domains on the same chain, forcing pairing with VH and VL domains on a different chain. Linkers between 3- and 12-residues form predominantly dimers. With linkers between 0 and 2 residues, trimeric (termed triabodies), tetrameric (termed tetrabodies) or higher oligomeric structures of scFvs are formed; however, the exact patterns of oligomerization appear to depend on the composition as well as the orientation of the V- domains, in addition to the linker length. The anti-hDR3 antibodies and antigen-binding fragments thereof of the present invention can be used to produce antigen-specific diabodies, triabodies and tetrabodies, which are multivalent but monospecific. The non-covalent association of two or more scFv molecules can form functional diabodies, triabodies and tetrabodies. Monospecific diabodies are homodimers of the same scFv, where each scFv comprises the VH domain from the selected antibody connected by a short linker to the VL domain of the same antibody. A diabody is a bivalent homodimer formed by the non-covalent association of two scFvs, yielding two Fv binding sites. A triabody results from the formation of a trivalent trimer of three scFvs, yielding three binding sites, and a tetrabody is a tetravalent tetramer of four scFvs, resulting in four binding sites. Several monospecific diabodies have been made using an expression vector that contains a recombinant gene construct comprising VH1-linker-VL1. Methods of constructing scFvs and multivalent, monospecific binding proteins based on scFv are known in the art. 26 241018 CMAL003WO1 DCA In a preferred embodiment of the invention, the antigen-binding protein is a multivalent, monospecific antibody or antigen-binding fragment thereof comprising antigen binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). In a preferred embodiment of the invention the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). In a preferred embodiment of the invention the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc capable of binding to FcγR (for FcR mediated cross-linking), e.g., (e.g., B9-hIgG1 (B9-H1)). In an alternative embodiment of the invention the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc that is silent (not capable of binding to FcγR), such as IgG1 N297A, N297Q, IgG1 LALA-PG, IgG1 N297A LALA-PG or IgG1 N297Q LALA-PG. In a preferred embodiment of the invention the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). In a preferred embodiment of the invention the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) or IgG1 Fc is absent. In a preferred embodiment of the invention the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human 27 241018 CMAL003WO1 DCA DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) and / or Fc is absent. In a preferred embodiment of the invention the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 N297A Fc in which Fc function is silent (does not bind FcγR), e.g., “Fc-silent” TET-B9-N297A. In an alternative embodiment of the invention the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen- binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc (capable of binding FcγR); e.g., tetravalent (TET) B9-H1 (TET-B9-H1). In an alternative embodiment of the invention the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent, tetravalent or pentavalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc. The invention provides a murine, chimeric, humanised or human antibody or antigen-binding fragment thereof, capable of competing with an antibody of the invention described herein (e.g., comprising a set of HCDR and LCDRs of Clone B9 or F10, the VH and VL amino acid sequences of Clone B9 or F10 or a humanised variant of the VH and VL amino acid sequences of Clone B9), for binding to an isolated recombinant peptide comprising an epitope, said peptide comprising or consisting of residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2), when assessed in a competition assay. Competition assays include cell-based and cell-free binding assays including an immunoassay such as ELISA (enzyme-linked immunosorbent assay), HTRF (homogeneous time-resolved fluorescence), flow cytometry, fluorescent microvolume assay technology (FMAT) assay, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, bio-layer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays. 28 241018 CMAL003WO1 DCA An antibody that binds to the same epitope as, or an epitope overlapping with, a reference antibody refers to an antibody that blocks binding of the reference antibody to its binding partner (e.g., an antigen or “target”) in a competition assay by 50% or more, and / or conversely, the reference antibody blocks binding of the antibody to its binding partner in a competition assay by 50% or more. Such antibodies are said to compete for binding to an epitope of interest, such as an epitope comprising or consisting of residues 37 – 45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). An antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the invention may be conjugated to a detectable label (for example, a radioisotope); or to a bioactive molecule. In this case, the antigen-binding protein, such as an antibody or antigen- binding fragment thereof may be referred to as a conjugate. Such conjugates may find application in the treatment and/or diagnosis of diseases as described herein. Such conjugates may find application for the detection (e.g., in vitro detection) of an epitope comprising or consisting of residues 37 – 45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). The antigen-binding proteins of the invention (including conjugates) may be useful in the detection (e.g., in vitro detection) of an epitope of the invention (an epitope present on an isolated recombinant peptide consisting of residues 37-45 (SEQ ID NO: 1) of human DR3 ; preferably said epitope is formed by residues of the amino acid sequence 37-45 of human DR3 (SEQ ID NO: 2). Thus, the present invention relates to the use of an antigen-binding protein of the invention for detecting the presence of the epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) in a sample. The antigen-binding protein may be conjugated to a detectable label as described elsewhere herein. In a preferred embodiment, the present invention relates to an in vitro method of detecting an epitope of the invention in a sample, wherein the method comprises incubating an antigen- binding protein of the invention with a sample of interest, and determining binding of the antigen-binding protein to an epitope of the invention present in the sample, wherein binding of the antigen-binding protein indicates the presence of an epitope of the invention in the sample. Methods for detecting binding of an antigen-binding protein to its target antigen are known in the art and include ELISA, ICC (immunocytochemistry), IHC (immunohistochemistry), immunofluorescence, western blot, IP (immunoprecipitation), SPR and flow cytometry. The sample of interest may be a sample obtained from an individual. The individual may be human. Samples include, but are not limited to, tissue such as brain tissue, cerebro-spinal 29 241018 CMAL003WO1 DCA fluid (CSF), primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, plasma, serum, blood-derived cells, urine, saliva, sputum, tears, perspiration, mucus, tumour lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumour tissue, cellular extracts, and combinations thereof. Following incubation, antigen-binding protein to antigen binding, e.g., antibody to antigen binding, is detected using an appropriate detection system. The method of detection can be direct or indirect, and may generate a fluorescent or chromogenic signal. Direct detection involves the use of primary antibodies that are directly conjugated to a label. Indirect detection methods employ a labelled secondary antibody raised against the primary antigen-binding protein, e.g., antibody, host species. Indirect methods may include amplification steps to increase signal intensity. Commonly used labels for the visualization (i.e., detection) of antigen-binding protein – antigen (e.g., antibody – epitope) interactions include fluorophores and enzymes that convert soluble substrates into insoluble, chromogenic end products. The term "detecting" is used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect. Suitable detectable labels which may be conjugated to antigen-binding proteins, such as antibodies, are known in the art and include radioisotopes such as iodine-125, iodine-131, yttrium-90, indium-111 and technetium-99; fluorochromes, such as fluorescein, rhodamine, phycoerythrin, Texas Red and cyanine dye derivatives for example, Cy7, Alexa750 and Alexa Fluor 647; chromogenic dyes, such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; phospho or laser dyes with spectrally isolated absorption or emission characteristics; electro-chemiluminescent labels, such as SULFO-TAG which may be detected via stimulation with electricity in an appropriate chemical environment; and chemical moieties, such as biotin, which may be detected via binding to a specific cognate detectable moiety, e.g., labelled avidin or streptavidin. An antigen-binding protein, such as an antibody or fragment thereof, of the invention may be conjugated to the detectable label by means of any suitable covalent or non-covalent linkage, such as a disulphide or peptide bond. Suitable peptide linkers are known in the art and may be 5 to 25, 5 to 20, 5 to 15, 10 to 25, 10 to 20, or 10 to 15 amino acids in length. 30 241018 CMAL003WO1 DCA The invention also provides a nucleic acid or set of nucleic acids encoding an antibody or antigen-binding fragment of the invention, as well as a vector comprising such a nucleic acid or set of nucleic acids. Where the nucleic acid encodes the VH and VL domain, or heavy and light chain, of an antibody molecule of the invention, the two domains or chains may be encoded on the same or on separate nucleic acid molecules. An isolated nucleic acid molecule may be used to express an antibody molecule of the invention. The nucleic acid will generally be provided in the form of a recombinant vector for expression. Another aspect of the invention thus provides a vector comprising a nucleic acid as described above. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Vectors may be plasmid vectors, or viral vectors, e.g., phage, or phagemid, as appropriate. A nucleic acid molecule or vector as described herein may be introduced into a host cell. Techniques for the introduction of nucleic acid or vectors into host cells are well established in the art and any suitable technique may be employed. A range of host cells suitable for the production of recombinant antibody molecules are known in the art, and include e.g., bacterial, yeast, insect or mammalian host cells. A preferred host cell is a mammalian cell, such as a CHO, NS0, or HEK cell, for example a HEK293 cell. A recombinant host cell comprising a nucleic acid or the vector of the invention is also provided. Such a recombinant host cell may be used to produce an antigen-binding protein (e.g., antibody) of the invention. Thus, also provided is a method of producing an antigen- binding protein, e.g., antibody, of the invention, the method comprising culturing the recombinant host cell under conditions suitable for production of the antigen-binding protein, e.g., antibody. The method may further comprise a step of isolating and/or purifying the antigen-binding protein, e.g., antibody. Thus the invention provides a method of producing an antigen-binding protein, e.g., antibody, of the invention comprising expressing a nucleic acid encoding the antigen-binding protein, e.g., antibody, in a host cell and optionally isolating and/or purifying the antigen-binding protein, e.g., antibody, thus produced. Methods for culturing host cells are well-known in the 31 241018 CMAL003WO1 DCA art. Techniques for the purification of recombinant antigen-binding proteins, e.g., antibodies, are well-known in the art and include, for example HPLC, FPLC or affinity chromatography, e.g., using Protein A or Protein L. In some embodiments, purification may be performed using an affinity tag on an antigen-binding protein, e.g., antibody. The method may also comprise formulating the antigen-binding protein, e.g., antibody, into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substance as described below. Antigen-binding proteins, e.g., antibodies, of the invention are expected to find application in therapeutic applications, in particular therapeutic applications in human patients, for example in the treatment of a cancer, including but not limited to, a cancer selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied. Also provided is a composition, such as a pharmaceutical composition, comprising an antigen- binding protein, e.g., antibody, according to the invention and an excipient, such as a pharmaceutically acceptable excipient. The invention further provides an antigen-binding protein, e.g., antibody, of the invention, for use in a method of treatment. Also provided is a method of treating a patient, wherein the method comprises administering to the patient a therapeutically-effective amount of an antigen-binding protein, e.g., antibody, according to the invention. Further provided is the use of an antigen-binding protein, e.g., antibody, according to the invention for use in the manufacture of a medicament. A patient, as referred to herein, is preferably a human patient. The invention also provides an antigen-binding protein, e.g., antibody, of the invention, for use in a method of treating a cancer, in a patient. Also provided is a method of treating a cancer, in a patient, wherein the method comprises administering to the patient a therapeutically- effective amount of an antigen-binding protein, e.g., antibody, according to the invention. Further provided is the use of an antigen-binding protein, e.g., antibody, according to the invention for use in the manufacture of a medicament for the treatment of a cancer, in a patient. Methods of treatment of the invention may comprise administration of nucleic acid sequences encoding an antigen-binding protein, e.g., antibody, of the invention. The invention provides a 32 241018 CMAL003WO1 DCA “vaccine” composition comprising nucleic acid sequence encoding an antigen-binding protein, e.g., antibody, of the invention. A “vaccine” composition of the invention comprising nucleic acid sequence encoding an antigen-binding protein, e.g., antibody, of the invention, may be in a form suitable for administration of the composition to an individual, such as a human. A “vaccine” composition of the invention may comprise a nucleic acid sequence encoding an antigen-binding protein, e.g., antibody, of the invention and a pharmaceutically acceptable excipient. The treatment may further comprise administering to the patient a second therapy, such as a second therapy selected from a chemotherapy, kinase inhibitor, vaccine, tumour targeting mAb (e.g., anti-CD20, anti-HER2, anti-VEGFR), antibody-drug conjugate, bispecific T cell engager, checkpoint inhibitor targeting an immune receptor (e.g., PD-1, PD-L1, CTLA-4, LAG3), mAb targeting other co-stimulatory receptors (e.g., CD27, GITR, OX40, 4-1BB, ICOS, CD28) and an agent targeting myeloid suppressor cells. The second therapy may be administered to the patient simultaneously, separately, or sequentially to the antigen-binding protein, e.g., antibody, of the invention. In embodiments of a method of treatment of the invention, an anti-hDR3 antibody of the invention is administered in combination with a second therapy selected from a mAb targeting an immune receptor (e.g., PD-1, PD-L1, CTLA-4, LAG3), and a mAb targeting a co-stimulatory receptors (e.g., CD27, GITR, OX40, 4-1BB, ICOS, CD28). In a preferred embodiment of a method of treatment of the invention, an anti- hDR3 antibody of the invention is administered in combination with a second therapy that is an anti-PD-1 antibody. In another aspect, the invention relates to an antigen-binding protein, e.g., antibody, of the invention for use in: a) treating a cancer, b) delaying progression of a cancer, c) prolonging the survival of a patient suffering from a cancer. The antigen-binding protein, e.g., antibody, as described herein may thus be for use for therapeutic applications, in particular for the treatment of a cancer; for example wherein the cancer is selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied. 33 241018 CMAL003WO1 DCA An antigen-binding protein, e.g., antibody, as described herein may be used in a method of treatment of the human or animal body. Related aspects of the invention provide; (i) an antigen-binding protein, e.g., antibody, described herein for use as a medicament, (ii) an antigen-binding protein, e.g., antibody, described herein for use in a method of treatment of a disease or disorder, (iii) the use of an antigen-binding protein, e.g., antibody, described herein in the manufacture of a medicament for use in the treatment of a disease or disorder; and, (iv) a method of treating a disease or disorder in an individual, wherein the method comprises administering to the individual a therapeutically effective amount of an antigen-binding protein, e.g., antibody, as described herein. The individual may be a patient, preferably a human patient. The individual may be an animal, such as a mouse, rat, guinea pig, rabbit, dog, cat, or primate. Treatment may be any treatment or therapy in which some desired therapeutic effect is achieved, for example, the inhibition or delay of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, cure or remission (whether partial or total) of the condition, preventing, ameliorating, delaying, abating or arresting one or more symptoms and/or signs of the condition or prolonging survival of an individual or patient beyond that expected in the absence of treatment. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, an individual susceptible to or at risk of the occurrence of a cancer, for example as described herein, may be treated as described herein. Such treatment may prevent or delay the occurrence of the disease in the individual. A method of treatment as described may comprise administering at least one further treatment to the individual in addition to the antigen-binding protein, e.g., antibody. The antigen-binding protein, e.g., antibody, described herein may thus be administered to an individual alone or in combination with one or more other treatments. When the antigen-binding protein, e.g., antibody, is administered to the individual in combination with another treatment, the additional treatment may be administered to the individual concurrently with, sequentially to, or separately from the administration of the antigen-binding protein, e.g., antibody. Where the additional treatment is administered concurrently with the antigen-binding protein, e.g., antibody, the antigen-binding protein, e.g., antibody, and additional treatment may be administered to the individual as a combined preparation. For example, the additional therapy may be a known therapy or therapeutic agent for the disease to be treated. The at least one 34 241018 CMAL003WO1 DCA further treatment may be one or more treatment selected from a vaccine, chemotherapy, kinase inhibitor, vaccine, tumour targeting mAb (e.g., anti-CD20, anti-HER2, anti-VEGFR), antibody-drug conjugate, bispecific T cell engager, checkpoint inhibitor targeting immune receptors (e.g., PD-1, PD-L1, CTLA-4, LAG3), mAb targeting other co-stimulatory receptors (e.g., CD27, GITR, OX40, 4-1BB, ICOS, CD28) and an agent targeting myeloid suppressor cells. In a preferred embodiment of a method of treatment of the invention, an anti-hDR3 antibody of the invention is administered in combination with an anti-PD-1 antibody. Whilst an antigen-binding protein, e.g., antibody, may be administered alone, antigen-binding proteins, e.g., antibodies, will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component (excipient) in addition to the antigen-binding protein, e.g., antibody. Another aspect of the invention therefore provides a pharmaceutical composition comprising an antigen-binding protein, e.g., antibody, as described herein. A method comprising formulating an antigen-binding protein, e.g., antibody, into a pharmaceutical composition is also provided. Pharmaceutical compositions may comprise, in addition to the antigen-binding protein, e.g., antibody, a pharmaceutically acceptable excipient, such as a diluent, carrier, buffer, stabilizer and / or other materials well known to those skilled in the art. The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each excipient must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or any other suitable route, as discussed below. For parenteral, for example subcutaneous or intravenous administration, e.g., by injection, the pharmaceutical composition comprising the antigen-binding protein, e.g., antibody, may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are able to prepare suitable solutions using, for example, isotonic vehicles, such as sodium chloride Injection, Ringer's Injection, or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be employed as required including buffers such as phosphate, citrate and other organic acids; antioxidants, such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium 35 241018 CMAL003WO1 DCA chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3’-pentanol; and m-cresol); low molecular weight polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagines, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, 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 as TWEEN^TM, PLURONICS^TM or polyethylene glycol (PEG). In some embodiments, antigen-binding proteins, e.g., antibodies may be provided in a lyophilised form for reconstitution prior to administration. For example, lyophilised antigen- binding proteins, e.g., antibodies may be reconstituted in sterile water or saline prior to administration to an individual. Administration may be in a "therapeutically-effective amount", this being sufficient to show benefit to an individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of antigen-binding protein, e.g., antibody,, the method of administration, the scheduling of administration and other factors known to medical practitioners. Prescription of treatment, e.g., decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and may depend on the severity of the symptoms and/or progression of a disease being treated. Appropriate doses of antigen-binding protein, e.g., antibodies, are well known in the art. A therapeutically effective amount or suitable dose of an antigen-binding protein, e.g., antibody, can be determined by comparing in vitro activity and in vivo activity in an animal model. Methods for extrapolation of effective dosages in mice and other test animals to humans are known. The precise dose will depend upon a number of factors, including whether the size and location of the area to be treated, and the precise nature of the antigen-binding protein, e.g., antibody. A typical antibody dose is in the range 100 µg to 1 g for systemic applications, and 1 µg to 1 mg for topical applications. An initial higher loading dose, followed by one or more lower doses, may be administered. This is a dose for a single treatment of an adult individual, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. 36 241018 CMAL003WO1 DCA Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. The treatment schedule for an individual may be dependent on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration and the nature of the condition being treated. Treatment may be periodic, and the period between administrations may be about two weeks or more, e.g., about three weeks or more, about four weeks or more, about once a month or more, about five weeks or more, or about six weeks or more. For example, treatment may be every two to four weeks or every four to eight weeks. Suitable formulations and routes of administration are described above. In a preferred embodiment, an antibody as described herein may be for use in a method of treating cancer. An antigen-binding protein, such as an antibody or an antigen-binding fragment thereof of the invention is not an anti-DR3 antibody described in: (a) WO0135995 (Tittle, et al.). (b) WO0064465 (Human Genome Sciences Inc). (c) "DR3/TNFRSF25 antibody" DR3/TNFRSF25 Antibody (1H2) (H00008718-M07): Novus Biologicals murine IgG2a anti-DR3 antibody, 1H2. (d) WO2011106707 (Human Genome Sciences Inc.). (e) WO2012117067 (Novo Nordisk AS). (f) WO2015152430 (Kyowa Hakko Kirin Co., Ltd). (g) WO2016081455 (Pelican Therapeutics, Inc) In preferred embodiments, an antigen-binding protein, such as an antibody or an antigen- binding fragment thereof of the invention does not bind to an epitope comprised in residues 1 to 32, 1 to 13, 64-69, or in CRD1 of human DR3. List of Figures Figure 1: The binding kinetics of anti-hDR3 mAb B9 and F10 to recombinant hDR3. The binding kinetics of anti-hDR3 mAb B9 and F10 to recombinant hDR3 were determined using SPR technology and the bivalent fit on the Biacore system. The KD indicated that B9 had the greatest avidity. The original parent mouse isotypes, (A) B9 mIgG2A (B9-M2A) and (B) F10 mIgG1 (F10-M1), are used in this illustrative analysis which is representative of data collected using the human IgG1 chimera. 37 241018 CMAL003WO1 DCA Figure 2: Characterisation of anti-hDR3 antibody specificity and T cell targets. (A) Mapping the B9 anti-hDR3 binding epitope using HEK293T cells transfected with pcDNA3.1 encoding N-terminal Rituximab epitope (Rtx) tagged CRD-truncated proteins and human-mouse CRD1- 2 chimeras of DR3 lacking the intracellular death domain (constructs illustrated above each histogram column, these data are also representative of F10 binding patterns). HEK293T cells were transfected with: vector control (pcDNA3.1(+)), full-length Rtx-hDR3 (SEQ ID NO: 81), Rtx-hCRD1-2 (SEQ ID NO: 82), Rtx-hCRD1-mCRD2 (SEQ ID NO: 83), Rtx-mCRD1-hCRD2 (SEQ ID NO: 84) and a murine edit of hCRD1 (h/mCRD1) attached to hCRD2 (Rtx-h/mCRD1- hCRD2 (SEQ ID NO: 85)). Each transfectant was screened by flow cytometry after labelling cells with: Rituximab (top row), R&D anti-hDR3 (αhDR32^nd row), B9 anti-hDR3 (B9 αhDR33^rd row) and finally LPA-2 anti-mouse DR3 (αmDR3) (4^th row). The grey peaks illustrate the isotype controls and positive binding is illustrated when there is a shift in the transparent peak. Figure 2: (A) illustrative of three experiments and representative of the F10 epitope mapping data. (B) Alignment of hDR3 hCRD1 with mCRD1 highlighting the species difference (Consensus sequence, SEQ ID NO: 86): the first 7 amino acid differences in hCRD1 (Positions 37-40, 42-43 and 45 in the hDR3 protein sequence (SEQ ID NO: 87) were replaced with their respective residues from mCRD1 (SEQ ID NO: 88) to create Rtx-h/mCRD1-hCRD2 used in Figure 2A. (C) Streptavidin-labelling of B9-biotin anti-hDR3 bound to CD4⁺, CD8⁺, CD25⁺CD127^LowCD4⁺ T regulatory cell (Treg), and CD25-CD127^HighCD4⁺ T cells in naïve PBMC. (D) Statistical comparisons of B9 anti-hDR3 labelling in CD4/CD8⁺ naïve (Tn = CD45RA⁺CCR7⁺), central memory (Tcm = CD45RA-CCR7⁺), effector memory (Tem = CD45RA-CCR7-) or effector memory RA⁺ (Temra = CD45RA⁺CCR7-) was calculated after subtracting the isotype control median fluorescence intensity and dividing by the median fluorescence intensity found in the total CD4⁺/CD8⁺ T cell population (each dot represents an individual donor). Statistical analysis was conducted using the 1-way ANOVA and Tukey’s multiple comparison test where *P<0.05 and ***P<0.001. Figure 3: The epitope of the B9 and F10 anti-hDR3 mAbs does not overlap or compete with the epitope of PTX-25 hIgG1 chimera (PBM01, WO2016081455A1 or US20180312599A1) anti-hDR3 mAb. hDR3+Jurkat cells were stained with biotinylated B9-mIgG2A (B9-M2A) or isotype control mAb (18B12-mIgG2A (M2A)) (A) and biotinylated F10-mIgG1 (F10-M1) or isotype control mAb (M63D10-mIgG1(M1)) (B) in the presence of titrated concentrations of B9-hIgG1, F10-hIgG1, PTX-25-hIgG1 (Pelican-H1) or the InVivoMAb BE0297 (BioXCell human IgG1 isotype control (H1)). The percentage of binding of each biotinylated antibody was determined after analysing the level of secondary labelling with Streptavidin-APC via the flow cytometer (A-B). See figure 3C for the position of the B9 (and F10) epitope compared to 38 241018 CMAL003WO1 DCA the PTX-25 epitope on the human (SEQ ID NO: 89) / cynomolgus (SEQ ID NO: 90) / mouse (SEQ ID NO: 91) DR3 overlay. Figure 4: B9-hIgG1 is a potent co-stimulator of human T cells in vitro when compared to B9- hIgG2/4 and clinically evaluated Urelumab. CFSE-stained PBMC were co-cultured for four days with 0.1ng/ml anti-CD3 antibody (OKT3) with/without: TL1A (0.25µg/ml) or mAb detailed on the X-axis (0.4µg/ml (A-C and F-G) or 0.0032µg/ml (D-E)). The different human IgG Fc regions (H) of each mAb are indicated with their respective numbers: H1, H2 and H4 (Urelumab = anti-4-1BB hIgG4). After four days, CFSE dilution (% CFSE^lo) was analysed in CD4^High cell (B/D/F) and CD8^High cell (C/E/G) populations by flow cytometry (illustrated by A). Data presented in A-E were derived from serial 1/5 antibody titration curves including six PBMC donors. The majority of the data (pooled from six donors) fitted a normal distribution pattern and mAbs were compared to each respective isotype control using the paired t-test. Different B9 IgG constructs were compared using the 1-way ANOVA and Tukey’s multiple comparison test. Statistical significance is marked by asterisks where: *P<0.05, **P<0.01 and ***P<0.001. Figure 5: The agonistic activity of different B9-hIgG1 Fc variants. CFSE-stained PBMC were co-cultured for four days with 0.1ng/ml OKT3 in the presence of 0.4µg/ml (A-B) or 0.0032µg/ml (C-D) mAb (concentrations derived from a serial 1/5 titration curve) detailed on the X-axis: different variants of human IgG1 Fc (parental H1 versus SELF/V11/N297A). After four days, CFSE dilution (% CFSE^lo) was analysed in CD4^High (A + C) and CD8^High (B + D) T cell populations using flow cytometry. The majority of the data pooled from five donors fitted a normal distribution pattern and was compared to the H1 isotype control using the paired t-test. Different B9 constructs were compared using the 1-way ANOVA and Tukey’s multiple comparison test. Statistical significance is marked by asterisks where: *P<0.05 and **P<0.01. Figure 6: T cell subsets activated by DR3 co-stimulation. CFSE-stained PBMC were co- cultured for 4 days in the presence of 0.1ng/ml OKT3 with 0.016µg/ml B9-hIgG1 (B9-H1) or hIgG1 (H1) mAb (taken from a serial 1/5 titration). (A) Representative histogram of Foxp3 labelling in proliferating (CFSE^lo) CD4 where the transparent peak illustrates the B9-H1 binding over the grey H1 peak. (B) Proliferating Foxp3-CD4⁺ T cell numbers collected using the Foxp3- gate (n=6). (C) Proliferating Foxp3⁺CD4⁺ T cell numbers collected using the Foxp3⁺ gate (n=6). (D) CFSE-stained PBMC were co-cultured for 4 days in the presence of OKT3 with 0.08µg/ml B9-H1/H1 mAb and analysed for CD8 proliferation (% CFSE^lo CD8 cells) in comparison with: (E) CFSE labelled autologous CD8⁺ T cells, co-cultured with irradiated autologous CD14⁺ 39 241018 CMAL003WO1 DCA monocytes, co-stimulated with the same OKT3 and 0.08µg/ml B9-H1/H1 (n=5) . Data were compared using the paired t-test: *P<0.05 and **P<0.01. Figure 7: DR3 is associated with better outcomes in head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM) human cancers as well as delaying an aggressive mouse melanoma model. (A) The positive association of high CD8⁺ T cell levels and high DR3 (TNFRSF25) expression with cumulative HNSC-HPV⁺ and SKCM-Metastasis survival was determined using the online “Tumor Immune Estimation Resource”. (B) TILs isolated from seven HPV⁺ HNSC tumour samples were stimulated for 4 days with plate-bound OKT3 in the presence of autologous irradiated peripheral CD14⁺ cells and 0.1µg/ml B9-hIgG1 (B9-H1) mAb compared to hIgG1 control and proliferation was analysed through measuring the incorporation of tritiated thymidine (paired Wilcoxon *P<0.05). (C) CD3⁺ TIL isolated from 6 Skin Squamous Carcinoma (SSC) patient tumours were challenged for 3 days with soluble 0.5ng/ml OKT3 and 0.1µg/ml H1/B9-H1 in the presence of autologous CD3-depleted irradiated-PBMCs and proliferation was analysed through measuring the incorporation of tritiated thymidine (paired t-test *P<0.05). (D-E) 3 groups of 5 homozygous hDR3-transgenic (hDR3-Tg) mice were challenged with 200,000 B16-OVA S.C. and vaccinated with 5mg OVA plus 200µg B9-mIgG1/mIgG1 (B9-M1/M1) I.V. the following day. OVA-specific CD8⁺ T cell immune responses in the blood were assessed 6 days after vaccination (D). (E) B16-OVA tumour growth (E – 2-way ANOVA & Bonferroni’s test *P<0.05) was monitored over time (error bars = SEM). Figure 8: Therapeutic combination of anti-hDR3 mAb with anti-PD-1 mAb delays B16-OVA growth in heterozygous hDR3-Tg mice. Therapeutic combination of anti-hDR3 mAb with anti- PD-1 mAb delays B16-OVA growth in heterozygous hDR3-Tg mice. Heterozygous hDR3-Tg mice were challenged on day zero with 5x10⁵ B16-OVA tumour cells S.C. Mice then received 200µg mAbs to a total dose of 400µg mAb per injection given I.P. on days three, five and seven. Tumour growth was measured over time where the error bars represent SEM. Figure 9: Potent and Fc-independent co-stimulation of human T cells by Fab:IgG tetravalent B9. (A) Illustration of the multimeric structure of the Fab:IgG tetravalent antibody (referred to as “TET”). (B-C) PBMC were co-stimulated with 0.1ng/ml OKT3 with/without titrated concentrations (serial 1/10 dilutions) of the different anti-hDR3 constructs (hIgG1:H1, N297A) compared to their respective isotype control. After four days, CFSE dilution (% CFSE^lo) was analysed in the CD4⁺ T cells (B) and CD8⁺ T cells (C) using flow cytometry. Figures D-E illustrate data pooled from 6 donors using the 0.01µg/ml dose in CD4⁺ T cells (D) and CD8⁺ T 40 241018 CMAL003WO1 DCA cells (E). Data were collected from 6 different donors and analysed using the 1-way ANOVA with Tukey’s post-hoc test where: *P<0.05, **P<0.01 and ***P<0.001. Figure 10: Fab:IgG tetravalent B9-mIgG1 inhibits lung metastasis of B16 melanoma in hDR3- Tg mice dependent on CD8 T cells. (A-B) The metastatic melanoma model was generated by injecting 5x10⁵ B16-OVA-GFP I.V. into hDR3-Tg mice and treating with 200µg AT171-2 isotype control (M1), B9 mIgG1 (B9-M1) or tetravalent B9 mIgG1 (TET-B9-M1) on days 1, 3 and 5. For CD8 depletion: mice were treated with 100µg YTS169-mIgG2a two days before and then one day after B16-OVA challenge. For Natural Killer cell (NK) depletion: mice were treated with 100µg PK136-mIgG2a both two days before and one day after B16-OVA challenge Mouse lungs were harvested the day after an average of >50 B16-OVA foci were visible on the surface of B16-OVA control mice. (A) B9-M1 treatment compared with TET-B9- M1 treatment, over the isotype control, including representative images and pooled data for B16-OVA foci counts on the surface of each mouse lung within each group (3 groups of n=5- 6), at day 22. (B) TET-B9-M1 treatment with/without CD8 depletion (YTS169) or NK depletion (PK136), compared to the isotype control, including representative images and pooled data for B16-OVA foci counts on the surface of each mouse lung within each group (4 groups of n=6), at day 16. Figure 11: Humanisation of the variable regions does not change the activity of B9-mIgG1 in vitro. CFSE-labelled splenocytes, harvested from the spleens of hDR3-Tg mice, were co- stimulated with 0.05µg/ml 2C11 (anti-CD3) and titrated concentrations of B9, humanised B9 (H1L11) or isotype control AT171-2 mIgG1 for 48 hours. CD4 (A) and CD8 (B) T cell proliferation (CFSE dilution) is illustrated by decreases in the median of FITC fluorescence compared with the 2C11/AT171-2 controls. Figure 12: Schematic representation of the DR3-8i protein (SEQ ID NO: 92). The full-length fusion protein consists of an N-terminal signal peptide, a His8-tag, maltose binding protein (MBP), a PreScission protease cleavage site (scissors). DR3-8i contains the extracellular domain (ECD) of human DR3 (25-195) consisting of four cysteine-rich subdomains, and the C-terminus of human DcR3 (194-300). The protein carries a C-terminal Avi-tag for site-specific biotinylation. Figure 13: Model B9 scFv selections. Round 1 endpoint PCR products were resolved on a TAE agarose gel. Outputs shown for DR3 agonist clones B9 and F10. Anti-insulin clone scFv- D3 was used as a positive control. B9 and F10 ribosome display selections were completed in the presence of DR3-Fc antigen (‘+’); D3 selections were completed with biotinylated insulin. 41 241018 CMAL003WO1 DCA No antigen control ‘-‘. RT-PCR controls: Neg1, Reverse Transcriptase reaction no template negative control; Neg2, B9 and F10 end-point PCR no template control; Neg3, D3 end-point PCR no template control. Figure 14: B9 ribosome display guided selection strategy. B9 VH-shuffle and B9 VL-shuffle libraries represent chimeras of the original mouse B9 V_L or mouse B9 V_H sequence coupled to a human naïve V_H or V_L gene sequence repertoire, respectively. Mouse::human chimeric clones are characterised for binding to target antigen, human DR3 (stage 1). An individual clone that retains DR3 binding is then used as template for incorporation of the reciprocal human naïve V_H or V_L domain repertoire (stage 2). Final clones are fully human and screened once again for human DR3 binding and agonism. Use of a non-proof reading polymerase during template amplification introduces additional non-targeted diversity across the entire scFv sequence (vertical lines). Figure 15: B9 guided selections using B9 VH-shuffle and B9 VL-shuffle libraries. Round 1 and round 2 endpoint PCR products were resolved on a TAE agarose gel. Anti-insulin clone scFv D3 was used as a positive control. B9 ribosome display guided selections were completed in the presence of DR3-Fc antigen (‘+’) at the concentrations shown; D3 selections were completed with biotinylated insulin antigen. No antigen control ‘-‘. RT-PCR controls: ‘Neg1’, reverse transcriptase reaction no template negative control; ‘Neg2’, end-point PCR no template control; ‘Pos’, 1.7ng of D3 mRNA used as RT-PCR template. Figure 16: B9 guided selections using a human::human B9 VL-shuffle library. Round 1 endpoint PCR products were resolved on a TAE agarose gel. Anti-insulin clone scFv D3 was used as a positive control. B9 ribosome display guided selections were completed in the presence of DR3-Fc antigen (‘+’) at the concentrations shown; D3 selections were completed with biotinylated insulin antigen. No antigen control ‘-‘. RT-PCR controls: ‘Neg1’, reverse transcriptase reaction no template negative control; ‘Neg2’, end-point PCR no template control; ‘Pos’, 1.7 ng of D3 mRNA used as RT-PCR template. Figure 17: Human V_H and V_L germline sequences chosen as candidate frameworks for B9 humanisation.13× VH and 13× VL human germlines (GL) were chosen based on their overall similarity to the original mouse B9 sequence or the most frequent human GL sequences obtained from the guided selection outputs. Two VL sequences obtained from phage ELISA (Example 6.1) screening on B9 guided selections were also included (VLs9 and VLs10). 42 241018 CMAL003WO1 DCA Figure 18: High throughput expression and screening of humanised B9 IgGs. Humanised variant B9 huIgG1s (n = 169) were expressed and subsequently screened for DR3 agonism using a human DR3 Jurkat reporter cell line (Example 6.2). Reporter cell line fluorescence signal (FLU) is plotted against IgG concentration (µg/mL). Non-humanised parental B9 is shown as a white diamond and the isotype control R347 (Paules, CI et al., 2017) as a black circle. Shaded regions define those clones taken forward as hits. Threshold A (right of the vertical dotted line) includes all hits with FLU >2/3 B9; threshold B encompasses those hits μg/ml <1/4 B9 (below the horizontal dash-dot line) and FLU >2/3 B9 (right of the vertical dashed line). Figure 19: Top thirty B9 humanised variant variable heavy chain and variable light chain amino acid sequences. Figure 20: Screening B9 humanisation clones as crude IgG (SiPF) for binding to human DR3, mouse DR3 and cynomolgus (cyno) DR3. Human DR3, mouse DR3 and cyno DR3 were all transiently expressed in mammalian Expi293T cells. Crude, unpurified IgGs were incubated at a single concentration with the transiently transfected cells and IgG binding was detected on a Mirrorball fluorescence cytometer using a goat anti-human IgG labelled with AlexaFluor647. Data from the top thirty B9 humanised variant clones is plotted here, alongside the non-humanised B9 parental molecule. Figure 21: Agonism of DR3 reporter cells by purified humanised B9 IgGs. The capacity of purified IgGs to agonise a DR3 expressing Jurkat reporter cell line was determined using a 10-point titration curve. Curves were fitted with a standard 4-parameter variable-slope agonist response model to determine an EC50. A list of associated EC50 values are provided in Table 10. All antibodies were tested as human IgG1. Irrelevant control represents a monoclonal antibody raised to human ARG2 (R347). Benchmark positive control for DR3 agonism is clone M5. The panel of 21 purified B9 humanised variant IgGs were split between two independent experiments. Figure 22: Relationship between agonism and affinity for B9 humanisation variant IgGs. Affinities determined by BioLayer Interferometry (Octet) measurements and derived for a panel of 21 prioritised humanised variant IgGs (Table 11), are plotted against the EC₅₀ values derived from the titrated DR3 reporter assay (Table 10). Hits from the ‘SiPF’-expression panel are denoted as grey diamonds and the B9 control as a black triangle. The shaded area contains clones (n = 14) that display improved affinity and agonism over the non-humanised B9 parent. 43 241018 CMAL003WO1 DCA Figure 23: B9 humanised variant clones VHg6/VLg13, VHg6/VLg11 and VHg1/VL11 bind cell expressed DR3 as detected by flow cytometry. The cells tested were the Jurkat reporter cell lines described in Example 6.2. R347 is a non-specific isotype control. An Alexa Fluor 647- labelled secondary antibody was used to stain for primary antibody binding. Figure 24: B9 epitope competition assay. (A) DyLight⁶⁵⁰ labelled non-humanised B9 IgG probe binds to recombinant human DR3-Fc protein in solution. Excitation at 337 nm promotes a fluorescence resonance energy transfer (FRET) between anti-His cryptate antibody and the bound DyLight⁶⁵⁰ labelled non-humanised B9. FRET signal was measured using a fluorescence plate reader measuring at 665 nm (B). Addition of competing unlabelled B9 or humanised variant IgG displaces the DyLight⁶⁵⁰ labelled non-humanised B9 probe leading to a reduction in FRET signal. Figure 25: B9 humanised variant clones VHg6/VLg13, VHg6/VLg11 and VHg1/VL11 share the same epitope as the original non-humanised B9 parent molecule. Epitope competition assay (Example 6.6; Figure 24) confirms that clones VHg6/VLg13, VHg6/VLg11 and VHg1/VL11 compete and thus share the same or a closely overlapping epitope with the original non-humanised, murine B9 parental antibody molecule. Data points and fitted curves from one of three independent experiments are shown. Mean IC50 values ± standard deviation (SD) from the three experiments for VHg1/VL11, VHg6/VLg11 and VHg6/VLg13, are 447 ± 71 pM, 287 ± 34 pM and 348 ± 37 pM, respectively. Unlabelled B9 has a mean IC50 of 252 ± 34 pM. All antibodies were titrated as purified, unlabelled human IgG1. R347 human IgG1 served as an isotype control. Examples Example 1: Generation and characterisation of murine and murine/human chimeric and humanised antibodies. Murine Monoclonal Antibody Generation Two murine anti-human DR3 monoclonal antibody (anti-hDR3 mAb) clones B9 and F10 were generated using mouse hybridoma technology. To generate the B9 anti-hDR3 mAb, a DR3 knock-out (KO) mouse was immunised with approximately 10⁸ RBL-hDR3 (rat basophil leukemia (RBL) cells expressing human DR3 (SEQ ID NO: 2)) delivered with 50µg CpG followed by four subsequent immunisations with RBL-hDR3 cells alone I.P., 2 weeks apart. To generate F10, a DR3 KO mouse was immunised with approximately 10⁸ RBL-hDR3 delivered I.P. by four separate injections 2 weeks apart. Spleens from immunised mice were then fused to NS1 myeloma cells in the presence of HAT media. Initially supernatants/Abs specificities were tested by hDR3 ELISA and binding to in vitro activated PBMC. Monoclonal 44 241018 CMAL003WO1 DCA hybridomas were subcloned 3-4 times before expansion, mAb concentration and mAb purification. Human DR3 specificity was checked using ELISA and binding to hDR3+RBL cells compared to controls (mDR3+RBL cells). The isotype of the mAbs was determined using the mouse mAb isotyping test kit (MMT1, Bio-rad). Their respective variable region amino acid sequences are described below (CDR = complementarity-determining region detected using Abysis (a web-based antibody research system). Binding profiles of antibodies When using surface SPR technology B9 was found to have a higher avidity for recombinant hDR3 when compared to F10 (Figure 1: Apparent KD calculated at 5.61x10^-10 M and 10.7x10- ¹⁰ M respectively). Epitope Specificity Using truncated variants of hDR3 cysteine-rich domain (CRD) and hybrids of human/mouse CRD1-CRD2, the epitopes of the murine B9 and F10 anti-hDR3 mAb were identified as being amino acids (AA) 37-45 (SEQ ID NO: 1) located within human DR3 (SEQ ID NO: 2) (Figure 2A-B: illustrating data for B9 – representative of F10). Binding of B9 to primary human T cells To determine if B9 anti-hDR3 mAbs recognised native hDR3, the binding pattern to human T- cell subsets was examined (Figure 2C). While absolute levels of DR3 expression varied within individuals, resting CD4+ T cells exhibited higher expression of DR3 when compared with resting CD8+ T cells. Within the resting CD4+ T cell compartment, CD25⁺CD127^low Treg cells exhibited slightly lower DR3 expression when compared with non-Treg cells (statistics not shown here). Comparing the epitopes of B9/F10 anti-hDR3 mAb and PTX-25 anti-hDR3 mAb To determine how unique the B9/F10 epitope is compared with the PTX-25 epitope: a competition assay was performed using hDR3+Jurkat cells (Figure 3). The lack of competition/inhibition between the PTX-25 and the B9 or F10 mAbs, for binding to hDR3+Jurkat cells, confirms that the B9 and F10 mAbs bind to a different epitope than the PTX-25 mAb. Figure 3C illustrates the position of each epitope in hDR3. T cell stimulatory effects of B9 and the influence of isotype To assess the role of isotype in the agonistic activity of anti-hDR3, chimeric versions of B9 were generated to express hIgG1 (binds all FcγR), hIgG2 (low affinity for FcγR) and hIgG4 (reduced affinity for FcγRIIA (activating) and no affinity for FcγRIIIB (inhibitory)) Fc regions. All 45 241018 CMAL003WO1 DCA isotype variants of anti-hDR3 mAbs retained their specificity and were tested for their capacity to co-stimulate T cells in a 4-day PBMC culture assay (Figure 4). All isotypes were able to promote T cell proliferation at the high concentration (0.4µg/ml – Figure 4B-C), but at lower concentration (e.g.0.0032µg/ml – Figure 4D-E) the hIgG2 (H2) and hIgG4 (H4) isotypes (as well as the F10-hIgG1/hIgG2 chimera, not shown) exhibited reduced activity. Previous studies have demonstrated the cross-linking requirement of the inhibitory FcуRIIB in driving the agonistic activity of antibodies targeting other members of the TNFRSF. Therefore, in an attempt to enhance the binding to hFcуRIIB and to evaluate FcγR independent agonism, chimeric mAbs were generated using different hIgG1 variants containing SELF, V11 and N297A mutations. Binding to hDR3 was retained by all the Fc variants. The relative affinity of each Fc variant for each hFcуR, in comparison to the parent hIgG1 isotype, has been determined previously, by Dahan et al. 2016, using SPR technology. Compared to unmodified hIgG1, the SELF variant has similar affinity for hFcуRI but enhanced affinity for hFcуRIIB and hFcуRIIA; V11 has almost negligible affinity for hFcуRI and low affinity for hFcуRIIA but markedly enhanced affinity for hFcуRIIB; N297A has negligible affinity for hFcуR. The B9 hIgG1 variants were evaluated in the T cell co-stimulation assay (Figure 5). Both hIgG1 and hIgG1 SELF displayed similar activity, which was more potent than that observed with either hIgG1 V11 or hIgG1 N297A at the lowest concentration (0.0032µg/ml - Figure 5C-D). The reduced T cell co-stimulatory activity seen with the N297A mutant suggests FcyR co- engagement contributes towards the potency of anti-hDR3 agonistic activity (Figures 5). Given that anti-hDR3 hIgG1 and hIgG1 SELF possess similar T cell stimulatory effects (Figures 5), and have comparable affinity for FcyRI, whereas the less agonistic hIgG V11 binds poorly to FcγRI, these data suggest that, contrary to expectations, FcyRI plays an important role in anti- hDR3 mAb agonism through mAb cross-linking. The impact of anti-hDR3 mAbs on Tregs versus CD4+ effectors and on CD8+ T cells without CD4+ T cell help Since DR3 is expressed on both effector and Tregs, we investigated the effects of DR3 agonism on the expansion of Treg versus CD4⁺ effector cells in the T cell proliferation assay (Figure 6A-C). We found there was no significance in the expansion of Foxp3⁺ Treg within the proliferating CD4⁺ population (Figure 6C) and the large proportion of these expanding cells were effectors (Figure 6B). 46 241018 CMAL003WO1 DCA Given that resting CD4⁺ T cells express significantly higher levels of DR3 than CD8⁺ T cells (Figure 2C), we evaluated the ability of anti-hDR3 mAb to activate CD8⁺ T cells in the absence of CD4+ T cell help (Figure 6D-E). To address this question in vitro, CFSE-labelled purified human CD8+ T cells were co-cultured with irradiated autologous CD14+ monocytes (providing FcγRI/II cross-linking) at a 4:1 ratio in the presence of anti-CD3 (OKT3) together with anti- hDR3/hIgG1 (Figure 6E). This was done in comparison to stimulation of CFSE-labelled PBMC with anti-CD3 (OKT3) and anti-hDR3/hIgG1 (Figure 6D). B9 anti-hDR3 mAb exhibited a significant ability to activate CD8⁺ T cells (Figure 6E), similar to F10 anti-hDR3 mAb which was less potent (data not shown). Responses were markedly lower in the purified CD8⁺ T cell culture (Figure 6E) than those observed with PBMC (Figure 6D). This may be attributed to the absence of CD4⁺ T cell help and other homeostatic feeder cells in the PBMC culture. Therapeutic potential of anti-hDR3 mAb Figure 7A shows that elevated levels of TNFRSF25/DR3 expression and CD8⁺ T cells were both significantly associated with superior overall survival in HPV⁺ HNSCC and SKCM. We demonstrated that anti-hDR3 mAb co-stimulated the proliferation of HNSC-HPV⁺ TIL (Figure 7B) and SSC TIL (Figure 7C). Taken together our data suggested that agonistic anti- hDR3 mAbs can stimulate tumour infiltrating T cells directly and thus potentially promote anti- tumour immune responses in cancer patients. The therapeutic potential of the B9 anti-hDR3 mAb was explored further using an aggressive solid tumour model in hDR3-Tg mice (Figure 7D-E). We found B9 mIgG1 was capable of promoting the expansion of OVA-specific CD8⁺ T cells (Figure 7D) and suppressing the growth of B16-OVA tumours (Figure 7E) in these hDR3-Tg mice. Furthermore, in a therapeutic model and in the absence of vaccination, agonistic anti-hDR3 mAb combined with anti-PD-1 mAb (anti-mouse PD-1 clone RMP1-14, commercially available) resulted in a statistically significant delay in the growth of B16-OVA tumours compared with single agent treatment (Figure 8). Overall, these data support the notion that agonistic anti-hDR3 mAb can be a useful adjunct to immune checkpoint therapies and anti-cancer vaccination strategies. Generation of Fcγ receptor independent B9 agonist A tetravalent Fab:IgG B9 antibody was produced by linking B9 Fabs recombinantly using a flexible linker. These antibodies were compared with their bivalent controls in the same T cell proliferation assay as described previously. The different chimeric Fc mAb include: hIgG1 isotype control (H1), B9-hIgG1 (B9-H1), “Fc-silent” B9-N297A, tetravalent (TET) B9-H1 (TET- 47 241018 CMAL003WO1 DCA B9-H1) and “Fc-silent” TET-B9-N297A. After four days, CFSE dilution (% CFSE^lo) was analysed in CD3⁺CD4^High cells (Figure 9B and 9D) and CD3⁺CD8^High cells (Figure 9C and 9E). We found significantly enhanced agonism to be associated with the tetravalent constructs (above the bivalent constructs): most starkly when comparing the potent effect of "Fc-silent” TET-B9-N297A with the negligible effect of “Fc-silent” B9-N297A (Figure 9B-E). In summary, tetravalency conferred FcyR-independent agonism on anti-hDR3 mAb and this agonism is significantly enhanced over the bivalent constructs. Interestingly, the Fab:IgG tetravalent B9-mIgG1 construct demonstrated very significant and consistent therapeutic effects in a metastatic melanoma model in hDR3-Tg mice (in contrast with B9-mIgG1) and this TET-B9-M1-dependent therapeutic effect was clearly driven by CD8⁺ T cells (Figure 10). Generation of B9 agonist with humanised variable regions The humanisation of the variable regions of B9 did not affect the capacity of B9 to activate hDR3⁺ T cells in vitro (Figure 11). This highlights the transferable nature of this agonist when considering the clinical setting. Materials and methods Mice All animals were bred using in-house facilities (Biomedical research facility, University of Southampton, UK). Wild-type (WT) C57BL/6 mice were maintained in-house. DR3 knock out (DR3KO) C57BL/6 mice were supplied by CRUK (Generated by E. Wang and M. Owen - Wang, E.C., et al., DR3 regulates negative selection during thymocyte development. Mol Cell Biol, 2001. 21(10): p. 3451-61). hDR3 BAC transgenic (hDR3-Tg) mice were generated in Taconic facilities by pronuclear injection of mixed nucleotide preparation (BAC CTD-2339I9: contains the fewest neighbouring genes alongside TNFRSF25 (Ensembl gene ID, human: ENSG00000215788; NCBI gene ID, human: 8718)) into the pronucleus of multiple C57B/6Ntac cell stage fertilized embryos (harvested 0.5 days post conception (dpc) from oviducts and placed in M2 medium under mineral oil). After recovery, 25-35 injected one cell stage embryos were transferred to one of the oviducts of 0.5 dpc pseudopregnant NMRI females. Genomic DNA was extracted from litter tail biopsies and transgene expression was analysed/confirmed by PCR using the following reaction mix: 5 µl PCR Buffer 10x (Invitrogen), 2 µl MgCl2 (50 mM), 1 µl dNTPs (10 mM), 1 µl Primer W (5 µM), 1 µl Primer X (5 µM), 1 µl Primer Y (5 µM), 1 µl Primer Z (5 µM), 0.2 µl Taq (5 U/µl, Invitrogen), 35.8 µl H2O and 2 µl DNA (Taconic primer combinations described below). PCRs were run using 95°C 5 minutes to start and 35 cycles of: 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 1 minute - with a final 72°C 10 minutes extension. The PCR amplicons were analysed by using a Caliper 48 241018 CMAL003WO1 DCA LabChip GX device. The following templates were used as PCR controls: H₂O and wildtype (WT) genomic DNA. The amplification of the internal control fragment (585 base pair (bp)), with oligos 1260_1 and 1260_2, confirms the presence of DNA in the PCR reactions (amplification of the CD79b wildtype allele, nt 17714036-17714620 on Chromosome 11). PCR analysing the 5’ end of the PNI fragment (362 bp) combined primers: 12624_1, 12624_2, 1260_1, and 1260_2. PCR analysing the presence of the transgene (255 bp) combined primers: 12626_7, 12626_6, 1260_1, and 1260_2. PCR analysing the human PLEKHG5 exon 11 in the transgene (394 bp) combined primers: 12627_9, 12627_10, 1260_1, and 1260_2. PCR analysing the human TNFRSF25 exons 6 and 7 in the PNI fragment (313 bp) combined primers: 12790_21, 12790_22, 1260_1 and 1260_2. PCR analysing the human ESPN exon 12 in the transgene (360 bp) combined primers: 12628_13, 12628_14, 1260_1 and 1260_2. PCR analysing the 3’ end of the PNI fragment (391 bp) combined primers: 12629_17, 12629_18, 1260_1 and 1260_2. PCR Primers: 1260_1: GAGACTCTGGCTACTCATCC (SEQ ID NO: 93) 1260_2: CCTTCAGCAAGAGCTGGGGAC. (SEQ ID NO: 94) 12624_1: CCACAGTAAATATATGCCAGACC (SEQ ID NO: 95) 12624_2: GGTTCGTCTTTGTGCATGC (SEQ ID NO: 96) 12626_7: CCTGTTTTCTTGAGACAGAAGGG (SEQ ID NO: 97) 12626_6: CGAGGCTGTGAGTGTGTAAGC (SEQ ID NO: 98) 12627_9: TGCATGCAGGTTGCTTATCC (SEQ ID NO: 99) 12627_10: ACCTGTGCTAATTCAGCTGAACC (SEQ ID NO: 100) 12790_21: TACCAGGGACACAATGACAGG (SEQ ID NO: 101) 12790_22: GAACACTGAAAGCAGCTGGTG (SEQ ID NO: 102) 12628_13: AGGGTAGTGTCCGATATTGGC (SEQ ID NO: 103) 12628_14: ATCTTGGGGTATGGTTGTCTTC (SEQ ID NO: 104) 12629_17: ACTGTGTATGCACTTAGTAACAGGG (SEQ ID NO: 105) 12629_18: TCTGGCTATCTTTCTGTTGTTGG (SEQ ID NO: 106) “Heterozygous” hDR3-Tg mice were initially bred in-house with WT C57BL/6 mice and phenotyped by detection of hDR3 protein expression on both CD8⁺ and CD4⁺ (as well as Foxp3⁺CD4⁺) blood-derived T cells using flow cytometry. “Homozygous” hDR3-Tg mice were generated by cross-breeding two “heterozygous” hDR3-Tg mice and selecting/breeding the mice which exhibited higher hDR3⁺ mean/median fluorescent intensities, on hDR3-labelled CD8⁺ and CD4⁺ cells, than the “heterozygous” hDR3-Tg mice. 49 241018 CMAL003WO1 DCA Cell-line culture Rat basophil leukaemia (RBL - American Type Culture Committee (ATCC)) cells were stably transduced with hDR3 cloned into the pMigR1 vector (kindly supplied by Prof P. Ashton- Rickardt, Imperial College London, UK), mDR3 cloned into pMigR1 or pMigR1 alone. RBL- hDR3⁺ and RBL-mDR3⁺ cells were generated by retroviral transduction using a method adapted from Kessels et al. (Kessels, H.W., et al., Immunotherapy through TCR gene transfer. Nat Immunol, 2001. 2(10): p. 957-61). Briefly, retroviral supernatants were obtained by transfecting Phoenix-ECO packaging cells (ATCC) with pCL-Eco (Addgene) and hDR3⁺/mDR3⁺ pMigR1 DNA (or empty pMigR1 vector), using Fugene HD (Promega). On day 2 post-transfection, RBL cells were resuspended at 1.2x10⁶ cells per ml in viral supernatant and 0.5 ml per well distributed in non-tissue culture-treated 24-well plates previously coated with Retronectin (Takara Clontech) and blocked with phosphate buffered saline (PBS) 2%BSA. Cells were centrifuged at 1800 rpm for 90 minutes at 32^oC, and further cultured for 24 hours in the viral supernatant. Transduced RBL were washed and GFP^high cells were sorted on a FACS ARIA II. All RBL cell-lines (RBL-hDR3⁺, RBL-mDR3⁺ and RBL-pMigR1) were cultured in complete DMEM (containing 10% FCS together with: 2 mM L-glutamine, 1 mM pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin (All from Gibco)). HEK293T cells (ATCC) and B16-OVA-GFP tumour cells (in house - acquired from Caetano’s lab (Buchan et al., 2018, Immunity; Buchan et al., 2018, Clin Cancer Res; Greenman et al., 1991, Mol Immunol)) were also cultured in the same complete DMEM medium. Jurkat reporter cells (NF- kB-GFP cassette already present, System Biosciences) were transfected with hDR3⁺pcDNA3.1 using the Amaxa nucleofection protocol. Colonies were screened using biotinylated anti-hDR3 antibody followed by streptavidin-APC. Positive wells were expanded and grown in complete RPMI (Gibco – including complete supplements listed above) media supplemented with 1mg/ml G418 (Life Technologies, ThermoFisher). Immune cell separation and culture Human peripheral blood mononuclear cells (PBMC) were isolated from anonymous leukocyte blood cones (NHS blood transfusion service, Southampton – approved by local ethics committee) using Lymphoprep (Stemcell Technologies). All immune cells were cultured in complete RPMI media (0.1mM 2-ME (Sigma-Aldrich) included for murine T cell culture). In vitro activation of PBMC for antibody screening involved 48-72 hours incubation with 2.5 µg/ml Phytohemagglutinin-L (PHA-L - 500x solution from Invitrogen, Thermo Fisher Scientific). PBMC cells were processed using EasySep immunomagnetic separation kits (STEMCELL Technologies) to: positively select CD14⁺ cells (95.2-98.1% purity) and negatively select total CD8⁺ T cells (93.2-98.9% purity - no CD4⁺ contamination detected). Mouse splenocytes were 50 241018 CMAL003WO1 DCA isolated from spleens of hDR3-Tg mice by pressurised homogenisation of the spleen in a sterile 100µm cell strainer (Fisher scientific) for the co-stimulation assay. Flow cytometry All cells were labelled in 0.1% BSA/PBS buffer. Background IgG/Fc-binding on cells was blocked by 15 minutes 4^oC incubation with: 10% AB serum (Sigma-Aldrich) on human cells; 10 µg/ml 2.4G2 (In-house) on mouse cells; or 1% BSA when there was conflict with the secondary antibody. Cells were labelled with primary monoclonal/polyclonal antibodies (mAb/pAb) for 30 minutes at 4^oC and washed twice in buffer. Murine red blood cells were lysed (Lysis buffer: 8.2 g NH₄Cl and 1 g KHCO₃ diluted in 1 L deionised H₂O) during the first wash. Antibody binding that lacked conjugation was detected via 30 minutes incubation (4^oC) with 1/200 conjugated secondary constructs: mIgG1 detected by Allophycocyanin-AffiniPure F(ab) Fragment Goat Anti-Mouse IgG and hIgG detected by Allophycocyanin-AffiniPure Goat Anti- Human IgG (Jackson ImmunResearch Laboratory INC, UK); biotinylated antibodies detected by eBioscience Streptavidin APC / PE-Cy7 Conjugate (Life Technologies, ThermoFisher). Cells were analysed on the BD FACSCanto II (BD Biosciences) using DIVA software (BD Biosciences) and further analysed/presented using Flowjo software (BD Biosciences). “Heterozygous/homozygous” hDR3-Tg mice were phenotyped by labelling blood samples with 2 µg/ml Human DR3/TNFRSF25 Biotinylated pAb, compared to the 2 µg/ml normal goat IgG biotinylated control pAb (both from R&D Systems), in the following panel sequence: primary labelling with FITC anti-Mo CD8a (Clone: 53-6.7 - Biolegend) and eFluor™ 450 anti- Mo CD4 (Clone: RM4-5 - Life Technologies, ThermoFisher); secondary labelling with streptavidin APC conjugate; and finally labelling with PE anti-Mo/Rt FOXP3 mAb (Clone: FJK- 16s - Life Technologies, ThermoFisher) after permeabilisation/fixation using the Foxp3 / Transcription Factor Staining Buffer Set (Life Technologies, ThermoFisher). B9 binding (DR3 expression) on CD4/Treg/CD8 T cells was analysed by labelling naïve PBMC with biotinylated B9-mIgG2A compared with biotinylated 18B12-mIgG2A (Expanded in-house - Sancho, et al., 2008, J Clin Invest ) isotype control (both biotinylated in-house using EZ-Link Sulfo-NHS-Biotin (Thermo Fisher Scientific) according to the manufacturer’s instructions) which was detected by APC streptavidin in the following panel: PerCP-Cy5.5 anti-Hu CD3 (Clone: UCHT1 - Biolegend), eFluor™ 450 anti-Hu CD4 (Clone: RPA-T4 – Life Technologies, ThermoFisher), APC-Cy7 anti-Hu CD8 (Clone: RPA-T8 – Biolegend), FITC anti-Hu CD127 mAb (Clone: HIL-7R-M21 – BD Pharminutesgen/Biosciences) versus FITC mouse IgG1 K isotype control (Clone: MOPC-21 – BD Pharminutesgen/Biosciences) and PE anti-Hu CD25 mAb (Clone: M-A251 – BD Biocience) versus PE Mouse IgG1 K Isotype control (MOPC-21 – BD Biocience). 51 241018 CMAL003WO1 DCA B9 binding (DR3 expression) on different memory subsets within CD4/CD8 T cells was analysed by labelling naïve PBMC with biotinylated B9-mIgG2A compared with biotinylated 18B12-mIgG2A which was detected by PE-Cyanine7/APC streptavidin depending on the fluorochromes used in the following panel: FITC Hu-CD3 (OKT3 – in-house) or eFluor™ 450 anti-Hu CD3 (Clone: UCHT1 – Life Technologies, ThermoFisher); eFluor™ 450 anti-Hu CD4 or eFluor™ 506 anti-Hu CD4 (Clone: RPA-T4 – Life Technologies Ltd); APC-Cyanine7 or PE- Cyanine7 anti-Hu CD8 (Clone: RPA-T8 – Biolegend); PerCP-Cy5.5 anti-Hu CCR7 (Clone: G043H7 - Biolegend); and APC anti-Hu CD45RA (Clone: HI100 – Life Technologies, ThermoFisher) or FITC anti-Hu CD45RA (Clone: T6D11 – MACS Miltenyi Biotec). Human CD8 T cell purity was confirmed by flow cytometry after primary labelling with: eFluor™ 450 anti-Hu CD3, eFluor™ 506 anti-Hu CD4 and PE-Cyanine7 anti-Hu CD8a. CD14⁺ cell purity was checked by flow cytometry after labelling with: PE anti-Hu CD14 (Clone: M5E2 – Biolegend). T cell panels used to analyse proliferating T cells without the CD3 marker include: eFluor™ 450 anti-Hu CD4, APC-Cyanine7 anti-Hu CD8a and APC anti-Hu CD25 (Clone: BC96 - Life Technologies, ThermoFisher) versus APC mouse IgG1 k Iso control (Clone: P3.6.2.8.1 - Life Technologies, ThermoFisher). T cell panels used to analyse proliferating T cells with the CD3 marker include: eFluor™ 450 anti-Hu CD3, eFluor™ 506 anti-Hu CD4 , PE-Cyanine7 anti-Hu CD8a and APC anti-Hu CD25 versus APC mouse IgG1 k Iso control. SIINFEKL-specific CD8⁺ T cells were analysed, in murine blood cells, by labelled with APC anti-Mo CD8a mAb (Clone: 53-6.7 – Life Technologies, ThermoFisher) and PE-labelled H- 2Kb/SIINKEFL tetramer construct (In house: Protein Core Facility, University of Southampton). CD8 depletion and NK depletion was checked in blood samples from DR3-Tg mice 4 days after B16-OVA I.V. injection using the following mastermix: FITC anti-Mo CD3ε (Clone: 145- 2C11 – Biolegend), eFluor™ 450 anti-Mo CD4 and PE-Cyanine7 anti-Mo CD335 (NKp46 – Clone: 29A1.4 - Biolegend). DR3 ELISA The ELISA for detecting hDR3-specific antibodies was conducted by coating Nunclon maxisorp plates, overnight, with 0.5 µg/ml recombinant hDR3/TNFRSF25 Fc chimera protein versus 0.5 µg/ml negative control recombinant mouse OX40/TNFRSF4 Fc chimera protein (Both from R&D Systems), in coating buffer (1.59 g Na₂CO₃ and 2.93 g NaHCO₃ in 1 L dH₂O 52 241018 CMAL003WO1 DCA – 50 µl/well at 4^oC). Washed (4x PBS/0.05%tween 200 µl/well) and blocked (1%BSA/PBS, 200 µl/well, 1 hour room temperature (RT)) plates were incubated for 2 hours (RT) with hybridoma supernatants compared to: blank media and 2 µg/ml Human DR3/TNFRSF25 Biotinylated pAb versus 2 µg/ml Normal Goat IgG Biotinylated Control in 0.1%BSA/PBS (50µl/well). Plates were washed and screened for bound antibodies using either 1/5000 Goat Anti-Mouse IgG (Fc specific)–Peroxidase (Sigma-Aldrich) or 1/1000 streptavidin horse radish peroxidase (HRP) (Sigma-Aldrich) in 1% BSA/PBS (100 µl/well, 1 hour, RT). The bound HRP constructs were incubated with 100 µl/well O-phenylenediamine dihydrochloride substrate (Tablets from Sigma-Aldrich – used according to manufacturer’s guidelines) for ~20 minutes (depending on RT) and the reaction was stopped using 40 µl/well 2.5M sulphuric acid. Changes in optical density were analysed using the Epoch plate-reader. Human DR3 specificity of B9 was later confirmed by lack of binding to the mDR3/TNFRSF25 Fc chimeric construct (R&D Systems) which was used to confirm the specificity of anti-mDR3 mAb clone LPA-2 for mDR3 (similar ELISA technique where BTLA-Fc was used as an alternative negative control). Development of hDR3-specific mAb and the different constructs The anti-hDR3 mAb clone B9 (murine) was raised using conventional hybridoma technology. To generate the B9 anti-hDR3 mAb: a DR3KO mouse was immunised with approximately 10⁸ RBL-hDR3⁺, delivered by intraperitoneal (I.P.) injection, with 50 µg CpG, followed by 4 subsequent immunisations with RBL-hDR3⁺ cells alone – 2 weeks apart. Spleen cells, harvested from immunised mice, were fused with NS-1 myeloma cells in the presence of hypoxanthine-aminopterin-thymidine (HAT) media (Gibco) and titrated down to 1 colony/well, over naïve thymocytes, in 96-well flat-bottom plates. Pre- and post-clonally selected hybridoma, supernatant-derived, antibody specificity was tested by DR3 ELISA and binding to PHA-L-activated PBMC. Monoclonal hybridomas were subcloned 3-4 times before: expansion and purification. Human DR3 specificity was confirmed via binding to hDR3Fc chimera by ELISA (data not shown) and binding to RBL-hDR3⁺ cells by flow (e.g., Figure 2A) - as opposed to mDR3 Fc chimera or RBL-mDR3⁺ cells. The B9 mIgG2A parental isotype was characterised, with its Kappa light chain, using the mouse mAb isotyping test kit (MMT1, Bio- rad). B9 variable regions were cloned from each of the hybridoma clones by lysing the cells in RLT buffer, homogenizing the lysate through QIAshredders and extracting the total RNA using the RNeasy Mini Kit (All from Qiagen) which was converted into cDNA by reverse transcriptase PCR (SuperScript III First-Strand Synthesis System – Thermo Fisher) on the Peltier Thermal Cycler (MJ research). The cDNA was screened for each variable heavy (VH) and variable light (VL) - plus Kappa constant - region using the VH reverse primer (My1/2R 114-120 – see below) with each of the respective VH forward primers (MHv1-12 – listed below) or the VL 53 241018 CMAL003WO1 DCA reverse primer (m-K const 3 EcoRI) with each of the respective VL forwards primers (Mkv1- 11 – listed below) in a PCR using high fidelity Pfu DNA polymerase enzyme (Promega: according to the manufacturer’s instructions – 55^oC annealing and 72^oC extension temperatures) and the S1000 Thermal Cycler (Thermo Fisher). Samples of each PCR reaction were run on a 1% Agarose gel by electrophoresis and PCR reactions containing positive bands (450bp VH band and 750bp VL plus Kappa constant band) were cleaned using a method adapted from the Mini Gel extraction kit (Qiagen kit) for DNA in solution. DNA sequences were cloned using the Zero Blunt TOPO PCR Cloning kit according to the manufacturer’s instructions (Invitrogen, Life Technologies). Plasmids were purified from transformed chemically competent TOP10 E.coli (Life Technologies, ThermoFisher) cultures using QIAprep Spin Miniprep (Qiagen) and checked for VH/VL expression using GoTaq Flexi DNA PCR (Promega) and their respective primers. Plasmids that gave a positive band, after running through 1% Agarose gel electrophoresis, were sent to Source BioScience (Invitrogen, Thermo Fisher) for sequencing and confirmed by sequence analysis using DNASTAR software. My1/2R 114-120 primer: mIgG1/2R (114-120aa): TGGATAGACAGATGGGGGTGTYGTTTTGGC (SEQ ID NO: 107) MHv1-12 primers: MHV-1: ACTAGTCGACATGAAATGCAGCTGGGTCATSTTCTTC (SEQ ID NO: 108) MHV-2: ACTAGTCGACATGGGATGGAGCTRTATCATSYTCTT (SEQ ID NO: 109) MHV-3: ACTAGTCGACATGAAGWTGTGGTTAAACTGGGTTTTT (SEQ ID NO: 110) MHV-4: ACTAGTCGACATGRACTTTGGGYTCCAGCTTGRTTT (SEQ ID NO: 111) MHV-5: ACTAGTCGACATGGACTCCAGGCTCAATTTAGTTTTCCTT (SEQ ID NO: 112) MHV-6: ACTAGTCGACATGGCTGTCYTRGSGCTRCTCTTCTCC (SEQ ID NO: 113) MHV-7: ACTAGTCGACATGGRATGGAGCKGGAWCTTTCWCTT (SEQ ID NO: 114) MHV-8: ACTAGTCGACATGAGAGTGCTGATTCTTTTGTG (SEQ ID NO: 115) MHV-9: ACTAGTCGACATGGMTTGGGTGTGGAMCTGCTATTCCTG (SEQ ID NO: 116) MHV-10: ACTAGTCGACATGGGCAGACTTACATTCTCATTCCTG (SEQ ID NO: 117) MHV-11: ACTAGTCGACATGGATTTTGGGCTGATTTTTTTTATTG (SEQ ID NO: 118) MHV-12: ACTAGTCGACATGATGGTGTTAAGTCTTCTGTACCTG (SEQ ID NO: 119) m-K const 3 EcoRI primer: AAGAATTCCTAACACTCATTCCTGTTGAAG (SEQ ID NO: 120) Mkv1-11 primers: 54 241018 CMAL003WO1 DCA MKV1: ACTAGTCGACATGAAGTTGCCTGTTAGGCTGTTGGTGCTG (SEQ ID NO: 121) MKV2: ACTAGTCGACATGGAGWCAGACACACTCCTGYTATGGGT (SEQ ID NO: 122) MKV3: ACTAGTCGACATGAGTGTGGCTCACTCAGGTCCTGGSGTTG (SEQ ID NO: 123) MKV4: ACTAGTCGACATGAGGRCCCCTGCTCAGMTTYTTGGMWTCTTG (SEQ ID NO: 124) MKV5: ACTAGTCGACATGGATTTWCAGGTGCAGATTWTCAGCTTC (SEQ ID NO: 125) MKV6: ACTAGTCGACATGAGGTKCCYTGYTCAGYTYCTGRGG (SEQ ID NO: 126) MKV7: ACTAGTCGACATGGGCWTCAAGATGGAGTCACAKWYYCWGG (SEQ ID NO: 127) MKV8: ACTAGTCGACATGTGGGGAYCTTKTTYCMMTTTTCAATTG (SEQ ID NO: 128) MKV9: ACTAGTCGACATGGTRTCCWCASCTCAGTTCCTTG (SEQ ID NO: 129) MKV10: ACTAGTCGACATGTATATATGTTTGTTGTCTATTTCT (SEQ ID NO: 130) MKV11: ACTAGTCGACATGGAAGCCCCATGCTCAGCTTCTCTTCC (SEQ ID NO: 131) The Fc region of B9-mIgG2A was class switched to generate a B9-mIgG1 chimeric antibody, using the pEE vector cloning system (Lonza) where the VL plus species-specific Kappa constant domain and VH plus species/isotype-specific constant domain sequences were cloned into the pEE12.4 and pEE6.4 expression vectors (Lonza, UK), respectively at the HindIII (N-terminus end) and EcoRI (C-terminus end) restriction sites. Isotype-specific VHpEE6.4 plasmids and species-specific VLpEE12.4 plasmids were used in species-specific combination to transfect ExpiCHO cells in the ExpiCHO expression system (Life Technologies, ThermoFisher). Both B9 Fc regions were class switched further to generate human IgG1, IgG2 and IgG4 chimeric antibodies, as well as SELF/V11/N297A human IgG1 Fc variants (Dahan et al., 2016, Cancer Cell), using the same technology. Tetravalent rFab:IgG B9-hIgG1/N297A/mIgG1 was generated using the monoepitopic Fab version in a method published by Yang et al. (Genentech 2019 (Yang et al., 2019, MAbs, 11(6): p.996- 1011)). All antibodies were purified on protein A columns (GE healthcare, UK) and checked to contain < 1% aggregate as determined by HPLC and tested to contain < 5EU endotoxin per 1 mg antibody assessed by the Endosafe-PTS portable test system (Charles River Laboratories, L’Arbresle, France). Development of mDR3-specific mAb The anti-mDR3 mIgG1 kappa light-chain mAb clone LPA-2 was raised using the conventional hybridoma technology (Lawrence Andrews, 2013, University of Southampton thesis). Briefly, DR3KO mice were immunised with two 100µl subcutaneous (S.C.) injections of 10 µg/ml mDR3/TNFRSF25 Fc chimera, diluted in complete Freund’s adjuvant (CFA - Becton Dickinson), at 2 sites. Two weeks later, a further injection of 20 µg mDR3/TNFRSF25 Fc chimera, in CFA, was administered I.P. Three days before harvesting spleens for cell fusion, 55 241018 CMAL003WO1 DCA mice were given a final dose of 25 µg mDR3/TNFRSF25 Fc chimera, in PBS, I.P. Supernatants from hybridomas were screened by ELISA using mDR3/TNFRSF25 Fc chimera coated plates where BTLA.Fc (R&D Systems) was used as a negative control. Supernatants that provided a positive ELISA signal were screened further by flow cytometry which analysed binding to RBL-mDR3⁺ cells over negative control RBL-pMigR1 cells (e.g., Figure 2A). Positive hybridoma cells were cloned and antibodies were purified/characterised as described before. Binding kinetics and Epitope mapping Binding kinetics were determined using surface plasmon resonance (SPR) technology by: capturing 10 µg/ml His-tagged hDR3/TNFRSF25 Fc chimera for 2 minutes with anti-His mAb immobilized on a CM5 chip and flowing 200 nM B9-mIgG2A over the chip, on the Biacore T100. The binding kinetics were analysed using the Bivalent model (e.g., Figure 2B). CRD- truncated constructs and human-mouse (Hu-Mo) CRD1-2 chimeras of DR3, lacking the intracellular death domain and encoding the N-terminal Rituximab epitope-tag, were designed with the help of DNAstar software and expressed in the pcDNA3.1(+) plasmid by GeneArt Gene Synthesis (Thermo Fisher Scientific). These constructs included: full-length HuDR3 (SEQ ID NO: 81), minimal HuCRD1-2 (Min HuCRD1-2) (SEQ ID NO: 82), HuCRD1+MoCRD2 (SEQ ID NO: 83), MoCRD1-HuCRD2 (SEQ ID NO: 84) and a murine edit of HuCRD1 attached to HuCRD2 (MoEdit of HuCRD1 +HuCRD2 (SEQ ID NO: 85)). MoEdit of HuCRD1 +HuCRD2 was generated by replacing amino acids 37-40, 42-43 and 45 in the hDR3 sequence with respective residues of dissimilarity from mDR3 (Figure 2D). Three days prior transfection, HEK293T cells were seeded 1x10⁵/well of a 6-well plate in antibiotic-free media. HEK293T cells were transfected with 3.33 µg plasmid DNA using Fugene HD reagent according to the manufacturer’s instructions (Promega). Transfectants were harvested and screened two days after transfection. Each transfectant was screened by flow cytometry after labelling cells with: 2.5 µg/mL anti-tag hIgG1 mAb Rituximab (gifted by Southampton General Hospital Oncology Pharmacy); 2 µg/mL Human DR3/TNFRSF25 Biotinylated pAb versus Normal Goat IgG Biotinylated Control; 2.5 µg/mL B9 hIgG1 anti-hDR3 mAb; and 2.5 µg/mL LPA-2 mIgG1 anti- mDR3 mAb. PBMC/splenocyte co-stimulation experiments In the human PBMC proliferation assay, 1x10⁵/well CFSE-labelled (</=1x10⁷ cells/mL 1µM CFSE (Sigma-Aldrich) for 10 minutes RT and washed 3x) PBMC were co-stimulated for 4 days with 0.1 ng/mL anti-CD3 (In-house clone: OKT3 - ATCC) together with titrated concentrations of different mAb versus their respective isotype controls, alongside positive control 0.25 µg/mL soluble carrier-free TL1A (R&D Systems), in 96-well U-bottom plates (200 µl/well). The isotype controls include: irrelevant control R347 hIgG1 (Greenman et al., 1991, 56 241018 CMAL003WO1 DCA Mol Immunol) for the Urelamab study (Urelumab hIgG4 and R347 hIgG1 were generated in house), non-specific hIgG1 control sourced from BioXCell (hIgG1 vs hIgG2 vs hIgG4 comparisons and the CD8 stimulation studies) and Varlimab-hIgG1 or AT171-2-hIgG1 sourced in-house (hIgG1-variants and Foxp3 study, respectively); non-specific hIgG2 control sourced from BioXCell; non-specific AT171-2-hIgG4 sourced in-house. CD8⁺ T cell proliferation assays adapted the 1x10⁵/well co-stimulation assay to include 20% irradiated (30 Gy, CSXF Faxitron MultiRad350 X-ray Irradiator) CD14⁺ cells (provides FcγR to drive cross- linking) and 80% CFSE-labelled purified CD8⁺ T cells. In the mouse splenocyte proliferation assay, 1x10⁵/well CFSE-labelled (</=5x10⁷ cells/ml 10µM CFSE (Sigma-Aldrich) for 5min at 37^oC and washed 2x) spleen cells were co-stimulated for 2-3 days with 0.01µg/ml Anti-Mo CD3 (In-house clone: 2C11) together with titrated concentrations of B9-mIgG1 (B9-M1) versus AT171-1-mIgG1 (M1, in-house clone) isotype control. HNSC carcinoma TIL processing and activation The study was approved by the Medical Research and Ethics committee (MREC 09/H0501/90) and written informed consent was obtained from all patients. Tumour biopsies and blood samples were obtained from 7 patients with HPV⁺ tumours at Poole Hospital NHS Foundation Trust (UK) (Table 4). Donor TNM site gender age hpv scc HN370 T1N2aM0 oropharynx female 42 + yes HN373 T1N1M0 oropharynx male 81 + yes HN379 T1N1M0 oropharynx male 55 + yes HN381 T3N1M0 oropharynx male 67 + yes HN389 T4N2bM1 oropharynx female 75 + yes HN399 T2N1M0 oropharynx male 65 + yes HN401 T4N2BM0 oropharynx female 58 + yes Table 4: Demographics for Head and Neck SCC. To isolate tumour-infiltrating lymphocytes (TIL), resected tumour samples were cut into small fragments in RPMI containing 0.15 Wu/ml of liberase DL and 800 units/ml of DNase I and incubated at 37^oC for 20 mins at 200rpm. RPMI containing 10% fetal calf serum was added to the digested fragments that were then dispersed through a 100 µm strainer. Cells were centrifuged and washed once before activation. For TIL proliferation assays, OKT3 was diluted at 500 ng/ml or 1 µg/ml in pH 9.650 mM bicarbonate coating buffer and immobilised on 96- 57 241018 CMAL003WO1 DCA well U-bottom plates for 3 hours and washed 3 times with PBS. Freshly isolated TILs were distributed at 7.5x10⁴ cells per well in triplicates and stimulated for 4 days with plate-bound OKT3 and 0.1 µg/ml of soluble hIgG1/B9-hIgG1, in the presence of irradiated (as described before) autologous CD14⁺ cells (2.5x10⁴ cells per well). SSC carcinoma TIL processing and activation Ethical approval for the study was provided by the South Central-Hampshire B NRES Committee (reference number 07/H0504/187). Skin squamous cell (SCC) carcinoma tumour samples were collected from 6 donors (Table 5). Donor Sex Age Tumour Site Histological Histological Depth diameter diagnosis differentiation of (mm) invasion (mm) 554 M 88 15 L elbow SCC well moderate 2.3 556 F 90 26 R jawline SCC well 2 563 F 82 16 R lower leg SCC poor 5 580.2 M 88 22 R posterior SCC poor 4.2 parietal scalp 581 M 88 15 R scalp SCC moderate 2 564 F 88 15 L forehead SCC moderate 3.5 Table 5: Demographics for Skin SCC. Tumour samples were finely cut into small pieces with scalpels and incubated at 37^oC for 1.5 hours in plain RPMI containing 1mg/ml collagenase IA and 10 µg/ml DNase I (Sigma-Aldrich enzymes). Digested tumours were filtered through 100 µm cell strainers and washed with complete RPMI media. Suspensions were centrifuged over an Optiprep density gradient (Sigma-Aldrich) and the lymphocyte layer extracted and washed with PBS prior to staining with anti-CD3 mAb (clone: UCHT1 – Biolegend) for FACS sorting (BD FACS Aria). FACS sorted CD3⁺ TILs were stimulated with soluble OKT3 (0.5 ng/mL) and hIgG1/B9-hIgG1 (0.1 µg/mL) in the presence of autologous CD3-depleted irradiated-PBMCs for 72 hours. 58 241018 CMAL003WO1 DCA Analysis of activated TIL proliferation Cells were pulsed with 1 uCi/ml tritiated thymidine (Perkin Elmer) for an additional 16 hours of culture, then harvested for radiation scintillation counting according to the local safety regulations and the manufacturers recommendations (filter plates, harvester, scintillation fluid, Top Count scintillation counter - all from Perkin Elmer). In vivo experiments Post-tumour vaccination therapy involved implanting 2x10⁵ B16-OVA-GFP cells S.C. into 3 groups of 5 “homozygous” hDR3-Tg and vaccinating with 5 mg OVA plus 200 µg 3G8/AT171- 2-mIgG1 isotype control or B9-mIgG1 in 200 µl PBS I.V., the following day. SIINFEKL-specific CD8⁺ T cell immune responses were analysed in the blood, six days after vaccination, by flow cytometry. Tumour growth was measured by callipers over time. Mice were culled when they reached the terminal end-point (15mm x 15mm and/or ulcer score >6). The lung metastatic melanoma model was generated through injecting 5x10⁵ B16-OVA-GFP I.V. and treated with 200µg AT171-2 isotype control, B9 mIgG1 or tetravalent B9 mIgG1 on days 1, 3 and 5 (3 groups of 5-6 mice). An additional group of B16-OVA control mice (6-9) were kept to assess the trajectory of B16-OVA growth on the lungs by culling 1-2 control mice every two days from day 15 onwards and counting B16-OVA foci (dark spots) on the surface of the lungs. Once the control mice exhibited an average >50 foci (black spots/tumours) on the surface of the lungs; the remaining mice were culled the following day for lung analysis as described. For CD8-depletion: mice were treated with 100µg in-house YTS169.4-mIgG2a (Cobbold, S.P., et al., Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Nature, 1984.312(5994): p.548-51) both two days before and one day after B16-OVA challenge. For NK-depletion: mice were treated with 100µg in-house PK136-mIgG2a (Koo, G.C., et al., The NK-1.1(-) mouse: a model to study differentiation of murine NK cells. J Immunol, 1986. 137(12): p. 3742-7) both two days before and one day after B16-OVA challenge. CD8/NK depletion efficacy was checked by flow cytometry using blood samples taken 4 days after B16-OVA challenge. Statistics Statistical data was collated in Microsoft EXCEL spreadsheets and analysed/presented using GrapPad Prism. HNSC clinical data was analysed using the online “Tumor IMmune Estimation Resource” (Li, T., et al., TIMER: A Web Server for Comprehensive Analysis of Tumor- Infiltrating Immune Cells. Cancer Res, 2017.77(21): p. e108-e110). Structural analysis 59 241018 CMAL003WO1 DCA Antibody structure was analysed using publicly available abYsis.org web-based antibody research system which includes pre-analysed sequence data from the European Molecular Biology Laboratory European Nucleotide Archive (EMBL-ENA) and Kabat as well as structure data from the Protein Data Bank (Swindell et al., 2017, J Mol Biol 429(3):356-364). Quote: “A defining characteristic of abYsis is that the sequences are automatically numbered with a series of popular schemes such as Kabat and Chothia and then annotated with key information such as complementarity-determining regions and potential post-translational modifications” (Swindell et al., 2017, J Mol Biol 429(3):356-364). Isolation of DR3 agonist mAbs by guided selection, humanisation of B9 tool mAb and via Phage Display selections Example 2. Production of recombinant DR3-8i protein 2.1 Cloning, expression, and purification of DR3-8i protein DR3-8i was produced as a fusion protein (SEQ ID NO: 92) comprising of human DR3’s ectodomain (cysteine-rich repeats 1-4 comprising amino acids 25 to 195 of human TNFRSF25; UniProt Q93038) and the C-terminus of human decoy receptor TNFRSF6B (DcR3; amino acids 194 to 300 of human TNFRSF6B; UniProt O95407) (Figure 12; Table 6). Note that fusing the DR3 ectodomain to the C-terminal portion of soluble DcR3 reduced formation of aberrant covalent dimers and dimerisation of DR3 ectodomain fusions to human antibody crystallisable fragments (DR3-Fc; see below) as observed by non-reducing sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) compared to other proteins comprising only the DR3 ectodomain. In addition, the full-length recombinant protein contains an N-terminal signal peptide (from TNRSF6B) for co-translational targeting to the secretory pathway, a His8-tag to allow for immobilised metal affinity chromatography, a solubility-enhancing maltose binding protein (MBP) domain, and a PreScission protease cleavage site. The protein-encoding sequence was obtained by gene synthesis and inserted downstream of a CMV promoter via the KpnI and EcoRI restriction sites into the pcDNA3.1 plasmid (Invitrogen) using standard molecular cloning techniques. A C-terminal Avi-tag- encoding sequence (for site-specific biotinylation of the protein) was added to the open reading frame (ORF) by site-directed mutagenesis. The amino acid sequences of the full- length fusion protein as well as the purified final product ‘DR3-8i’ after site-specific proteolytic cleavage are presented in Table 7. Human DR3 MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLF (TNFRSF25) CCRGCPAGHYLKAPCTEPCGNSTCLVCPQDTFLAWENHHNSECAR (SEQ ID NO: 2) CQACDEQASQVALENCSAVADTRCGCKPGWFVECQVSQCVSSSPF YCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVSCPTS TLGSCPERCAAVCGWRQMFWVQVLLAGLVVPLLLGATLTYTYRHCW PHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQL 60 241018 CMAL003WO1 DCA VGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESP AGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVE IGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSR LQRGP Human DcR3 MRALEGPGLSLLCLVLALPALLPVPAVRGVAETPTYPWRDAETGERL (TNFRSF6B) VCAQCPPGTFVQRPCRRDSPTTCGPCPPRHYTQFWNYLERCRYCN (SEQ ID NO: VLCGEREEEARACHATHNRACRCRTGFFAHAGFCLEHASCPPGAGV 132) IAPGTPSQNTQCQPCPPGTFSASSSSSEQCQPHRNCTALGLALNVP GSSSHDTLCTSCTGFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRL LQALEAPEGWGPTPRAGRAALQLKLRRRLTELLGAQDGALLVRLLQA LRVARMPGLERSVRERFLPVH Table 6: DR3 and DcR3 protein sequences. Primary amino acid sequences of human DR3 (SEQ ID NO: 2) and human DcR3 (SEQ ID NO: 132) obtained from UniProt (IDs: Q93038 and O95407, respectively; www.uniprot.org) Recombinant DR3-8i protein was produced by Expi293F mammalian cells as a secreted fusion protein using the Expi293 Expression System Kit (Gibco by Life Technologies; Cat# A14635). Cells were grown in a 200 ml suspension culture and transfected with 200 µg pcDNA3.1-DR3-8i plasmid DNA according to manufacturer’s instructions. The cells were incubated for four days in a cell culture incubator (37°C, 80% humidity, 8% CO₂), shaking at 120 rpm. Afterwards, the cells were pelleted by centrifugation in a table-top centrifuge for 5 minutes at 300 × g followed by centrifugation for 15 minutes at 2800 × g. The clarified cell culture medium (CCM) containing the recombinant protein was filtered using a 0.2-µm pore- size vacuum filter unit (Nalgene; Cat# 10421791). The CCM was then concentrated and diafiltrated with PBS pH 6.8 by tangential flow filtration using a 30-kDa molecular weight cut- off (MWCO) membrane cassette (Pall; Cat# OA030C12). The recombinant protein was then purified by nickel affinity chromatography using a 5-ml HisTrap HP column (Cytiva; Cat# 17- 5248-02) attached to an ÄKTAxpress liquid chromatography system (Cytiva) and equilibrated with PBS pH 6.8 at a flow rate of 2.5 ml/minute. After a wash step with PBS pH 6.8, bound protein was eluted applying a step gradient of 5 column volumes (CV) of 10% solution B (PBS pH6.8 containing 400 mM imidazole), 10 CV of 30% B, and 10 CV of 100% B. The DR3-8i fusion protein eluted at 100% B and corresponding fractions were pooled. Proteolytic cleavage of the fusion protein with glutathione S-transferase (GST)-tagged PreScission protease (Cytiva; Cat# 27-0843-01) was carried out in a 10-kDa MWCO membrane cassette (ThermoFisher Scientific; Cat# 66830) during dialysis against a total volume of 5 litres buffer solution (25 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM EDTA) over 16 hours at 4°C. Uncleaved full-length protein as well as His8-MBP cleavage product and GST-tagged protease were removed by affinity chromatography using a 1-ml GSTrap FF column (Cytiva; Cat# 17-5130- 02) attached in series to a 1-ml HisTrap HP column (GE Healthcare; Cat# 17-5247-01) and 61 241018 CMAL003WO1 DCA equilibrated with 25 mM Tris-HCl pH 8, 300 mM NaCl, 1 mM MgCl₂, and 15 mM imidazole. The flow-through fraction containing unbound protein was then concentrated using a 10-kDa MWCO centrifugal filter (Merck Millipore; Cat# UFC901024) and subjected to size-exclusion chromatography (SEC) using a HiLoad 16/600 Superdex 200 pg column (Cytiva; Cat# 28- 9893-35) equilibrated with 25 mM Tris-HCl pH 8 and 150 mM NaCl. The run was performed on an ÄKTAxpress liquid chromatography system at a flow rate of 1 ml/minute. The elution fractions containing pure DR3-8i protein were pooled and concentrated using 10-kDa MWCO centrifugal filters (Merck Millipore; Cat# UFC801024). The concentrated protein was stored frozen in aliquots at -70°C. Depending on the downstream applications, DR3-8i was produced either as non-biotinylated or biotinylated protein. In the latter case, site-specific biotinylation of DR3-8i on its Avi-tag (Figure 12; Table 7) was performed during the purification process. For that, DR3-8i protein obtained after the preparative SEC step was concentrated and biotinylated using recombinant BirA enzyme (Sigma; Cat# SRP0417) and a commercial biotinylation kit (Avidity, Cat# BULKBIRA) according to manufacturer’s instructions. DR3-8i MRALEGPGLSLLCLVLALPALLPVPAVRGHHHHHHHHMGIEEGKLVI (SEQ ID NO: 92) WINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGD GPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNG KLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFN LQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLV DLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNY GVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEG LEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQM SAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGGGSLEVLFQG PQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGN STCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVA DTRCGCKPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLC SRRDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVCTSCT GFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPEGWG PTPRAGRAALQLKLRRRLTELLGAQDGALLVRLLQALRVARMPG LERSVRERFLPVHGLNDIFEAQKIEWHE N-terminal signal MRALEGPGLSLLCLVLALPALLPVPAVRG peptide (SEQ ID NO: 133) 62 241018 CMAL003WO1 DCA His8-tag HHHHHHHH (SEQ ID NO: 134) Maltose binding MGIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEK protein (MBP) FPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFT (SEQ ID NO: 135) WDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKA KGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAG AKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWS NIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFL ENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQK GEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSGG PreScission LEVLFQ/GP protease cleavage site (SEQ ID NO: 136) human DR3^25-195 QGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCGNST (SEQ ID NO: 137) CLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADT RCGCKPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSR RDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVC human DcR3^194- TSCTGFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPE 3⁰⁰ (SEQ ID NO: GWGPTPRAGRAALQLKLRRRLTELLGAQDGALLVRLLQALRVARM 138) PGLERSVRERFLPVH Avi-tag at the C- GLNDIFEAQKIEWHE terminus (SEQ ID NO: 139) The sequence of GPQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAPCTEPCG the final ‘DR3-8i’ NSTCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAV purified product ADTRCGCKPGWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLL (GPQ…WHE CSRRDTDCGTCLPGFYEHGDGCVSCPTSTLGSCPERCAAVCTSC (SEQ ID NO: TGFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPEGW 140)) GPTPRAGRAALQLKLRRRLTELLGAQDGALLVRLLQALRVARMP GLERSVRERFLPVHGLNDIFEAQKIEWHE Table 7: DR3-8i protein sequence. The full-length DR3-8i fusion protein (SEQ ID NO: 92) consists of an N-terminal signal peptide (MRA…VRG SEQ ID NO: 133) followed sequentially by a His8-tag (SEQ ID NO: 134), maltose binding protein (MBP) (MGI…SGG (SEQ ID NO: 135)), and a PreScission protease cleavage site (LEVLFQ/GP (SEQ ID NO: 136)). DR3-8i 63 241018 CMAL003WO1 DCA consists of human DR3^25-195 (QGG…AVC (SEQ ID NO: 137)) and human DcR3^194-300 (TSC…PVH (SEQ ID NO: 138) and an Avi-tag (GLNDIFEAQKIEWHE (SEQ ID NO: 139)) at the C-terminus. The sequence of the final ‘DR3-8i’ purified product (GPQ…WHE (SEQ ID NO: 140)) is highlighted in bold italics. Example 3. B9 Guided Selections 3.1 Conversion of B9 mouse tool monoclonal antibody to ribosome display format The DNA sequences encoding the corresponding variable heavy chain (V_H) and light chain (V_L) domains of the hybridoma-derived DR3 agonist B9 tool monoclonal antibody (mAb) were subcloned as a scFv into the pUC-RD vector for ribosome display (RD) selections. For that, the B9 scFv-encoding sequence was synthesised de novo and codon-optimised for E. coli expression; V_H and V_L sequences were separated by a (G₄S)₃ linker-encoding sequence. The B9 scFv-encoding sequence was ligated into the ribosome display vector pUC-RD using standard molecular biology techniques. The integrity of the open reading frame was confirmed by Sanger sequencing. 3.2 Model B9 guided selections Model RD selections were performed to assess the parental B9 scFv’s suitability for use with RD. Selections were completed as described by Thom, G. and Groves, M. (2012) Ribosome Display in Proetzel G., Ebersbach H. (eds) Antibody Methods and Protocols, Methods in Molecular Biology, Vol 901. Humana Press, Totowa, NJ. A single round of ribosome display was completed using 100 nM or 50 nM of the target antigen, recombinant human DR3-Fc (RnD Systems; Cat# 943-D3). Protein G beads (Invitrogen; Cat# 10004D) were used to pull down DR3-Fc/B9-scFv/mRNA/ribosome complexes. Streptavidin beads (Invitrogen; Cat# 11205D) were used to pull down the positive control biotinylated Insulin/D3- scFv/mRNA/ribosome complexes. Purified mRNA was reverse transcribed and then amplified by reverse transcription PCR (RT-PCR). The antigen-specific enrichment of the parental clone was visualised by resolving samples of each RT-PCR product on an agarose gel and comparing it directly to the corresponding ‘no antigen control’ selection (Figure 13). 3.3 Guided selection B9 shuffle library builds Guided selection B9 V_H and V_L shuffle libraries were created according to Osbourn, J., et al. 2005 (Osbourn J, Groves M, and Vaughan T. (2005) From rodent reagents to human therapeutics using antibody guided selections. Methods. Vol.36 (1), 61 - 68). The parental clone B9 was used as a starting template, into which a repertoire of naïve human VH or VL antibody sequences were introduced, creating either a B9 V_H shuffle or B9 V_L shuffle library, respectively (Figure 14). The human naïve antibody repertoire was amplified from the 64 241018 CMAL003WO1 DCA AstraZeneca ‘Combined Spleen’ phage display library (Lloyd C, Lowe D, Edwards B, Welsh F, Dilks T, Hardman C, Vaughan T. (2009) Modelling the human immune response: performance of a 10¹¹ human antibody repertoire against a broad panel of therapeutically relevant antigens. Protein Eng. Des. Sel. Vol.22, 159–68). The CS VH repertoire was amplified using a pool of germline-specific ‘SDCAT_XX’ primers and the scFv linker-specific primer ‘H- Link’ (Table 8). The CS VL repertoire was amplified using the forward ‘mycRestore’ and reverse ‘L-Link’ primer combination (Table 8). The DNA template used for these PCRs was a purified plasmid preparation derived from a CS library glycerol stock. B9 V_H was PCR amplified using ‘SDCAT-B9F10’ and ‘H-Link’ primers. The B9 V_L was PCR amplified using ‘mycRestore’ and ‘L-Link’ primers (Table 8). Pull-through recombination PCR was used to join the B9 V_H gene with the CS V_L repertoire (B9 V_L shuffle library) and the CS V_H repertoire with the B9 V_L gene (B9 V_H shuffle library). Full-length scFv-encoding DNA inserts were obtained by using pooled ‘SDCAT_XX’/’mycRestore’ primer combinations (B9 V_H-shuffle library) or ‘SDCAT_B9F10’/’mycRestore’ primer pairs (B9 V_L shuffle library). Each recombination PCR product was resolved on a TAE agarose gel, corresponding bands were excised and DNA was extracted. A gene III tether fragment (G3T) was PCR amplified from an empty pCantab6 vector using primers ‘geneIIIfor2’ and ‘mycG3SA’ (Table 8). The G3T fragment was joined to the B9 VH- shuffle and B9 VL-shuffle libraries using pull-through recombination PCR. Full-length scFv- G3T inserts were PCR amplified using pooled ‘SDCAT_XX’/’T8te’ primer pairs or ‘SDCAT_B9F10’/’T8te’ primer pairs for the B9 VH-shuffle library and B9 VL shuffle library, respectively. Each recombination PCR product was resolved on a TAE agarose gel, before excising the corresponding bands and DNA extraction. This B9 VH-shuffle and B9 VL-shuffle amplified DNA was subsequently digested and ligated into the pCantab6 vector and transformed into TG1 E. coli cells. A panel of 44 clones from each library were sequenced to confirm overall library integrity and diversity. The two libraries were amplified one further time using ‘T7B’ and ‘T6te’ primer pairs to generate double-strand DNA template for RD selections (Table 8). Vent high-fidelity DNA polymerase was used for this final PCR amplification (New England Biolabs; Cat# M0254S). Primer name Primer sequence (5’ – 3’) SDCAT_H1 AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA (SEQ ID NO: 141) AGAAGGAGATATATCCATGGCCCAGRTGCAGCTGGTGCART 65 241018 CMAL003WO1 DCA SDCAT_H2 AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA (SEQ ID NO: 142) AGAAGGAGATATATCCATGGCCSAGGTCCAGCTGGTRCAGT SDCAT_H3 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 143) AGAAGGAGATATATCCATGGCCCAGRTCACCTTGAAGGAGT SDCAT_H4 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 144 ) AGAAGGAGATATATCCATGGCCSAGGTGCAGCTGGTGGAG SDCAT_H5 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 145) AGAAGGAGATATATCCATGGCCCAGGTGCAGCTACAGCAGT SDCAT_H6 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 146) AGAAGGAGATATATCCATGGCCCAGGTGCAGCTACAGCAGT SDCAT_H7 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 147) AGAAGGAGATATATCCATGGCCGARGTGCAGCTGGTGCAG SDCAT_H8 (SEQ ID AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA NO: 148) AGAAGGAGATATATCCATGGCCCAGSTGCAGCTGCAGGAGTC SDCAT_DP47 (SEQ AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA ID NO: 149) AGAAGGAGATATATCCATGGCCGAGGTGCAGCTGTTGGAGT SDCAT_EG3 (SEQ AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA ID NO: 150) AGAAGGAGATATATCCATGGCCGAGGTGCAGCTGGTGGAG H-Link (SEQ ID NO: ACCGCCAGAGCCACCTCCGCC 151) L-Link (SEQ ID NO: GGCGGAGGTGGCTCTGGCGGT 152) mycRestore (SEQ ID ATTCAGATCCTCTTCTGAGATGAG NO: 153) geneIIIfor2 (SEQ ID CCGTCACCGACTTGAGCC NO: 154) mycG3SA (SEQ ID ATCTCAGAAGAGGATCTGAATGGTGGCGGCTCCGGTTCCGGT NO: 155) GAT T8te (SEQ ID NO: CACCAGTAGCACCATTACCATTAGCAAGG 156) T7B (SEQ ID NO: ATACGAAATTAATACGACTCACTATAGGGAGACCACAACGG 157) T6te (SEQ ID NO: CCGCACACCAGTAAGGTGTGCGGTATCACCAGTAGCACCATT 158) ACCATTAGCAAG SDCAT-B9F10 AGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTA (SEQ ID NO: 159) AGAAGGAGATATATCCATGGCCGAAGTGCAGC Table 8. Guided selection shuffle library PCR primers. 3.4 Ribosome display-based guided selections 66 241018 CMAL003WO1 DCA An overview of the B9 RD guided selection strategy is provided shown in Figure 14. Parallel RD selections with B9 V_H-shuffle and V_L-shuffle libraries (Example 3.3) were completed as described in Example 3.2. The target antigen was recombinant human DR3-Fc (RnD Systems; Cat# 943-D3). Two rounds of selection were completed, using 50 nM antigen at round 1 and 30 nM at round 2 (Figure 15). The round 2 B9 VH-shuffle and VL-shuffle library outputs were subcloned into pCantab6 plasmid using standard molecular biology techniques and a representative panel was sequenced. B9 V_H-shuffle outputs contained full-length scFv sequences, all of which contained a human V_H sequence coupled to the original parent mouse B9 V_L sequence. In contrast, B9 V_L-shuffle outputs had mostly reverted to the original fully mouse B9 parent scFv sequence, suggesting that light-shuffle sequences were inefficiently selected even in the presence of trace parental sequence found within the original B9 V_L- shuffle library. Due to the failure of the B9 V_L-shuffle guided selections, only the B9 V_H-shuffle outputs were taken forward and recombined with the human VL repertoire (stage 2; Figure 16). Construction of the human::human VL-shuffle library was completed as described for the generation of the chimeric human::mouse VL-shuffle library (Example 3.3). Guided ribosome display selections using the human::human VL-shuffle library were performed as described in Example 3.2. A single round of selection was completed, using 150 nM or 50 nM of the target antigen protein, DR3-Fc (RnD Systems; Cat# 943-D3). Resolving end-point PCRs on a 1% TAE agarose gel revealed that no target-specific enrichment had occurred with this library. Example 4. Rational design of B9 humanised germline variants 4.1 Germline choice and complementarity-determining region (CDR) grafting A panel of 13 VH and 13 VL human germline sequences were chosen as frameworks for B9 humanisation. Human germline choice was based on (i) sequences that were enriched in B9 guided selection outputs (Example 3.4) and (ii) which human germline sequences were most similar in sequence to the original mouse B9 sequence (www.IMGT.org/). Human germline sequences were engineered to be either ‘3× VH CDR grafts’, ‘3× VL CDR grafts’ (in which all three CDRs were grafted) or ‘single VHCDR3’ grafts (where only CDR3 was grafted). Two VL variant sequences, identified by phage ELISA screening (Example 6.1) of B9 guided selection outputs, were also included in the panel of 13 V_L sequences (Figure 17). Each V_H- and V_L- encoding sequence was synthesised de novo and cloned into the appropriate IgG expression vector (Persic, L. et al., 1997) using standard molecular biology techniques. Example 5. High-throughput IgG expression 67 241018 CMAL003WO1 DCA 5.1 IgG high-throughput expression for B9 humanisation The panel of humanised B9 clones was expressed as human IgG1 in a high-throughput manner (Screening in Product Format, ‘SiPF’) (PMID: 28613102) using mammalian Expi293F cells following Life Technologies Expi293 Expression System protocol for 96-well microtiter plates (Protocol # CO25793 0912). Each of the 13 humanised B9 V_H and V_L constructs (Example 4.1; Figure 17) were expressed as a matrix in a 96 deep-well plate generating all 169 possible V_H × V_L combinations. Culture medium containing secreted IgGs was collected 4 days after transfection and used as crude preparations of unpurified IgGs in subsequent assays. See Example 5.2 for IgG quantification in culture medium. 5.2 High-throughput IgG expression quantification The concentration of high-throughput-expressed B9 humanised variant IgGs (Example 5.1) was determined by BioLayer Interferometry (BLI) using the OctetRED96 platform (Pall Fortébio) (Figure 18, y-axis of the graph). Protein A biosensors (Pall FortéBio; Cat# 18-5010) were used to capture expressed IgGs for quantification using a standard curve (0.4 – 200 µg/ml non-humanised B9 as human IgG1 isotype). Example 6. Screening of antibodies from guided selections (B9 humanisation) 6.1 Specificity phage ELISA with clones from guided selections A panel of sequence-unique scFv clones were picked from stage 1 human::mouse chimeric VH and VL shuffle selection outputs (Figures 15 & 16). Specifically, 88 and 10 clones were chosen from round 1 and round 2 VL shuffle outputs, respectively (see Example 3.4). A further 44 clones were chosen from the round 2 VH-shuffle outputs. The binding specificity of each clone was determined by generating phage that display the phagemid-encoded scFv on their surface as a gene III fusion, before assessing phage binding to a panel of human DR3 and irrelevant proteins. In brief, individual bacterial clones were grown in 500 µl of 2× TYAG media (2× TY plus 100 µg/ml ampicillin and 2% (w/v) glucose) to mid-log phase in a 2-ml 96-well plate (Greiner Bio-one; Cat# 780270-FD). K07 M13 helper phage was added, and the cultures were left to grow for a further hour at 37°C while shaking at 150 rpm. The bacterial cultures were pelleted at 3200 rpm for 10 minutes in a benchtop centrifuge before the media was replaced with 500 µl of 2× TYAK media (2× TY plus 100 µg/ml ampicillin and 50 µg/ml kanamycin). Plates were cultured overnight at 25°C shaking at 280 rpm. Sixteen hours later, an equal volume of 6% (w/v) skimmed milk powder (Marvel) in 2× PBS was added to block the phage cultures before incubating at room temperature (RT) for 1 hour. Test proteins were adsorbed to Nunc MaxiSorp plates overnight at 4°C at a coating concentration of 2.5 µg/ml. Recombinant proteins screened included DR3-Fc (RnD Systems; 68 241018 CMAL003WO1 DCA Cat# 943-D3) and DR3-8i (Example 2.1; Figure 12). The antigen-coated plates were washed once with PBS, before blocking with Marvel-PBS (3% (w/v) skimmed milk powder in 1× PBS) for 1 hour at RT. Each 96-well plate was spun for 10 minutes at 3200 rpm in a benchtop centrifuge to pellet the bacteria and to recover a clarified phage supernatant. The blocked antigen plates were washed once with PBS before 50 µl of the clarified phage supernatant was added. The phages were allowed to bind for 1 hour at RT before unbound phage were removed by washing three times with 1× PBS, 0.1% (v/v) Tween-20 solution. Washed wells were incubated with the anti-M13-HRP secondary antibody (Sino Biologicals; Cat# 11973- MM05T-H) for 1 hour at room temperature. Unbound secondary antibody was removed by washing five times with 1× PBS, 0.1% (v/v) Tween-20 solution. Bound phages were detected by incubating with HRP substrate 3,3’,5,5”-tetramethylbenzidine (TMB) and the reaction was stopped with the addition of an equal volume of 0.5 M H₂SO₄. All plates were read using an Envision plate reader (PerkinElmer) measuring absorbance at 450 nm. 6.2 Jurkat human DR3 reporter cell assay An engineered fluorescent NF-κB transcriptional reporter cell line stably expressing the human DR3 was used as a functional assay for DR3 agonism. Three cell lines were established for this process, designated DR3^hi, DR3^med and DR3^no based on their relative surface expression of human DR3 (high, medium, and not detectable, respectively). Upon incubation with a DR3 activator (such as the natural ligand, TLA1, or an agonist IgG) the DR3^hi and DR3^med cells produce GFP, which in turn can be detected using a fluorescence cytometer. DR3^no cells express GFP at baseline level. IgGs from high-throughput IgG expression outputs (Example 5), were mixed with reporter cells and incubated at 37°C to allow GFP expression to occur. Cell-free control wells were included to account for media autofluorescence. Multi-well assay plates were then read on a Mirrorball fluorescence cytometer, using the 640 nm laser to detect the number and intensity of fluorescent objects in the green channel (488 – 540 nm). Functional agonism is reported as the number of GFP-positive cells multiplied by the median mean fluorescence intensity and corrected for background fluorescence by subtraction of control well signal from each experimental well. For the B9 humanisation approach, Table 9 show corrected reporter assay fluorescence data from human DR3 positive cells, which were also matched with the high throughput quantification data from the same expression plate (Figure 18) (Example 5.2). All results with fluorescence greater than or equal to two thirds of the B9 positive control were considered ‘hits’. Outputs with expression levels lower than one quarter of the B9 positive control levels 69 241018 CMAL003WO1 DCA were revisited and included as hits if the fluorescence signal from the reporter assay was also greater than or equal to one third of the B9 control signal. Any outputs with an expression level too low to be quantified were eliminated from the hit pool. As a final filter, the ratio of corrected fluorescence signal in the DR3^no cells relative to the human DR3 positive cells was calculated for each output and any samples with a high background (control greater than 10% of human DR3-positive for DR3^hi cells, and greater than 50% of human DR3-positive for DR3^med cells) were eliminated as hits. Germline Signal (FLU) Concentration (μg/mL) no/hi (%) VHg13/mVL 4702775 68.60 3.64% VHg10/VLg12 3562775 19.19 0.86% mVH/VLg2 3512775 62.90 6.73% mVH/mVL 3452775 15.20 3.20% 30nM B9 IgG 3430900 8.23 2.01% VHg9/mVL 3352775 5.07 3.74% VHg9/VLg13 3327775 4.09 1.24% mVH/VLg6 3297775 1.56 2.71% VHg9/VLg11 3272775 10.20 1.57% VHg10/VLg13 3267775 17.60 1.74% VHg10/VLg11 3237775 26.30 1.46% VHg10/mVL 3197775 3.10 2.75% VHg10/VLg4 3167775 4.71 1.42% mVH/VLg4 3117775 4.10 1.73% mVH/VLg5 3102775 7.23 1.45% VHg10/VLg5 3077775 8.39 2.58% mVH/VLg6 3062775 2.49 1.27% VHg9/VLg5 3052775 2.61 1.91% VHg13/VLg5 3027775 55.10 1.41% VHg9/VLg4 2957775 1.48 1.49% VHg2/VLg13 2937775 4.67 1.82% VHg2/VLg11 2902775 16.30 1.68% VHg13/VLg11 2877775 93.00 1.78% VHg9/VLg2 2877775 20.10 6.18% VHg3/mVL 2862775 16.40 1.09% VHg6/VLg5 2847775 3.61 1.34% VHg10/VLg2 2837775 32.80 3.27% 70 241018 CMAL003WO1 DCA VHg13/VLg4 2812775 40.90 1.14% VHg1/VLg13 2812775 5.36 1.69% VHg13/VLg3 2807775 21.20 2.65% VHg7/VLg5 2782775 0.98 1.19% mVH/VLg13 2782775 65.50 1.74% VHg5/VLg2 2762775 2.80 1.23% VHg1/VLg12 2757775 7.30 1.92% VHg6/VLg13 2747775 24.80 2.13% VHg10/VLg6 2747775 1.31 3.36% VHg13/VLg12 2742775 51.64 1.84% VHg5/VLg13 2697775 1.28 1.46% VHg5/mVL 2692775 0.59 1.50% VHg13/VLg13 2687775 50.60 1.29% VHg6/VLg4 2682775 1.39 1.16% VHg2/VLg12 2682775 15.20 1.66% VHg3/VLg13 2662775 28.90 1.78% VHg7/VLg13 2662775 13.50 2.40% mVH/VLg5 2642775 7.23 1.09% VHg7/VLg12 2622775 17.50 1.39% VHg5/VLg12 2617775 2.66 0.92% VHg10/VLs9 2607775 22.80 2.55% VHg6/mVL 2602775 10.90 1.33% VHg3/VLg12 2602775 24.60 1.69% VHg6/VLs10 2592775 1.06 2.45% mVH/VLg12 2577775 17.83 3.84% VHg6/VLs9 2572775 3.27 1.55% VHg2/mVL 2567775 3.19 1.63% VHg10/VLg1 2542775 0.80 5.74% mVH/VLg11 2537775 18.40 2.18% VHg2/VLg5 2517775 1.63 2.10% VHg10/VLg3 2517775 0.66 2.38% VHg2/VLg2 2502775 17.80 1.68% VHg2/VLs9 2482775 4.89 1.62% VHg1/VLg11 2472775 7.92 1.98% VHg6/VLg12 2467775 28.10 1.18% 71 241018 CMAL003WO1 DCA VHg4/VLg13 2462775 19.30 1.48% VHg3/VLg1 2457775 4.44 1.95% VHg6/VLg11 2447775 30.30 1.63% mVH/VLg4 2442775 3.11 1.42% VHg6/VLg2 2442775 33.50 2.06% VHg1/VLg2 2437775 3.08 2.46% VHg7/VLg11 2432775 14.60 1.23% VHg3/VLg5 2432775 5.87 1.72% VHg1/mVL 2427775 1.67 1.57% VHg5/VLg5 2422775 0.49 1.68% VHg7/VLg2 2412775 14.10 1.73% mVH/VLs9 2402775 18.10 1.60% VHg5/VLg11 2397775 2.54 0.84% VHg10/VLs10 2397775 9.99 1.32% VHg3/VLg11 2382775 38.00 2.49% VHg7/mVL 2372775 1.74 1.91% VHg13/VLg6 2357775 43.10 4.66% VHg2/VLs10 2342775 2.25 2.73% VHg3/VLg4 2322775 5.72 1.66% VHg9/VLg6 2317775 0.49 5.97% mVH/VLg12 2312775 62.50 1.64% VHg7/VLs9 2227775 1.23 1.87% VHg2/VLg4 2212775 0.71 2.21% VHg7/VLg4 2202775 0.69 1.89% VHg3/VLg3 2182775 1.97 1.97% VHg6/VLg1 2157775 0.54 2.57% VHg1/VLg4 2092775 0.64 2.10% VHg9/VLs10 1992775 1.56 2.02% VHg7/VLs10 1992775 0.67 5.47% VHg1/VLg5 1987775 0.58 2.51% mVH/VLg3 1977775 0.50 3.13% VHg4/mVL 1967775 1.38 2.41% VHg1/VLs9 1957775 0.94 2.45% VHg1/VLs10 1897775 0.62 1.98% mVH/VLg1 1872775 0.56 3.15% 72 241018 CMAL003WO1 DCA VHg9/VLg1 2232775 0.27 10.14% VHg13/VLg1 2857775 22.90 19.59% mVH/VLg7 1007275 0.27 49.83% VHg9/VLg12 681775 0.84 5.41% VHg13/VLg7 408275 1.30 9.14% VHg4/VLs9 375775 0.38 17.14% VHg13/VLg8 403775 2.50 3.17% VHg11/VLg8 404775 2.79 20.24% VHg11/VLg7 386775 2.89 18.08% VHg9/VLs9 2247775 3.01 2.42% VHg12/VLg8 394775 3.07 14.42% VHg3/VLg6 2047775 3.12 2.17% mVH/VLs10 2232775 4.18 0.74% VHg4/VLg2 2062775 6.02 2.13% mVH/VLs10 2102775 6.22 1.01% VHg12/VLg3 1247775 9.29 9.45% VHg3/VLs10 1727775 12.90 2.12% mVH/VLg13 2067775 14.30 2.12% mVH/VLg2 2262775 16.10 2.14% VHg12/VLg1 1157775 18.40 53.15% VHg12/VLg4 952775 19.60 4.61% VHg12/VLg6 602775 19.90 36.82% VHg12/VLg5 802275 20.10 9.03% VHg12/mVL 1672775 20.60 12.94% mVH/mVL 2107775 20.70 1.22% VHg4/VLg12 2142775 22.50 1.52% VHg3/VLs9 1762775 22.70 3.06% VHg12/VLs10 384275 23.50 11.30% VHg4/VLg11 2082775 24.40 1.85% mVH/VLs9 2012775 25.10 1.44% VHg12/VLg13 528275 27.10 8.12% VHg11/VLg3 498775 36.10 23.44% VHg12/VLg11 1387775 36.70 2.16% VHg3/VLg2 2227775 36.70 1.53% VHg11/VLg1 854775 39.70 103.29% 73 241018 CMAL003WO1 DCA VHg12/VLg12 781775 40.10 3.76% VHg11/mVL 554775 41.50 49.64% VHg12/VLs9 424275 46.60 12.59% VHg13/VLs10 413775 47.70 8.51% VHg12/VLg2 561775 54.40 17.87% VHg13/VLg2 2032775 58.30 2.97% R347 IgG 384625 60.74 12.40% VHg11/VLg4 661275 64.50 8.61% VHg11/VLs10 371275 70.50 8.26% VHg11/VLg6 667275 74.80 49.74% VHg11/VLg5 747275 83.00 18.79% VHg11/VLg13 451275 86.00 17.26% mVH/VLg11 2017775 87.30 1.53% VHg11/VLg12 469275 89.43 8.10% VHg11/VLg11 460275 124.60 6.52% VHg13/VLs9 435275 129.50 15.95% VHg11/VLg2 595275 158.10 57.52% VHg11/VLs9 416275 203.80 26.28% mVH/VLg1 1557775 n.d. 5.16% mVH/VLg3 1567775 n.d. 2.22% mVH/VLg7 369775 n.d. 7.25% mVH/VLg8 435275 n.d. 9.63% mVH/VLg8 344775 n.d. 8.36% VHg1/VLg1 796775 n.d. 8.52% VHg1/VLg3 455775 n.d. 10.51% VHg1/VLg6 1502775 n.d. 5.05% VHg1/VLg7 376775 n.d. 10.86% VHg1/VLg8 369775 n.d. 12.96% VHg10/VLg7 413275 n.d. 24.42% VHg10/VLg8 404775 n.d. 16.65% VHg12/VLg7 362275 n.d. 7.87% VHg2/VLg1 401775 n.d. 9.11% VHg2/VLg3 718275 n.d. 6.88% VHg2/VLg6 1617775 n.d. 4.11% VHg2/VLg7 372275 n.d. 14.48% 74 241018 CMAL003WO1 DCA VHg2/VLg8 373275 n.d. 10.37% VHg3/VLg7 375275 n.d. 11.10% VHg3/VLg8 358775 n.d. 12.24% VHg4/VLg1 414775 n.d. 10.43% VHg4/VLg3 410775 n.d. 14.59% VHg4/VLg4 634275 n.d. 8.18% VHg4/VLg5 607275 n.d. 6.57% VHg4/VLg6 422275 n.d. 8.77% VHg4/VLg7 394775 n.d. 13.40% VHg4/VLg8 369275 n.d. 9.09% VHg4/VLs10 422775 n.d. 14.05% VHg5/VLg1 410275 n.d. 9.17% VHg5/VLg3 370775 n.d. 9.63% VHg5/VLg4 1442775 n.d. 2.76% VHg5/VLg6 386775 n.d. 11.09% VHg5/VLg7 432775 n.d. 8.38% VHg5/VLg8 433275 n.d. 11.29% VHg5/VLs10 451775 n.d. 13.93% VHg5/VLs9 762775 n.d. 5.69% VHg6/VLg3 1782775 n.d. 3.00% VHg6/VLg6 1962775 n.d. 1.43% VHg6/VLg7 455775 n.d. 9.85% VHg6/VLg8 494275 n.d. 10.60% VHg7/VLg1 689775 n.d. 9.41% VHg7/VLg3 1019275 n.d. 3.63% VHg7/VLg6 1742775 n.d. 1.89% VHg7/VLg7 425775 n.d. 7.38% VHg7/VLg8 375775 n.d. 10.58% VHg8/mVL 503275 n.d. 12.10% VHg8/VLg1 469775 n.d. 13.60% VHg8/VLg11 489775 n.d. 8.22% VHg8/VLg12 760275 n.d. 5.97% VHg8/VLg13 382275 n.d. 9.46% VHg8/VLg2 487775 n.d. 38.22% VHg8/VLg3 455275 n.d. 10.64% 75 241018 CMAL003WO1 DCA VHg8/VLg4 460775 n.d. 9.04% VHg8/VLg5 471275 n.d. 7.44% VHg8/VLg6 446275 n.d. 13.76% VHg8/VLg7 497275 n.d. 72.18% VHg8/VLg8 426275 n.d. 11.36% VHg8/VLs10 399775 n.d. 2.77% VHg8/VLs9 772275 n.d. 2.87% VHg9/VLg3 1702775 n.d. 3.93% VHg9/VLg7 509275 n.d. 14.91% VHg9/VLg8 495775 n.d. 10.57% Table 9: B9 humanisation ‘SiPF’ reporter assay output. Reporter assay fluorescence data from human DR3-positive cells and high throughput quantification data from the same expression plate. This data is shown in Figure 18. Hits based on the reporter assay signal FLU > (2/3) B9 are in bold. Additional hits, with μg/ml < (1/4) B9 and FLU > (1/3) B9, are in bold italics. mVH and mVL represent parental mouse B9 VL and VH chains; n.d. = not detectable. A panel of thirty B9 humanised variant clones (see Figure 19 and Table 1 for sequences) were prioritised for small-scale IgG expression (Example 8.1). The triage process was guided by each clone’s performance in the Jurkat DR3 reporter cell assay and their relative expression levels (Figure 18), as well as a requirement for cross-reactivity to cell surface- expressed human and Cynomolgus DR3 (Example 6.3.3; Figure 20), all as unpurified ‘SiPF’- expressed human IgG1. An in silico assessment for the presence of non-germline residues and potential sequence-dependent developability liabilities in these clones was also completed (Example 7.1). Twenty-one of the thirty clones expressed at sufficiently high levels to allow testing in the Jurkat DR3 reporter cell assay, but this time using titrated concentrations of purified IgG (Figure 21). The EC50 values for each of these IgGs are recorded in Table 10. Clone EC₅₀ (pM) R squared VHg1/VLg2 336 0.894 VHg1/VLg11 331 0.994 VHg1/VLg12 322 0.919 VHg2/VLg2 103 0.980 VHg2/VLs10 644 0.981 VHg2/VLg11 136 0.885 76 241018 CMAL003WO1 DCA VHg2/VLg12 117 0.820 VHg3/VLg11 1232 0.988 VHg3/VLg12 1481 0.991 VHg3/VLg13 1771 0.990 VHg6/VLg2 118 0.945 VHg6/VLg4 115 0.988 VHg6/VLg11 130 0.910 VHg6/VLg12 134 0.622 VHg6/VLg13 141 0.813 VHg9/VLg11 261 0.910 VHg10/VLg4 570 0.985 VHg10/VLg5 634 0.994 VHg10/VLg6 n/a 0.952 VHg10/VLg11 245 0.880 VHg10/VLg12 132 0.771 Non-humanised B9 457 0.907 Table 10: Agonism of DR3 reporter cells by purified humanised B9 IgGs. The capacity of purified IgGs to agonise a DR3 expressing Jurkat reporter cell line was determined using a 10-point titration curve (Figure 21). Curves were fitted with a standard 4-parameter variable- slope agonist response model to determine an EC₅₀. 77 241018 CMAL003WO1 DCA K _D (M) Germ line Experiment 1 Expriment 2 Average Non-humanised B9 3.27E-10 1.90E-10 2.59E-10 VHg1/VLg11 1.00E-12 1.00E-12 1.00E-12 VHg1/VLg12 1.00E-12 4.45E-11 2.28E-11 VHg2/VLg4 2.48E-11 2.48E-11 VHg2/VLg11 6.36E-11 4.35E-11 5.36E-11 VHg6/VLg1 7.60E-11 7.60E-11 VHg5/VLg11 8.81E-11 8.81E-11 VHg2/VLg2 1.00E-12 2.10E-10 1.06E-10 VHg2/VLg12 6.44E-11 1.77E-10 1.21E-10 VHg5/VLg13 1.24E-10 1.24E-10 VHg5/VLg12 1.26E-10 1.26E-10 VHg5/VLg2 1.32E-10 1.32E-10 VHg6/VLg11 1.90E-10 9.99E-11 1.45E-10 VHg2/VLg13 1.57E-10 1.57E-10 VHg6/VLg12 2.27E-10 8.76E-11 1.57E-10 VHg1/VLg2 3.16E-10 2.02E-12 1.59E-10 VHg6/VLg13 2.25E-10 9.56E-11 1.60E-10 VHg10/VLg12 1.75E-10 1.88E-10 1.81E-10 VHg9/VLg11 1.92E-10 1.84E-10 1.88E-10 VHg6/VLg2 2.27E-10 1.59E-10 1.93E-10 VHg1/VLg4 1.96E-10 1.96E-10 VHg6/VLg4 2.50E-10 1.46E-10 1.98E-10 VHg10/VLg4 2.42E-10 1.67E-10 2.04E-10 VHg10/VLg11 1.66E-10 2.44E-10 2.05E-10 VHg10/VLg5 2.33E-10 2.59E-10 2.46E-10 VHg10/VLg6 4.18E-10 2.34E-10 3.26E-10 VHg3/VLg4 3.85E-10 3.85E-10 VHg3/VLg12 3.90E-10 4.09E-10 4.00E-10 VHg2/VLs10 4.10E-10 4.20E-10 4.15E-10 VHg3/VLg13 4.64E-10 3.89E-10 4.27E-10 VHg3/VLg11 3.64E-10 6.18E-10 4.91E-10 Table 11: Affinities for the binding of DR3-8i to a prioritised panel of humanised B9 variant IgGs. The binding interaction between immobilised candidate antibodies (IgGs) and purified monomeric DR3-8i antigen was analysed in solution using kinetic Bio-layer interferometry (BLI) (Example 6.5). Dissociation constants (K_D) based on the corresponding association and dissociation rate constants (k_on and k_off) obtained by global fitting of the data, assuming a 1:1 binding mode. Thirty B9 humanised variant IgGs were analysed, representing 78 241018 CMAL003WO1 DCA a prioritised panel taken from the starting panel of 169 clones expressed and screened for agonism and DR3 orthologue binding (Example 6.3.3). Where expression levels allowed, two independent experiments were completed, and their average affinity K_D derived. Affinity values (Table 11) and EC50 values (Table 10) for the panel of 21 humanised B9 variant IgGs were plotted against each other (Figure 22), highlighting a subset of 14 clones that displayed improvements in affinity and agonism, relative to the original non-humanised parental B9 antibody. 6.3 Cell-based DR3 binding assay using microplate cytometry (Mirrorball assay) 6.3.1 Cloning of human, mouse and Cynomolgus DR3 death domain deletion constructs For cell-based assays, DR3 was expressed on the surface of Expi293T cells. Sequences encoding amino acids 1 to 244 (SEQ ID NO: 160) of human DR3 (UniProtKB - Q93038 (SEQ ID NO: 2)), amino acids 1 to 219 (SEQ ID NO: 161) of mouse DR3 (UniProtKB - Q8VD70 (SEQ ID NO: 162) and aa 1 to 247 (SEQ ID NO: 163) of cynomolgus DR3 (UniProtKB - A0A2K5WIZ0 (SEQ ID NO: 164)) were cloned into pcDNA3.1 plasmid with a C-terminal linker, an Avi-tag, and a tandem His8-tag using standard molecular biology techniques. All DR3 orthologue constructs lack the cytoplasmic death domain. 6.3.2 Transient transfection of Expi293F cells with DR3 death domain deletion constructs The human, mouse and cynomolgus DR3 death domain deletion constructs (Section 6.3.1) were transiently transfected into Expi293F cells using the Expi293F expression system kit (Gibco by Life Technologies; Cat# A14635) according to the manufacturer’s instructions. Briefly, on the day before transfection, Expi293F cells were seeded at a density of 2× 10⁶ cells/ml in Expi293 Expression Medium and grown overnight. The cells were counted and adjusted to 2.9× 10⁶ cells/ml in a volume of 25.5 ml. Thirty micrograms plasmid DNA was diluted in Opti-MEM I Reduced Serum Medium in 1.5 ml and mixed. Afterwards, 81 µl of ExpiFectamine 293 Reagent was diluted in Opti-MEM I Reduced Serum Medium to a total volume of 1.5 ml, mixed, and incubated for five minutes at room temperature. The diluted DNA and diluted ExpiFectamine 293 Reagent were then combined and mixed resulting in a total volume of 3 ml. This mixture was incubated for 20 minutes at room temperature and added to the cells, resulting in a total culture volume of 28.5 ml. The cells were incubated in a cell culture flask for 20 hours at 37°C and 8% CO2 on an orbital shaker platform, shaking at 125 rpm. Afterwards, 150 µl ExpiFectamine 293 Transfection Enhancer 1 and 1.5 ml ExpiFectamine 293 Transfection Enhancer 2 were added to the flask, adding up to a final volume of 30 ml. The cells were incubated for a further 24 hours before being collected and cryopreserved for use in antibody binding assays. Cells were cryopreserved at 5× 10⁶ cells/ml in complete 79 241018 CMAL003WO1 DCA growth medium plus 5% (v/v) DMSO. This process was repeated for each DR3 death domain deletion construct. 6.3.3 Mirrorball DR3 orthologue screen Species cross reactivity profiles for the entire panel of 169 B9 humanised antibody variants were determined by testing crude, unpurified (‘SiPF’-expressed; Example 5.1; Figure 18) IgGs for binding to human, mouse and cynomolgus DR3 expressed in Expi293F cells (Example 6.3.2). Crude, unpurified IgGs were incubated at a single concentration with Expi293F cells transiently transfected with either human, mouse or cynomolgus DR3. Binding of IgG to DR3- expressing cells was detected using a goat anti-human IgG (H+L) cross-adsorbed secondary antibody labelled with Alexa Fluor 647 (Invitrogen; Cat# A21445). Ten microliters of crude, unpurified IgG were added to a 384-well assay plate (Greiner Bio- one; Cat# 781906). This was followed by the addition of 10 μl of antibody detection mix containing 8 nM goat anti-human IgG (H+L) cross-adsorbed secondary antibody labelled with Alexa Fluor 647 and 20 µl cell suspension at 75,000 cells/ml. To gauge non-specific binding of the detection antibody, wells containing a negative control unpurified IgG (isotype control) in place of the test IgG were defined for each plate. Clones binding non-specifically to cells were identified in a parallel assay with mock transfected Expi293F cells (i.e., plasmid DNA was omitted during transfections). All dilutions were performed in Hanks’ balanced salt solution (Sigma; Cat# H8264) containing 0.1% (v/v) bovine serum albumin (BSA) (Sigma; Cat# A9576) (‘assay buffer’). Assay plates were incubated at room temperature in the dark for 4 hours prior to reading on a Mirrorball fluorescence cytometer (SPT Labtech) using a 640 nm laser for excitation and measuring emission in the FL-4 channel (650 nm and 690 nm). Data was analysed as Count × median mean intensity (× FLU) (Figure 20). 6.4 Flow cytometry DR3 cell binding assay Humanised B9 variant IgGs VHg6/VLg13, VHg6/VLg11 and VHg1/VL11 were evaluated in separate experiments for binding to cell expressed DR3 by flow cytometry (Figure 23). The modified Jurkat reporter cell lines designated DR^no (no detectable DR3 expressed) and DR3^hi (high level DR3 expression) (Example 6.2) were incubated with primary test antibodies that were in turn stained using an Alex Fluor 647-labelled secondary antibody (Invitrogen; #A- 21445). Fluorescence was detected using an ACEA NovoCyte flow cytometer. Specific 80 241018 CMAL003WO1 DCA binding to cell expressed DR3 is evident where a high fluorescence signal was seen in the DR3^hi, but not the DR3^no cells. 6.5 Bio-layer interferometry (Octet) affinity determinations The binding interaction between immobilised candidate IgG antibodies and purified monomeric DR3-8i antigen protein (Example 2.1) in solution was analysed using kinetic Bio- layer interferometry (BLI), performed on an 8-channel OctetRED96 system (Pall Fortébio). All samples were prepared in assay buffer (1× PBS supplemented with 0.01% (v/v) BSA and 0.002% (v/v) Tween 20) by diluting IgGs and antigen from concentrated stock solutions. Prior to each experiment, protein A-coated biosensors (FortéBio; Cat# 18-5010) were hydrated in assay buffer solution for 10 minutes. All BLI solutions were transferred onto a 96-well microplate (Greiner Bio-one; Cat# 655209) at volumes of 200 µl/well. Data acquisition was conducted at 25°C and a shake speed of 1000 rpm. The following assay steps were performed: After (1) an equilibration step in assay buffer solution, the IgGs (each at 0.002 mg/ml in assay buffer) were (2) captured on the protein A sensors for 5 minutes. The sensors were then (3) washed in assay buffer to establish a stable baseline signal and subsequently (4) dipped for 10 minutes into solutions containing DR3-8i protein at different concentrations to measure association (0, 3.75, 7.5, 15, 30, 60, 120, and 240 nM). The sensors were then (5) dipped for 15 minutes into protein-free assay buffer to measure dissociation. The signal from a reference well containing assay buffer only (without antigen) was subtracted from the binding data. The Octet data analysis software (Version 10.0.3.1; Pall FortéBio) was used to calculate dissociation constants (KD) based on the corresponding association and dissociation rate constants (kon and koff) obtained by global fitting of the data, assuming a 1:1 binding mode. The obtained binding affinities are summarised in Table 11. 6.6 Epitope competition assay Humanised B9 variant IgGs VHg6/VLg13, VHg6/VLg11 and VHg1/VL11 were tested in a B9 epitope competition assay (ECA) against non-humanised parental B9 IgG1. The ability of the humanised clones to compete with parental B9 IgG for binding to DR3-Fc (recombinant human DR3 extracellular domain fused to human IgG1 Fc and a C-terminal His6-tag; RnD Systems; Cat# 943-D3) was determined using a homogeneous time-resolved fluorescence (HTRF; Cisbio International) assay. FRET was measured between anti-His cryptate (Cisbio; Cat# 61HISKLA) associated with the His6-tag on DR3-Fc and bound parental B9 IgG labelled with DyLight⁶⁵⁰ (‘probe’; DyLight Microscale Antibody Labelling Kit, Thermo Scientific, Cat# 84536), in the presence of non-labelled humanised B9 variant antibody at different concentrations (Figure 24A). Occupation of the B9 IgG epitope by a humanised B9 variant IgG results in a reduction in FRET signal (Figure 24B). 81 241018 CMAL003WO1 DCA A ‘Maximum’ binding signal was determined by analysing the binding of fluorescent B9 IgG probe to recombinant human DR3-Fc in the absence of competitor IgG. The ‘Sample’ signals were obtained from samples containing B9 IgG probe and DR3-Fc in the presence of a test IgGs at different concentrations. Finally, a ‘Background’ signal was derived from the fluorescence generated in the absence of B9 IgG probe. The potency of each purified test IgG to displace the B9 IgG probe was determined by applying a dilution series of the IgG (typically 1.5 pM – 300 nM) to the epitope competition assay. All reagents were diluted from concentrated stock solutions with assay buffer composed 0.4 M potassium fluoride and 0.1% (v/v) BSA (Sigma; Cat# A9576) in phosphate buffered saline (ThermoFisher Scientific; Cat# 14190-094). Test IgGs were diluted in assay buffer and 2.5 µl of each dilution were transferred to the ‘Sample’ wells of a 384-well assay plate (Greiner BioOne; Cat# 784076) using an electronic multichannel pipette. The remaining reagents were added to the assay plate as follows: 2.5 µl of 5.33 nM anti-His cryptate (to all wells; final 1.33 nM), 2.5 µl of 30 nM recombinant human DR3-Fc (to all wells; final 7.5 nM), 2.5 µl of 1 nM fluorescent B9 IgG probe (to ‘Sample’ and ‘Maximum’ wells; final 0.25 nM), and 2.5 µl assay buffer (to background wells). Assay plates were sealed and incubated overnight at 4°C in the dark, prior to measuring time-resolved fluorescence at 620 nm and 665 nm emission wavelengths upon excitation at 337 nm on a fluorescence plate reader (Pherastar™, BMG Labtech). Data were analysed by calculating the HTRF ratio (Equation 1) and Delta F% values (Equation 2) for each sample. Equation 1: HTRF ratio = (665nm counts / 620nm counts) ×10000 Equation 2: Delta F%= ((Sample HRTF ratio−Background HTRF ratio) / Background HTRF ratio) ×100 Note: The background HTRF ratio was calculated from wells without fluorescent B9 probe. Delta F% values were plotted against test IgG concentration and IC50 values were calculated by non-linear regression analysis using GrahpPad Prism software (version 8.2.0; GraphPad) (Figure 25). 82 241018 CMAL003WO1 DCA Example 7. Developability 7.1 Clone triaging based on sequence liability and germline requirements B9 humanised variant IgGs were triaged in silico for the presence of high or medium risk sequence liabilities. Clones with high or medium risk sequence liabilities, especially within CDRs, were deprioritised. High risk sequence liabilities include unpaired canonical cysteines, non-canonical cysteines, N-linked glycosylation sites, and asparagine deamidation motifs. Clones with high numbers of non-germline-encoded changes located within their frameworks, excluding Vernier regions, were also deprioritised. Example 8. Small- and large-scale IgG expression 8.1 Small-scale (10 ml) IgG1 expression and purification Antibody clones (as human IgG1 isotype) were expressed using the ExpiCHO Expression System Kit (ThermoScientific; Cat# A29133) according to manufacturer’s instructions in 10 ml cell cultures. The corresponding heavy and light chain-encoding IgG expression vectors were co-transfected into ExpiCHO cells leading to transient human IgG1 expression and secretion into the medium. After 9 days, the cells were removed by centrifugation in a table-top centrifuge for 5 minutes at 300 × g, followed by centrifugation for 15 minutes at 2800 × g. The clarified cell culture medium (CCM) containing the antibodies was filtered using a 0.2-µm pore- size vacuum filter unit (Nalgene; Cat# 10421791). The IgG1s were purified by protein A affinity chromatography using MabSelect SuRe antibody purification resin (MabSelect SuRe™ columns, Cytiva; Cat# 17543801). For that, CCM was mixed with 500 µl affinity resin pre- equilibrated in 25 mM Tris-HCl pH 7.4, 50 mM NaCl and incubated rotating at room temperature. Afterwards, the resin was washed three times with buffer solution and bound IgGs were eluted from the resin using 0.1 M citrate pH 3.0, 100 mM NaCl. The purified IgGs were then buffer exchanged into PBS using NAP-10 desalting columns (Cytiva; Cat# 17-0854- 02). The concentration of the purified IgGs was determined by absorbance at 280 nm using an extinction coefficient defined by the amino acid sequence of the IgG being quantified. Purified IgGs were analysed for aggregation or degradation using SDS-PAGE techniques and stored at 4°C. 8.2 Large-scale (100 ml) IgG1 expression and purification The prioritised panel of 30 humanised variant B9 clones (Figure 19 and Table 1) was reduced to 10 clones, which were produced in 100 ml expression cultures as described in Example 8.1. The 10 clones chosen for large-scale expression were: VHg1/VLg11, VHg2/VLg2, VHg2/VLs10, VHg2/VLg11, VHg6/VLg2, VHg6/VLg4, VHg6/VLg11, VHg6/VLg13, VHg9/VLg11 and VHg10/VLg5. 83 241018 CMAL003WO1 DCA Antibody clones as human IgG1 were expressed using the ExpiCHO Expression System Kit (ThermoScientific; Cat# A29133) according to manufacturer’s instructions in 100 ml cell cultures. The corresponding heavy and light chain-encoding IgG expression vectors were co- transfected into ExpiCHO cells leading to transient human IgG1 expression and secretion into the medium. After 9 days, the cells were removed by centrifugation in a table-top centrifuge for 5 minutes at 300 × g followed by centrifugation for 15 minutes at 2800 × g. The clarified cell culture medium (CCM) containing the antibodies was filtered using a 0.2 µm pore-size vacuum filter unit (Nalgene; Cat# 10421791). Large-scale batches of human IgG1 antibodies were purified by protein A affinity chromatography using 1 ml HiTrap MabSelect SuRe columns (Cytiva; Cat# 29-0491-04) attached to an ÄKTAxpress liquid chromatography system (Cytiva). Cleared CCM was loaded onto affinity columns pre-equilibrated in 25 mM Tris-HCl pH 7.4, 50 mM NaCl. After a wash step with binding solution, bound IgGs were eluted using 0.1 M Citrate pH 3.0, 100 mM NaCl. The purified IgGs were desalted using PD-10 columns (Cytiva; Cat# 17-0851-01) equilibrated in PBS. The concentration of the purified IgGs was determined by absorbance at 280 nm using an extinction coefficient defined by the amino acid sequence of the IgG being quantified. Purified IgGs were analysed for aggregation or degradation using reducing and non-reducing SDS-PAGE. The purified IgGs were filtered using a 0.2 µM PES syringe filter (Starlab, Cat# E4780-1226) and stored at 4°C. References Kabat et al., 1991, J Immunol 147(5): 1709-19). Lefranc et al., 2005, Dev Comp Immunol 29(3): 185-203. Swindells et al., 2017, J Mol Biol, abYsis: Integrated Antibody Sequence and Structure— Management, Analysis, and Prediction. Feb 3; 429(3):356-364. doi: 10.1016/j.jmb.2016.08.019. Epub 2016 Aug 22. PMID: 27561707. Hu et al. (1996) Cancer Res 56(13): 3055-61. Altschul et al. (1990) J. MoI. Biol.215: 405-410). Pearson and Lipman (1988) PNAS USA 85: 2444-2448. Smith and Waterman (1981) J. MoI Biol.147: 195-197). psi-Blast algorithm Nucl. Acids Res. (1997) 253389-3402. 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PMID: 27265505. Taraban, V.Y., et al., Sustained TL1A expression modulates effector and regulatory T-cell responses and drives intestinal goblet cell hyperplasia. Mucosal Immunol, 2011.4(2): p.186- 96. Wang et al., 2001, Mol Cell Biol, DR3 regulates negative selection during thymocyte development. May; 21(10):3451-61. doi: 10.1128/MCB.21.10.3451-3461.2001. PMID: 11313471. Kessels et al., 2001, Nat Immunol, Immunotherapy through TCR gene transfer. Oct; 2(10):957-61. doi: 10.1038/ni1001-957. PMID: 11577349. Buchan et al., 2018, Immunity, Antibodies to Costimulatory Receptor 4-1BB Enhance Anti- tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function. Nov 20;49(5):958-970.e7. doi: 10.1016/j.immuni.2018.09.014. Epub 2018 Nov 13. PMID: 30446386. Buchan et al., 2018, Clin Cancer Res, PD-1 Blockade and CD27 Stimulation Activate Distinct Transcriptional Programs That Synergize for CD8(+) T-Cell-Driven Antitumor Immunity. May 15; 24(10):2383-2394. doi: 10.1158/1078-0432.CCR-17-3057. PMID: 29514845. Sancho, et al., 2008, J Clin Invest, Tumor therapy in mice via antigen targeting to a novel, DC- restricted C-type lectin. Jun; 118(6):2098-110. doi: 10.1172/JCI34584. PMID: 18497879. Yang et al., 2019, MAbs, Tetravalent biepitopic targeting enables intrinsic antibody agonism of tumor necrosis factor receptor superfamily members. Aug/Sep; 11(6):996-1011. doi: 10.1080/19420862.2019.1625662. Epub 2019 Jun 20. PMID: 31156033. Greenman et al., 1991, Mol Immunol, Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors. Nov; 28(11):1243-54. doi: 10.1016/0161-5890(91)90011-8. PMID: 1835758. Cobbold et al., 1984, Nature, Therapy with monoclonal antibodies by elimination of T-cell subsets in vivo. Dec; 312(5994):548-51. doi: 10.1038/312548a0. PMID: 6150440. 85 241018 CMAL003WO1 DCA Koo, et al., 1986, J Immunol. The NK-1.1(-) mouse: a model to study differentiation of murine NK cells. Dec 15; 137(12):3742-7. PMID: 3782794. Li et al., 2017, Cancer Res, TIMER: A Web Server for Comprehensive Analysis of Tumor- Infiltrating Immune Cells.77(21): p. e108-e110. Nov 1; 77(21):e108-e110. doi: 10.1158/0008- 5472.CAN-17-0307. PMID: 29092952. Wang, E.C., et al., DR3 regulates negative selection during thymocyte development. Mol Cell Biol, 2001.21(10): p.3451-61. Lloyd C, Lowe D, Edwards B, Welsh F, Dilks T, Hardman C, Vaughan T. (2009) Modelling the human immune response: performance of a 10¹¹ human antibody repertoire against a broad panel of therapeutically relevant antigens. Protein Eng. Des. Sel. Vol.22, 159–68. Osbourn J, Groves M, and Vaughan T. (2005) From rodent reagents to human therapeutics using antibody guided selections. Methods. Vol.36 (1), 61-68. Persic L, Roberts A, Wilton J, Cattaneo A, Bradbury A, Hoogenboom HR. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. Vol.187.9–18. PMID: 9073060. Thom G. and Groves M. (2012) Ribosome Display. In: Proetzel G., Ebersbach H. (eds) Antibody Methods and Protocols. Methods in Molecular Biology, Vol 901. Humana Press, Totowa, NJ. Paules CI, Lakdawala S, McAuliffe JM, Paskel M, Vogel L, Kallewaard NL, Zhu Q, Subbarao K. (2017) The hemagglutinin a stem antibody MEDI8852 prevents and controls disease and limits transmission of pandemic influenza viruses. J. Infectious Diseases. Vol.216, 356-265. (PMID: 28633457) Screening in Product Format, ‘SiPF’ (PMID: 28613102. Sequence Listing Information The sequence listing submitted herewith forms part of the specification as filed. SEQ ID NO: 1 amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 UniProt Q93038, (Ensembl gene ID, human: ENSG00000215788; NCBI gene ID, human: 8718): GDFHKKIGL. SEQ ID NO: 2 human DR3: UniProt Q93038, (Ensembl gene ID, human: ENSG00000215788; NCBI gene ID, human: 8718) human DR3: MEQRPRGCAAVAAALLLVLLGARAQGGTRSPRCDCAGDFHKKIGLFCCRGCPAGHYLKAP CTEPCGNSTCLVCPQDTFLAWENHHNSECARCQACDEQASQVALENCSAVADTRCGCKP GWFVECQVSQCVSSSPFYCQPCLDCGALHRHTRLLCSRRDTDCGTCLPGFYEHGDGCVS CPTSTLGSCPERCAAVCGWRQMFWVQVLLAGLVVPLLLGATLTYTYRHCWPHKPLVTADE 86 241018 CMAL003WO1 DCA AGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWS WDQLPSRALGPAAAPTLSPESP B9 clone CDRs SEQ ID NO: 3 HCDR1 SYAMS SEQ ID NO: 4 HCDR2 TISDGDSYSYFPDSVKD SEQ ID NO: 5 HCDR3 DRIYGSVQYYAMDY SEQ ID NO: 6 LCDR1 RASESVEFSGTSLMQ SEQ ID NO: 7 LCDR2 AASNVES SEQ ID NO: 8 LCDR3 QQSRKLPYT F10 Clone CDRs SEQ ID NO: 9 HCDR1 AYAMS SEQ ID NO: 10 HCDR2 TISDGDPYTYYPDNVKG SEQ ID NO: 11 HCDR3 ERNDYDQYYTMDY SEQ ID NO: 12 LCDR1 RASKSVSTSGYNYLH SEQ ID NO: 13 LCDR2 LASNLES SEQ ID NO: 14 LCDR3 QHSRELPWT SEQ ID NO: 15 B9 Variable Heavy chain sequence: EVQLVESGGGLVKPGGSLKLSCSASGFTFSSYAMSWVRQTPEKRLEWVATISDGDSYSYF PDSVKDRFIISRDNAKNNLFLQMSHLRSEDTAMYYCTRDRIYGSVQYYAMDYWGQGTSVT VSS. SEQ ID NO: 16 B9 Variable Kappa light chain sequence: DIVLTQSPASLAVSLGQRATISCRASESVEFSGTSLMQWYQQRPGLPPKLLIYAASNVESGV PARFSGSGSGTDFSLNIHPVEEDDIAMYFCQQSRKLPYTFGGGTKLEIN. SEQ ID NO: 17 F10 Variable Heavy chain sequence: EVQLVESGGDLVKPGGSLKLSCAASGFSFSAYAMSWVRQTPEKILEWVATISDGDPYTYYP DNVKGRFTISRDNARNNLYLQMSHLRSEDTAIYYCTRERNDYDQYYTMDYWGQGTSVTVS S. SEQ ID NO: 18 F10 Variable Kappa light chain sequence: DIVLTQSPASLVVSLGQRATISCRASKSVSTSGYNYLHWYQQTPGQPPKLLIYLASNLESGV PARFSGSGSGTDFTLNIHPVEEEDVATYYCQHSRELPWTFGRGTKLEIKRT. 87 241018 CMAL003WO1 DCA SEQ ID NO: 19 VHg1/VLg11 (H1L11) heavy chain sequence: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSTISDGDSYSY FPDSVKDRFTISRDNSKNTLYLQMNSLRAEDTSVYYCARDRIYGSVQYYAMDYWGQGTLV TVSS SEQ ID NO: 20 VHg1/VLg11 (H1L11) kappa light chain sequence: DIVMTQSPDSLAVSLGERATINCRASESVEFSGTSLMQWYQQKPGQPPKLLIYAASNVESG VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSRKLPYTFGQGTKLEIK DR3 construct sequences inserted into pcDNA3.1 vector for HEK293 transfections (Figure 2): SEQ ID NO: 81 Full-length Rtx-hDR3 from 5’ HindIII to 3’ EcoRI: AAGCTTATGGAACAGCGGCCTAGAGGATGTGCCGCTGTTGCTGCTGCTCTGCTGCTGG TTCTGCTGGGAGCTAGAGCCGCCTGTCCTTACAGCAATCCTAGCCTGTGTGCCCAAGG CGGCACCAGATCTCCCAGATGTGATTGTGCCGGCGACTTCCACAAGAAGATCGGCCTG TTCTGCTGCAGAGGCTGTCCTGCCGGACACTATCTGAAGGCCCCTTGCACAGAGCCCT GCGGCAATAGCACATGTCTCGTGTGCCCTCAAGACACCTTCCTGGCCTGGGAGAACCA CCACAATAGCGAGTGTGCCAGATGCCAGGCCTGTGATGAACAGGCTTCTCAGGTGGCC CTGGAAAACTGTAGCGCCGTGGCCGATACAAGATGCGGCTGCAAACCTGGATGGTTCG TGGAATGCCAGGTGTCCCAGTGCGTGTCAAGCAGCCCCTTCTACTGCCAGCCTTGCCT GGATTGTGGCGCCCTGCACAGACACACCAGACTGCTGTGTAGCAGACGGGACACCGA TTGCGGAACATGCCTGCCTGGCTTTTACGAGCATGGCGACGGCTGTGTGTCTTGCCCT ACAAGCACACTGGGCAGCTGTCCCGAAAGATGCGCTGCTGTTTGTGGCTGGCGGCAG ATGTTCTGGGTGCAAGTGCTTCTGGCTGGCCTGGTGGTTCCTCTGCTTCTGGGAGCCA CACTGACCTACACCTACAGACACTGCTGGCCCCACAAGCCTCTGGTCACAGCTGATGA GGCCGGCATGGAAGCTCTGACACCTCCACCAGCCACACACCTGTCTCCACTGGATTCT GCCCACACTCTGCTGGCTCCTCCAGACAGCAGCGAGAAGATCTGTACCGTGCAGCTCG TGGGCAACAGCTGGACACCAGGCTACCCTGAGACACAAGAGGCCCTGTGTCCTCAAGT GACCTGGTCCTGGGATCAGCTGCCTTCTAGAGCCCTTGGACCTGCCGCTGCTCCTACA CTGTCTCCCGAATCTCCATGAGAATTC. SEQ ID NO: 82 Rtx-hCRD1-2 from 5’ from HindIII to 3’ EcoRI: AAGCTTATGGAACAGCGGCCTAGAGGATGTGCCGCTGTTGCTGCTGCTCTGCTGCTGG TTCTGCTGGGAGCTAGAGCCGCCTGTCCTTACAGCAATCCTAGCCTGTGTGCCCAAGG CGGCACCAGATCTCCCAGATGTGATTGTGCCGGCGACTTCCACAAGAAGATCGGCCTG TTCTGCTGCAGAGGCTGTCCTGCCGGACACTATCTGAAGGCCCCTTGCACAGAGCCCT 88 241018 CMAL003WO1 DCA GCGGCAATAGCACATGTCTCGTGTGCCCTCAAGACACCTTCCTGGCCTGGGAGAACCA CCACAATAGCGAGTGTGCCAGATGCCAGGCCTGTGATGAACAGGCTTCTCAGGTGGCC CTGGAAAACTGTAGCGCCGTGGCCGATACAAGATGCGGCTGGGTTCAAGTGCTGCTG GCTGGACTGGTTGTGCCTCTGCTTCTGGGAGCCACACTGACCTACACCTACAGACACT GCTGGCCCCACAAGCCTCTGGTCACAGCTGATGAGGCCGGCATGGAAGCTCTGACAC CTCCACCAGCCACACACCTGTCTCCACTGGATTCTGCCCACACTCTGCTGGCTCCTCC AGACAGCAGCGAGAAGATCTGTACCGTGCAGCTCGTGGGCAACAGCTGGACACCTGG CTACCCTGAGACACAAGAGGCCCTGTGTCCTCAAGTGACCTGGTCCTGGGATCAGCTG CCTTCTAGAGCACTGGGACCTGCCGCTGCTCCTACACTGTCTCCAGAGTCTCCATGAG AATTC. SEQ ID NO: 83 Rtx-hCRD1-mCRD2 from 5’ HindIII to 3’ EcoRI: AAGCTTGCCACCATGGAACAGCGGCCTAGAGGATGTGCCGCTGTTGCTGCTGCTCTGC TGCTGGTTCTGCTGGGAGCTAGAGCCGCCTGTCCTTACAGCAATCCTAGCCTGTGTGC CCAAGGCGGCACCAGATCTCCCAGATGTGATTGTGCCGGCGACTTCCACAAGAAGATC GGCCTGTTCTGCTGCAGAGGCTGTCCTGCCGGACACTATCTGAAGGCCCCTTGCACAG AGCCCTGCGGCAATAGCACATGTCTCCCCTGCCCCTCCGACACCTTCCTGACCCGCGA CAACCACTTCAAGACCGACTGCACCCGCTGCCAGGTGTGCGACGAGGAGGCCCTGCA GGTGACCCTGGAGAACTGCTCCGCCAAGTCCGACACCCACTGCGGCTGGGTGCAAGT GCTTCTGGCTGGCCTGGTGGTTCCTCTGCTTCTGGGAGCCACACTGACCTACACCTAC AGACACTGCTGGCCCCACAAGCCTCTGGTCACAGCTGATGAGGCCGGCATGGAAGCT CTGACACCTCCACCAGCCACACACCTGTCTCCACTGGATTCTGCCCACACTCTGCTGG CTCCTCCAGACAGCAGCGAGAAGATCTGTACCGTGCAGCTCGTGGGCAACAGCTGGA CACCAGGCTACCCTGAGACACAAGAGGCCCTGTGTCCTCAAGTGACCTGGTCCTGGGA TCAGCTGCCTTCTAGAGCCCTTGGACCTGCCGCTGCTCCTACACTGTCTCCCGAATCTC CATGAGAATTC SEQ ID NO: 84 Rtx-mCRD1-hCRD2 from 5’ HindIII to 3’ EcoRI: AAGCTTGCCACCATGGAACAGCGGCCTAGAGGATGTGCCGCTGTTGCTGCTGCTCTGC TGCTGGTTCTGCTGGGAGCTAGAGCCGCCTGTCCTTACAGCAATCCTAGCCTGTGTGC CCAGGGCGGCATGTCCGGCCGCTGCGACTGCGCCTCCGAGTCCCAGAAGCGCTACG GCCCCTTCTGCTGCCGCGGCTGCCCCAAGGGCCACTACATGAAGGCCCCCTGCGCCG AGCCCTGCGGCAACTCCACCTGCCTGGTGTGCCCTCAAGACACCTTCCTGGCCTGGGA GAACCACCACAATAGCGAGTGTGCCAGATGCCAGGCCTGTGATGAACAGGCTTCTCAG GTGGCCCTGGAAAACTGTAGCGCCGTGGCCGATACAAGATGCGGCTGGGTGCAAGTG CTTCTGGCTGGCCTGGTGGTTCCTCTGCTTCTGGGAGCCACACTGACCTACACCTACA GACACTGCTGGCCCCACAAGCCTCTGGTCACAGCTGATGAGGCCGGCATGGAAGCTC 89 241018 CMAL003WO1 DCA TGACACCTCCACCAGCCACACACCTGTCTCCACTGGATTCTGCCCACACTCTGCTGGC TCCTCCAGACAGCAGCGAGAAGATCTGTACCGTGCAGCTCGTGGGCAACAGCTGGACA CCAGGCTACCCTGAGACACAAGAGGCCCTGTGTCCTCAAGTGACCTGGTCCTGGGATC AGCTGCCTTCTAGAGCCCTTGGACCTGCCGCTGCTCCTACACTGTCTCCCGAATCTCC ATGAGAATTC. SEQ ID NO: 85 Rtx-h/mCRD1-hCRD2 from 5’ HindIII to 3’ EcoRI: AAGCTTGCCACCATGGAACAGCGGCCTAGAGGATGTGCCGCTGTTGCTGCTGCTCTGC TGCTGGTTCTGCTGGGAGCTAGAGCCGCCTGTCCTTACAGCAATCCTAGCCTGTGTGC CCAAGGCGGCACCAGATCTCCCCGCTGCGACTGCGCCTCCGAGTCCCAGAAGCGCTA CGGCCCCTTCTGCTGCAGAGGCTGTCCTGCCGGACACTATCTGAAGGCCCCTTGCACA GAGCCCTGCGGCAATAGCACATGTCTCGTGTGCCCTCAAGACACCTTCCTGGCCTGGG AGAACCACCACAATAGCGAGTGTGCCAGATGCCAGGCCTGTGATGAACAGGCTTCTCA GGTGGCCCTGGAAAACTGTAGCGCCGTGGCCGATACAAGATGCGGCTGGGTGCAAGT GCTTCTGGCTGGCCTGGTGGTTCCTCTGCTTCTGGGAGCCACACTGACCTACACCTAC AGACACTGCTGGCCCCACAAGCCTCTGGTCACAGCTGATGAGGCCGGCATGGAAGCT CTGACACCTCCACCAGCCACACACCTGTCTCCACTGGATTCTGCCCACACTCTGCTGG CTCCTCCAGACAGCAGCGAGAAGATCTGTACCGTGCAGCTCGTGGGCAACAGCTGGA CACCAGGCTACCCTGAGACACAAGAGGCCCTGTGTCCTCAAGTGACCTGGTCCTGGGA TCAGCTGCCTTCTAGAGCCCTTGGACCTGCCGCTGCTCCTACACTGTCTCCCGAATCTC CATGAGAATTC. 90 241018 CMAL003WO1 DCA

Claims

Claims 1. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, capable of binding specifically to an epitope formed by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2).

2. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof of claim 1, capable of binding specifically to an epitope formed by residues of the amino acid sequence 37-45 of human DR3 (SEQ ID NO: 1) and thereby agonising a DR3 pathway, wherein optionally agonism may be assessed in a T cell proliferation assay.

3. An antigen-binding protein of claim 1 or claim 2, such as an antibody or antigen-binding fragment thereof, wherein the antigen binding protein is a murine, chimeric, humanised, or human antibody or antigen-binding fragment thereof.

4. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, of any preceding claim, comprising an antigen-binding site comprising framework sequences (FW1 to FW4) and CDRs (HCDR1, HCRD2, HCDR3, LCDR1, LCDR2 and LCDR3, respectively) selected from: (a) SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 (Clone B9); or (b) SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14 (Clone F10), wherein the sequences are defined according to Kabat nomenclature.

5. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof, of any preceding claim, wherein the antigen-binding site comprises the VH and / or VL domain sequence of, or a VH and / or VL domain sequence with at least 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identity to, a clone selected from: (a) humanised Clone B9 of VHg1/VLg11 (H1L11) of VH SEQ ID NO: 19 and VL SEQ ID NO: 20, respectively; (b) murine Clone B9 of VH SEQ ID NO: 15 and VL SEQ ID NO: 16, respectively; (c) murine Clone F10 of VH SEQ ID NO: 17 and VL SEQ ID NO: 18, respectively; and (d) a humanised version of Clone B9 of Figure 19 and Table 1. wherein the sequences are defined according to Kabat nomenclature. 91 241018 CMAL003WO1 DCA

6. An antigen-binding protein, such as a humanised or murine antibody or antigen-binding fragment thereof, of any preceding claim, wherein the antibody comprises the VH and / or VL domain of: (a) humanised Clone B9 VHg1/VLg11 (H1L11) of VH SEQ ID NO: 19 and VL SEQ ID NO: 20, respectively; (b) murine Clone B9 of VH SEQ ID NO: 15 and VL SEQ ID NO: 16, respectively; (c) murine Clone F10 of VH SEQ ID NO: 17 and VL SEQ ID NO: 18, respectively; or (d) a humanised version of Clone B9 shown in Figure 19 and Table 1. wherein the sequences are defined according to Kabat nomenclature.

7. An antigen-binding protein, such as an antibody or antigen-binding fragment thereof capable of competing with an antibody according to any one of claims 1 to 6 for binding to an epitope formed by residues of the amino acid sequence 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) when assessed in a competition assay.

8. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims comprising a human Fc selected from hIgG1, hIgG1-SELF, hIgG2, hIgG4, IgG1V11, IgG1 N297A, IgG1 N297Q, IgG1 N297A LALA-PG, IgG1 N297Q LALA-PG and IgG1 LALA-PG.

9. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, characterised in that the antigen binding protein is monovalent, bivalent, trivalent, or tetravalent for binding human DR3 (SEQ ID NO: 2).

10. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a multivalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2).

11. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2). 92 241018 CMAL003WO1 DCA

12. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc capable of binding to FcγR (for FcR mediated cross-linking), e.g., (e.g., B9-hIgG1 (B9-H1)).

13. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a bivalent, monospecific antibody or antigen-binding fragment thereof comprising two antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc that is silent (not capable of binding to FcγR), such as IgG1 N297A.

14. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a trivalent or tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2).

15. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) or IgG1 Fc is absent.

16. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc in which Fc function is silent (does not bind FcγR) and / or Fc is absent.

17. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human 93 241018 CMAL003WO1 DCA DR3 (SEQ ID NO: 2) and an IgG1 N297A Fc in which Fc function is silent (does not bind FcγR), e.g., “Fc-silent” TET-B9-N297A.

18. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a tetravalent, monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG1 Fc (capable of binding FcγR); e.g., tetravalent (TET) B9- H1 (TET-B9-H1).

19. An antigen binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a multivalent (e.g., bivalent, trivalent or tetravalent), monospecific antibody or antigen-binding fragment thereof comprising antigen-binding sites having affinity toward the DR3 epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2) and an IgG Fc.

20. An antigen-binding protein, such as an antibody or antigen-binding fragment of any one of the preceding claims, wherein the antigen-binding protein comprises a Fab:IgG tetravalent construct.

21. A multivalent antigen-binding protein of any one of claims 1 to 20 for use to agonise DR3 without cross-linking by Fc receptors.

22. A multivalent antigen-binding protein of any one of claims 1 to 21 for use to agonise DR3 wherein the multivalent antigen-binding protein lacks effector function.

23. A hybridoma comprising an antigen-binding protein, such as an antibody or antigen- binding fragment thereof, according to any one of the preceding claims.

24. A composition comprising an isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof, according to any one of claims 1 to 22, and an excipient.

25. An isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof, according to any one of claims 1 to 22.

26. An isolated recombinant DNA sequence of claim 25 which is a vector. 94 241018 CMAL003WO1 DCA

27. An isolated recombinant DNA sequence of claim 26 which is an expression vector.

28. An isolated recombinant DNA sequence of claim 26 or 27 encoding an antibody or antigen- binding fragment thereof, according to any one of claims 1 to 22 under control of a promoter.

29. A host cell comprising a DNA or RNA sequence according to any one of claims 25 to 28.

30. A host cell of claim 29 capable of expressing an isolated antibody or antigen-binding fragment thereof, of any one of claims 1 to 22.

31. A method of making an isolated antibody or antigen-binding fragment thereof, of any one of claims 1 to 22 comprising culturing a host cell according to claim 29 or 30 in conditions suitable for expression of the isolated antibody or antigen-binding fragment thereof.

32. A composition comprising: (a) an isolated antibody or antigen-binding fragment thereof, according to any one of claims 1 to 22 and an excipient, preferably a pharmaceutically-acceptable excipient, or (b) an isolated recombinant DNA or RNA sequence comprising a sequence encoding an isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 22 and an excipient, preferably a pharmaceutically-acceptable excipient.

33. An antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or composition of claim 24 or 32 for use as a medicament or for use in diagnosis.

34. An antibody or antigen-binding fragment thereof any one of claims 1 to 22, or a composition of claim 24 or 32, for use in the prophylactic or therapeutic treatment of a cancer, for example wherein the cancer is selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin’s lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma, head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied.

35. An antibody or antigen-binding fragment thereof any one of claims 1 to 22, or a composition of claim 24 or 32, for use in the prophylactic or therapeutic treatment of cancer in combination with one or more 2^nd therapy optionally selected from an anti-PD-1 mAb, vaccine, 95 241018 CMAL003WO1 DCA chemotherapy, kinase inhibitor, tumour targeting mAb (e.g., anti-CD20, anti-HER2, anti- VEGFR), antibody-drug conjugate, bispecific T cell engager, checkpoint inhibitors targeting immune receptors (e.g., PD-1, PD-L1, CTLA-4, LAG3), mAb targeting other co-stimulatory receptors (e.g., CD27, GITR, OX40, 4-1BB, ICOS, CD28), STING agonists and agents targeting myeloid suppressor cells.

36. An antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or a composition of claim 24 or 32, for use to stimulate the proliferation and differentiation of T cells into effector, cytotoxic and memory T cells.

37. An antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or a composition of claim 32, for use to identify human DR3 protein (SEQ ID NO: 2) comprising an epitope formed by residues 37-45 (SEQ ID NO: 1) of human DR3 (SEQ ID NO: 2),

38. An antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or a composition of claim 32, for use in a diagnostic test for a cancer, for example wherein the cancer is selected from haematological and solid cancers, including breast cancer, bladder cancer, cervical cancer, colon cancer, head and neck cancer, Hodgkin’s lymphoma, liver cancer, lung cancer, renal cell cancer, skin cancer (e.g., melanoma, squamous cell carcinoma head and neck squamous cell carcinoma (HNSC) and skin cutaneous metastasis (SKCM)), stomach cancer, rectal cancer and any solid tumour that is not able to repair errors in its DNA that occur when the DNA is copied.

39. A diagnostic kit comprising an antibody or antigen-binding fragment thereof of any one of claims 1 to 22, or a composition of claim 32, and a reagent capable of detecting an immunological (antigen-antibody) complex which contains said antibody or antigen-binding fragment thereof, wherein optionally said antibody or antigen-binding fragment is immobilized on a solid support (e.g., microplate well), and / or wherein optionally said immunological complex which contains said antibody or antigen-binding fragment is detectable by ELISA or an alternative immunoassay method or by lateral flow.

40. A diagnostic kit according to claim 39, further comprising one or more control standards and / or specimen diluent and / or washing buffer. 96 241018 CMAL003WO1 DCA