Therapeutic antibodies

Abstract

The present invention relates to bispecific binding molecules comprising a first antigen-binding region that binds human interleukin-1 receptor accessory protein (IL1RAP) and a second antigen binding region that binds human B7H4. The invention also relates to methods of treatment and diagnosis using said bispecific binding molecules.

Description

[0001] Therapeutic Antibodies

[0002] Background

[0003] Ovarian cancer (OC) is the most common cause of mortality for women with gynaecological cancers. The majority of instances are discovered when the disease has already progressed, which leads to poor outcomes whilst the limited predictive value of the current screening tests adds to this anguish. The most common early detection methods, including a detailed gynaecological examination, transvaginal ultrasound, and laboratory markers have not significantly reduced the morbidity or death of this malignancy over the past decade. Despite screening programmes, most women are asymptomatic and diagnosed only late in the cancer's progression, with 5-year survival rates of only 18-45% in stages III— IV. A high rate of recurrence or relapse after the initial treatment has also been noted. The majority of these relapsed cases have a lower chance of recovery and are known to have a higher rate of treatment failures in future therapy. The later OC is being diagnosed and treatment started, the higher the rate of relapse; analysis of large data pools suggest that patients diagnosed at Stage 3 have a 70-90% chance of recurrence whereas stage 4 patients have a 90-95% relapse rate. In summary, unfortunately few therapies are effective and most women ultimately relapse and die of the disease.

[0004] Epithelial ovarian cancer (EOC) is a heterogenous disease and can be classed into five major histologically distinct subtypes: high-grade serous (HGSOC), low-grade serous (LGSOC), endometrioid (ENOC), clear cell (CCOC) and mucinous (MOC). Amongst those, HGSOC is the most prevalent subtype representing 70-80% of all OC cases.

[0005] B7-H4 also known as VTCN1 (V-Set Domain Containing T Cell Activation Inhibitor 1) is a transmembrane protein that negatively regulates T cell immune responses and promotes immune escape by inhibiting the proliferation, cytokine secretion, and cell cycle of T cells. It has been shown that B7H4 is abnormally expressed in tumours, inflammation and autoimmune diseases. Here, B7-H4 is up-regulated on the surface of cancer cells and immunosuppressive tumour-associated macrophages (TAMs) and therefore exhibits a significant immune- suppressive role in the etiology of a number of solid cancers. Moreover, B7-H4 plays an extremely important role in tumourigenesis and tumour development including cell proliferation, invasion, metastasis and anti-apoptosis activity. B7-H4 is now considered a validated target in High-Grade Serous Ovarian Carcinoma (Gitto et al Cancer Res (2023) 83 (7_Supplement): 1133).

[0006] B7-H4 expression levels inversely correlate with patient survival in ovarian cancer. As B7-H4 is overexpressed in breast and ovarian cancers, investigators examined its expression in high- grade serous ovarian carcinoma (HGSOC) tumours at diagnosis and following resistance to platinum or PARP inhibitor treatment. Here, B7-H4 was over-expressed in 92% of HGSOC tumours at diagnosis (n = 12). Its expression persisted in recurrent matched samples after platinum treatment and was expressed at similar levels at multiple metastatic sites after acquired multidrug resistance.

[0007] In healthy individuals, B7-H4 mRNA is detected at low levels in many tissues (Sica GL, et al. B7-H4, a molecule of the B7 family, negatively regulates T cell immunity. Immunity. 2003;18:849-61 , Choi IH, et al. Genomic organization and expression analysis of B7-H4, an immune inhibitory molecule of the B7 family. J Immunol. 2003;171 :4650-4) however, B7-H4 protein expression seems to be limited due to tight translational control in peripheral tissues in humans. This regulation of restricted and inducible B7-H4 expression, notably, does not take place in cancer. Instead, B7-H4 is overexpressed in multiple stages of cancer and various types including ovarian, uterine, and endometrial cancers with prognostic value. mAbs against human B7-H4 showed no staining of normal human tissue by IHC, even though they detected mRNA in multiple tissues. However, they did show staining in 85% (22 of 26) of freshly isolated ovarian tumours and 31 % (5 of 16) of lung cancer tissue. Microscopically, B7-H4 was found either in cytoplasm or plasma membrane. B7-H4 can also exist in a soluble form in serum of diseased patients. Levels of soluble, serum B7-H4 correlate with tumour stage, poor prognosis, and pathological types. As a Type 1 TM anchor protein, it has been shown that B7H4 does internalise rapidly into the lysosome as has been determined by FRET probes. This supports its use as an ADC target for specific toxin delivery.

[0008] Current clinical development candidates are all either monospecific for B7H4 or T-cell engagers. Based on its biological role and tissue / cell expression pattern, this bares some risks: Systemic depletion of macrophages (TAMs) can promote tumour progression rather than regression due to their role in recruiting and activating adaptive immune cells to kill tumour. Furthermore, excessive macrophage depletion can also lead to increased chances of infection and / or autoimmunity. Both pose significant treatment-related risks that will have to be monitored in future larger clinical trials.

[0009] IL-1 RAP is involved in the pro-inflammatory response through NF-KB and AP-1 pathways mediated by interleukins 1 , 33, and 36 (IL-1 , IL-33, IL-36) signalling. Inflammation is now recognized as a hallmark of carcinogenesis, suggesting that IL-1 RAP could play a role in cancer development and progression. As a consequence, IL1 RAP was widely explored within the tumour microenvironment and at the surface of tumour cells during the last decade (Frenay et al., Int. J. Mol. Sci. 2022, 23(23), 14918). IL-1 are pleiotropic cytokines that induce synthesis of acute phase and proinflammatory proteins during infection, tissue damage, or stress. Cytokines are known to exert a critical function in malignancies, influencing the tumour microenvironment and promoting both cancer initiation and progression. Consequently, IL1 RAP aberrant expression and signaling has been considered to play a central role in the pathogenesis of chronic inflammatory diseases. Today, an overexpression of IL-1 RAP has been described in a number of haematological and solid types of cancer including ovarian and non-small cell lung cancer (NSCLC) thus confirming its potential involvement in carcinogenesis.

[0010] IL1 RAP is a single pass, type I transmembrane glycoprotein which internalises rapidly. Like other family members, IL1 RAP contains 3 immunoglobulin (Ig)-like domains in the extracellular region and an intracellular TIR (Toll-like receptor / IL-1 receptor) signaling domain.

[0011] A number of soluble forms exists dependent on the physiological state of the cell. sIL-I RAP p has been described as a soluble isoform and it was suggested that under stress conditions, the splicing machinery was shifted to produce sIL-I RAP instead of membrane-bound mlL-1 RAP. To date, it is unclear whether this could constitute a mechanism of evasion and resistance brought about by certain classes of anti-cancer therapies. As IL1 RAP forms a vital receptor complex with IL1 R for interleukin-1 alpha (ILI)-dependent protective immune responses, its targeting on healthy cells I tissues could interfere with its normal physiological function. IL1 signalling stimulates the activity of genes involved in inflammation and immunity. This cytokine signalling plays a critical role in protecting the body from foreign invaders such as bacteria and viruses and a mono-specific therapy could result in an imbalance in this protective system.

[0012] Therefore, there is a need for treatments of cancer, in particular ovarian cancer. The invention is aimed at addressing this need.

[0013] Summary of the Invention

[0014] The inventors have evaluated co-expression of antigens on tumour associated cells and healthy cells. The inventors have surprisingly found that tumour associated cells express both IL1 RAP and B7H4 antigens on their cell surface whilst healthy cells do not express both antigens.

[0015] Advantageously, the present inventors have identified that tumour cells presenting both IL1 RAP and B7H4 can be selectively targeted with a binding agent that includes a binding portion that binds IL1 RAP and a binding portion that binds B7H4 and thus bispecifically targets cells that express both antigens. This makes it possible to selectively target tumour associated cells. This leads to reduction of unwanted side effects, e.g. cytotoxicity.

[0016] In one aspect, the invention therefore relates to a binding agent comprising a first antigenbinding region that binds human IL1 RAP and a second antigen binding region that binds human B7H4, wherein the first antigen binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 1 15 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 119 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 112 or a sequence comprising 1 , 2, or 3 amino acid differences.

[0017] In one embodiment, the antigen binding region comprises an antibody or antigen binding fragment thereof.

[0018] In one embodiment, the antigen binding fragment is selected from a Fab, scFv, F(ab')2, single domain antibody or single chain antibody.

[0019] In one embodiment, the antigen-binding region that binds IL1 RAP is a Fab and the antigen binding region that binds B7H4 is a Fab.

[0020] In one embodiment, the binding agent comprises an Fc region.

[0021] In one embodiment, the binding agent is capable of selectively targeting cells expressing IL1 RAP and B7H4, compared to cells expressing IL1 RAP- / B7H4+ or cells expressing IL1 RAP+ / B7H4-.

[0022] In one embodiment, the antigen-binding region that binds IL1 RAP and the antigen binding region that binds B7H4 are linked by a linking portion.

[0023] In one embodiment, the binding agent comprises a payload.

[0024] In one embodiment, the antibody or antigen binding fragment is conjugated to a payload.

[0025] In one embodiment, the payload is a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

[0026] In one embodiment, the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

[0027] In one embodiment, the cell killing agent comprises a cytotoxic moiety.

[0028] In one embodiment, the cytotoxic moiety is selected from a radioisotope, peptide toxin or chemical toxin. In one embodiment, the cytotoxic moiety is selected from an auristatin, maytansinoid, tubulysin, RNA polymerase II inhibitor, transcription inhibitor, calicheamicin, duocarmycin, pyrrolobenzodiazepine, camptothecin analogue, topoisomerase inhibitor or doxorubicin.

[0029] In one embodiment, the binding agent has an IC50 of 0.1 nM to 5 nM.

[0030] In one embodiment, the first and / or second antigen binding region is linked to the payload with a linker.

[0031] In one embodiment, the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a pH sensitive linker, a redox sensitive linker.

[0032] In one embodiment, the binding agent is capable of being internalised.

[0033] In one embodiment, the binding agent comprises an additional moiety selected from a half-life extending moiety and / or a label.

[0034] In one embodiment, said binding agent comprises a T cell and / or a NK cell.

[0035] In another aspect, the invention relates to a nucleic acid encoding the binding agent as described herein.

[0036] In another aspect, the invention relates to a nucleic encoding a chimeric antigen receptor (CAR) comprising a) an extracellular antigen-binding domain comprising first antigen-binding region that binds human IL1 RAP as defined herein and a second antigen binding region that binds human B7H4 as defined herein; b) a co- stimulatory signaling domain and c) a cytoplasmic signaling domain.

[0037] In another aspect, the invention relates to a vector comprising the nucleic acid as described herein.

[0038] In another aspect, the invention relates to a host cell comprising the nucleic acid or the vector as described herein.

[0039] In one embodiment, the host cell is a bacterial cell, viral cell, plant cell or mammalian cell.

[0040] In another aspect, the invention relates to a CAR-T or NK cell comprising the binding agent as described herein.

[0041] In another aspect, the invention relates to an antibody drug conjugate (ADC) comprising the binding agent as described herein.

[0042] In another aspect, the invention relates to a pharmaceutical composition comprising the binding agent, cell or ADC as described herein and a pharmaceutical excipient.

[0043] In another aspect, the invention relates to the binding agent or cell as described herein for use in treating a disease.

[0044] In another aspect, the invention relates to the binding agent, cell, ADC or pharmaceutical composition as described herein for use in a method of treating a disease associated with cells expressing IL1 RAP and B7H4.

[0045] In another aspect, the invention relates to the binding agent, cell, ADC or pharmaceutical composition as described herein for use in a method for reducing or preventing progression of a tumour or treating cancer in a subject. In another aspect, the invention relates to a method of treating a disease associated with cells expressing IL1 RAP and B7H4 in a subject comprising administering a binding agent, cell, ADC or pharmaceutical composition as described herein to said subject.

[0046] In another aspect, the invention relates to a method of reducing or preventing progression of a tumour or treating cancer in a subject comprising administering a binding agent, cell, ADC or pharmaceutical composition as described herein to said subject.

[0047] In one embodiment, said cancer is a solid tumour.

[0048] In one embodiment, the solid tumour is selected from tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain / CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head / neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, bile duct cancer and sarcomas.

[0049] In one embodiment, the cancer is selected from ovarian cancer, breast cancer, liver cancer, bile duct cancer, pancreatic cancer and lung cancer.

[0050] In one embodiment, the ovarian cancer is selected from ovarian adenocarcinoma.

[0051] In one embodiment, the haematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

[0052] In another aspect, the invention relates to a use of the binding agent, cell, ADC or pharmaceutical composition as described herein for the manufacture of a medicament for the treatment of a disease associated with cells expressing IL1 RAP and B7H4.

[0053] In one embodiment, the pharmaceutical composition or said binding agent is administered together with another therapy.

[0054] In one embodiment, said administration is sequentially or concurrently.

[0055] In one embodiment, said other therapy is selected from chemotherapy, radiotherapy or therapy with a checkpoint inhibitor.

[0056] In another aspect, the invention relates to a method of producing the binding agent described herein comprising culturing the host cell as described herein.

[0057] In another aspect, the invention relates to a method for targeting tumour cells that express both IL1 RAP and B7H4 comprising administering a binding agent, cell, ADC or pharmaceutical composition as described herein.

[0058] In another aspect, the invention relates to an in vitro, ex vivo or in vivo method of delivering a payload to IL1 RAP+ / B7H4+ cells within a biological sample, comprising obtaining or providing a biological sample contacting said biological sample with a binding agent as described herein.

[0059] In another aspect, the invention relates to a method for reducing off target toxicity of a cancer treatment comprising administering to a subject a binding agent, cell, ADC or pharmaceutical composition as described herein. In another aspect, the invention relates to a kit comprising the binding agent, cell, ADC or pharmaceutical composition as described herein and instructions for use.

[0060] In another aspect, the invention relates to a method for identifying a patient that responds to therapy with a binding agent as described herein comprising analysing tumour cells for coexpression of IL1 RAP and B7H4.

[0061] In one embodiment, the method comprises (a) providing a tumour sample of cells from a patient to be tested; (b) optionally, extracting and / or purifying the cells present in the sample; (c) analysing tumour cells for co-expression of IL1 RAP and B7H4.

[0062] In another aspect, the invention relates to a method for identifying a tumour that responds to therapy with a binding agent as described herein comprising analysing tumour cells for coexpression of IL1 RAP and B7H4.

[0063] In one embodiment, the tumour cells are obtained from a patient or a cell line.

[0064] In another aspect, the invention relates to an in vitro, ex vivo or in vivo method of detecting IL1 RAP+ B7H4+ cells in a biological sample, comprising obtaining a biological sample contacting said biological sample with a binding molecule as described herein.

[0065] In another aspect the invention relates to a binding agent comprising an antigen-binding region that binds human IL1 RAP comprising; a. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 1 19 or a sequence comprising

[0066] 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising

[0067] 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising

[0068] 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; h. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 112 or a sequence comprising 1 , 2, or 3 amino acid differences.

[0069] Figures Figure 1. Cell kill assay results of multiple anti-B7H4 x anti-IL1 RAP bispecific ADCs which contain different combinations of anti-IL1 RAP and anti-B7H4 sequences. Average dose response curves ±SEM (n=2, biological repeats, 2 technical replicates) where multiple anti-B7H4 x anti- 1 L1 RAP bispecific ADCs have been tested with different combinations of anti- 1 L1 RAP and anti-B7H4 sequences. IC50 value for B7H4+ / IL1 RAP+ target cells reported in Figure 1 , with all other cell lines being reported as >100nM.

[0070] Figure 2. B7H4+ / IL1 RAP+ cell selectivity of anti-B7H4 x anti-IL1 RAP Bi-Fab ADCs containing different antilLI RAP sequences. Selectivity index of mcMMAF conjugated bispecific ADCs when comparing target B7H4+ / IL1 RAP+ SK-BR3 (A) and B7H4+ / IL1 RAP+ OVCAR-3 (B) cells to nontarget single antigen expressing (B7H4+ / IL1 RAP- or B7H4- / IL1 RAP+) cells, ZR-75-1 , EFO-21 and SK-OV-3. A value of 1 indicates equitoxicity of the ADC, a value of <1 indicates no preferential cytotoxicity to double positive antigen expressing cells and a value of >1 indicates preferential cytotoxicity to double positive antigen expressing cells.

[0071] Figure 3. Binding of different anti-IL1 RAP Fabs to human IL1 RAP antigens by ELISA. Curve showing binding of eight different anti-l L1 RAP Fabs to human IL1 RAP antigens.

[0072] Figure 4. Binding of BVX02-a0155-AB4A and BVX01 -a0161-AB4A bispecific ADCs to human B7H4 and human IL1 RAP antigens by ELISA. Curve showing binding of BVX01-a0155-AB4A and BVX02-a0161-AB4A bispecific ADCs to human B7H4 and human IL1 RAP antigens.

[0073] Figure 5. Binding of bispecific ADCs to cell lines expressing B7H4 and / or IL1 RAP. Median fluorescent intensity normalised to IL1 RAP receptor number of bispecific ADCs bound to B7H4+ / IL1 RAP+ cell line (SK-BR-3), B7H4- / IL1 RAP+ cell lines (EFO-21 and SK-OV-3).

[0074] Figure 6. Schematic of antibody formats tested. AntiB7H4 x a nti I L1 RAP antibody formats which were conjugated to mcMMAF to produce the ADC.

[0075] Figure 7. Cell kill assay results of different anti-B7H4 x anti-IL1 RAP bispecific ADC formats.

[0076] Average dose response curves ±SEM (n=2, biological repeats, 2 technical replicates) where multiple anti-B7H4 x anti-IL1 RAP bispecific ADC formats have been tested, Bi-Fab-Fc (A,B), Asymmetric (C, D) Asymmetric extended 4xG4S ScFv (E, F). The Bi-Fab-Fc in A and B differ in their DAR, 4 and 6 respectively. The asymmetric in C and D or E and F differ in their antil L1 RAP sequences. A, B, D and F contain the same anti- 1 L 1 RAP sequence as BVX02-a0161 -AB4A, and C and E contain the same anti-IL1 RAP sequence as BVX02-a0155- AB4A. IC50 value for B7H4+ / IL1 RAP+ target cells reported in figure (n=2, biological replicates), with all other cell lines being reported as >30nM. Figure 8. B7H4+ / IL1 RAP+ cell selectivity of different anti-B7H4 x anti-IL1 RAP bispecific ADC formats. Selectivity index of mcMMAF conjugated bispecific ADCs when comparing target B7H4+ / IL1 RAP+ SK-BR3 (A) and B7H4+ / IL1 RAP+ OVCAR-3 (B) cells to non-target single antigen expressing (B7H4+ / IL1 RAP- or B7H4- / IL1 RAP+) cells, ZR-75-1 , EFO-21 and SK-OV-3. A value of 1 indicates equitoxicity of the ADC, a value of <1 indicates no preferential cytotoxicity to double positive antigen expressing cells and a value of >1 indicates preferential cytotoxicity to double positive antigen expressing cells. Overall, all anti-B7H4 x anti-IL1 RAP ADCs showed good potency and good selectivity in targeting double antigen positive B7H4+ / IL1 RAP+ target cells (SK-BR-3, OVCAR-3) vs single antigen positive non-target cells.

[0077] Figure 9. Schematic of bi-specific ADC DAR 4 and monospecific Fab ADCs DAR 2.

[0078] Figure 10. Synergistic cell kill of BVX02-a0161-AB4A and BVX02-a0155-AB4A Bi-Fab ADC, which differ in their anti-IL1 RAP sequences, compared to the corresponding, mono-specific anti- B7H4-mcMMAF Fab ADC (BVX02-d0110-AB2A) and anti-IL1 RAP-mcMMAF Fab ADC (BVX02- dO153-AB2A or BVX02-d0147-AB2A) as a mixture, respectively. Average dose response curves of BVX02-a0161-AB4A (A, B) or BVX02-a0155-AB4A (C, D) bispecific ADC compared to anti- B7H4-mcMMAF Fab ADC (BVX02-d0110-AB2A) and anti-IL1 RAP-mcMMAF Fab ADC (BVX02- dO153-AB2A or BVX02-d0147-AB2A) as a mixture in SK-BR-3 and OVCAR-3 (B7H4+ / IL1 RAP+) cell lines. ADC concentration is normalised to the number of equivalent Fab arms.

[0079] Figure 11. Bar charts showing average IC50 ± SEM of BVX02-a0161-AB4A (A) and BVX02- aO155-AB4A (B) bispecific ADCs in double positive antigen expressing cells (SK-BR-3 and OVCAR-3) compared to anti-B7H4-mcMMAF Fab ADC (BVX02-d0110-AB2A) and anti-IL1 RAP- mcMMAF Fab ADC (BVX02-d0153-AB2A or BVX02-d0147-AB2A) alone and as a mixture. IC50 data are obtained by an average of two separate experiments.

[0080] Figure 12. Dose Response curves and IC50s of Bi-Fab Fc format ADCs. Dose response curves of cell kill assay conducted using a 9-point dose response of anti-B7H4 x anti- 1 L1 RAP Bi-Fab Fc ADCs (A,B) Error bars represent the standard error of the mean of 2 biological repeats (n=2) performed in duplicate. IC50 values presented in table format.

[0081] Figure 13. Anti-B7H4 x anti-IL1 RAP bispecific ADC (BVX02-b0184-AA4A) showed good efficacy in target OVCAR-3 (B7H4+|OW / IL1 RAP+) xenograft model. ADC was dosed intravenously at 10, 5 or 2.5 mg / kg once weekly for 4 weeks. At 10 mg / kg or 5 mg / kg BVX02-b0184-AA4A ADC showed >100% tumour growth inhibition (TGI), at 2.5 mg / kg 78.3% TGI was observed (***p<0.001). BVX02-b0184-AA4A ADC showed 63.1 % and 46.2 % tumour regression at 10 mg / kg and 5 mg / kg, respectively. Figure 14. The average ± SEM percentage change in body weight of mice during and after dosing relative to start body weight on day 0. Dosing completion represented by dotted line.

[0082] Figure 15. Mean tumour volume ± SEM in mice during and after dosing once weekly with BVX2184-vcMMAE ADC (2.5, 5 and 10 mg / kg, respectively) and BVX2175-vcMME ADC (8 mg / kg). Table indicates tumour growth inhibition (TGI) and tumour regression for each TA (determined at day 29). NB isotype control data TGI and regression are not shown as it is not a suitable comparator (see below). % TGI was assessed by comparison of the geometric mean change in tumour volume for the control vs treated groups. Tumour regression was calculated as the percentage reduction in tumour volume from baseline value % Regression = (1 - RTV) x 100%, where RTV is the geometric mean relative tumour volume. Statistical significance was evaluated using a one-tailed t-test.

[0083] Figure 16. Seagen’s CLDX study in OVCAR-3 murine model. Plot showing mean tumour volume in mice during dosing with monospecific SGN-B7H4-ADC. Non-binding isotype control ADC in comparison with Untreated vehicle (Seagen INC, 2023; WO2023056362A1).

[0084] Figure 17. Anti-B7H4 x anti-IL1 RAP bispecific antibody showed synergistic binding to B7H4+ / IL1 RAP+cell lines compared to the monovalent monospecific counterparts, alone and in combination.

[0085] Figure 18. Anti-B7H4 x anti-IL1 RAP bispecific antibody conjugated to AF488 showed synergistic internalisation into B7H4+ / IL1 RAP+SK-BR-3 cells compared to the bivalent monospecific antibodies conjugated to AF488, alone or in combination.

[0086] Figure 19. Anti-B7H4 x anti-IL1 RAP bispecific ADC showed synergistic cell killing of B7H4+ / IL1 RAP+cell lines compared to bivalent monospecific ADCs, alone or in combination.

[0087] Detailed Description

[0088] The embodiments of the invention will now be described. In the following passages, different embodiments are described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0089] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012) Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0090] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties of the molecules disclosed herein are also described in the examples.

[0091] Binding Agents

[0092] The inventors have shown that cancer cells express both IL1 RAP+B7H4+. The inventors have also surprisingly shown that targeting both IL1 RAP and B7H4 on tumour-associated cells provides an effective way of treating cancer as the dual targeting of this specific antigen pair targets tumour associated cells, but not healthy cells.

[0093] These results presented herein show that targeting IL1 RAP and B7H4 with a bispecific antibody or antigen binding fragment thereof targets tumour-associated cells expressing both IL1 RAP and B7H4 whilst avoiding targeting healthy cell populations.

[0094] Then inventors have thus shown that a binding agent that binds both IL1 RAP and B7H4, that is bispecifically binds these targets, shows selectivity for cells expressing IL1 RAP and B7H4 (IL1 RAP+ B7H4+ cells) compared to cells that express only IL1 RAP or B7H4 (IL1 RAP+ B7H4- / IL1 RAP- B7H4+ cells).

[0095] Therefore, targeting both IL1 RAP and B7H4 surprisingly avoids or reduces off-target cytotoxic effects. Off-target cytotoxicity that targets healthy cells can lead to common side effects of anticancer therapy, e.g. impaired immune function. Thus, the present invention relates to a binding agent comprising a first antigen-binding region that binds human interleukin-1 receptor accessory protein (IL1 RAP) and a second antigen binding region that binds human B7H4. Thus, the binding agent is therefore bispecific and binds to the two antigens IL1 RAP and B7H4. As explained below, in one embodiment, the binding agent may comprise additional binding moieties.

[0096] The inventors have shown that bispecific binding agents of the invention comprising a payload demonstrate preferential cell kill of IL1 RAP+ B7H4+ cells.

[0097] As used herein, "IL1 RAP expressing cell" and “IL1 RAP + cell” refers to a cell that expresses IL1 RAP as a surface antigen. As used herein, " B7H4 expressing cell" and “B7H4+ cell” refers to a cell that expresses B7H4 as a surface antigen. As used herein, " IL1 RAP and B7H4 expressing cell" and “IL1 RAP+ B7H4 cell” refers to a cell that expresses both IL1 RAP and B7H4 as surface antigens.

[0098] As used herein "target cell" refers to a cell or cell-type characterized by the expression or overexpression of the target molecule B7H4 and IL1 RAP. Any type of malignant cell expressing both B7H4 and IL1 RAP may be envisaged as a target cell for treatment with the binding agent of the invention. In certain embodiments, the cell is a tumour cell as explained further herein.

[0099] The terms “cell selectivity” or “selectivity for cells” as used herein refer to the ability of a binding agent, antibody or fragment thereof to target a specific target cell, i.e. a malignant cell, over a non-target cell, i.e. a healthy cell. The terms may refer to selective binding and / or selective cell killing. In the present invention, the binding agents demonstrate selectivity for cells expressing both IL1 RAP and B7H4 compared to non-target cells which do not express IL1 RAP and B7H4. In particular, the binding agents of the invention show higher selectivity for cells expressing both IL1 RAP and B7H4 compared to non-target cells expressing IL1 RAP or cells expressing B7H4 respectively.

[0100] In one embodiment, cell selectivity to IL1 RAP+ B7H4+ cells may be cell killing and may be 1.1 to more than 260-fold higher, e.g. at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250 fold higher compared to cells expressing IL1 RAP or cells expressing B7H4 respectively.

[0101] In one embodiment, cell selectivity to IL1 RAP+ B7H4+ cells may be cell binding and may be 2 to 50 fold, e.g. 2, 5, 20, 25, 20, 25, 3, 35, 40, 45 fold higher compared to cells expressing IL1 RAP or cells expressing B7H4 respectively. In one embodiment, a binding agent of the invention has a IC50 value in the low nanomolar subnanomolar range, e.g. 0.1 nM to 5 nM, e.g. 0.5, 1 , 1 .5, 2.5, 3, 3.5, 4, 4.5 nM.

[0102] The inventors have also surprisingly shown that bispecific binding agents of the invention comprising a payload have a greater cell killing effect of IL1 RAP+ B7H4+ cells compared to anti- IL1 RAP antibody or B7H4 antibody combined as a mixture or alone. In one embodiment, the cell killing effect is additive. In one embodiment, the cell killing effect is synergistic.

[0103] The bispecific antigen binding agents of the invention have the potential to target tumour heterogeneity, where different populations of cancer cells within a patient can express target antigens at both, high and low levels. Tumour heterogeneity currently represents a substantial hurdle for drug developers in the antibody-based therapeutic sector.

[0104] Thus, binding agents of the present invention may comprise one or more of the following features i) are capable of binding I bind to cells expressing both human IL1 RAP and human B7H4; ii) target cells expressing both IL1 RAP and B7H4 at varying levels; iii) have greater selectivity for target IL1 RAP+B7H4+ cells compared to non-target cells that do not express both IL1 RAP and B7H4, e.g. express IL1 RAP or B7H4; iv) demonstrate cell selectivity for IL1 RAP+B7H4+ cells compared to non-target cells that do not express both IL1 RAP and B7H4, e.g. express IL1 RAP or B7H4 with a fold selectivity as shown in the examples; v) demonstrate the ability to induce cell selective cell death of IL1 RAP+B7H4+ cells; vi) demonstrate cell killing of IL1 RAP+B7H4+ cells with an IC50 as shown in the examples; vii) demonstrate greater cell killing effect of IL1 RAP+ B7H4+ cells compared to individual anti-l L1 RAP antibody or B7H4 antibody combined as a mixture or alone. In particular, the cell killing effect may be synergistic; viii) show good efficacy in vitro against a cell line displaying the cancer-specific IL1 RAP+ and B7H4+ target fingerprint expressing the targets at different levels as shown in the examples; ix) have an IC50 in the low nanomolar subnanomolar range, e.g. 0.1 nM to 5 nM as measured in the examples x) demonstrate tumour growth inhibition for example as measured by mean tumour volume. In particular demonstrate tumour growth inhibition in OVCAR-3 (B7H4+|OW / IL1 RAP+) xenograft model; xi) demonstrate greater internalisation into IL1 RAP+ B7H4+ cells compared to individual anti-l L1 RAP antibody or B7H4 antibody combined as a mixture or alone. In particular the internalisation effect may be synergistic; xii) demonstrate greater binding to IL1 RAP+ B7H4+ cells compared to individual anti- IL1 RAP antibody or B7H4 antibody combined as a mixture or alone. In particular the binding effect may be synergistic..

[0105] The term binding agent as used herein may include a binding molecule or a cell. A bispecific binding agent as used herein binds to two antigens, human IL1 RAP and human B7H4.

[0106] The present invention relates to a binding agent comprising a first antigen-binding region that binds human ILI RAPand a second antigen binding region that binds human B7H4, wherein the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 1 12 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 1 19 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0107] In an embodiment the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 21 , and a VL CDR1 comprising SEQ ID NO: 24, a VL CDR2 comprising SEQ ID NO: 26, a VL CDR3 comprising SEQ ID NO: 28; b. a VH CDR1 comprising SEQ ID NO: 31 , a VH CDR2 comprising SEQ ID NO: 33, a VH CDR3 comprising SEQ ID NO: 35, and a VL CDR1 comprising SEQ ID NO: 38, a VL CDR2 comprising SEQ ID NO: 40, a VL CDR3 comprising SEQ ID NO: 42; c. a VH CDR1 comprising SEQ ID NO: 45, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 49, and a VL CDR1 comprising SEQ ID NO: 52, a VL CDR2 comprising SEQ ID NO: 54, a VL CDR3 comprising SEQ ID NO: 56; d. a VH CDR1 comprising SEQ ID NO: 59, a VH CDR2 comprising SEQ ID NO: 61 , a VH CDR3 comprising SEQ ID NO: 63, and a VL CDR1 comprising SEQ ID NO: 66, a VL CDR2 comprising SEQ ID NO: 68, a VL CDR3 comprising SEQ ID NO: 70; e. a VH CDR1 comprising SEQ ID NO: 73, a VH CDR2 comprising SEQ ID NO: 75, a VH CDR3 comprising SEQ ID NO: 77, and a VL CDR1 comprising SEQ ID NO: 80, a VL CDR2 comprising SEQ ID NO: 82, a VL CDR3 comprising SEQ ID NO: 84; f. a VH CDR1 comprising SEQ ID NO: 87, a VH CDR2 comprising SEQ ID NO: 89, a VH CDR3 comprising SEQ ID NO: 91 , and a VL CDR1 comprising SEQ ID NO: 94, a VL CDR2 comprising SEQ ID NO: 96, a VL CDR3 comprising SEQ ID NO: 98; g. a VH CDR1 comprising SEQ ID NO: 101 , a VH CDR2 comprising SEQ ID NO: 103, a VH CDR3 comprising SEQ ID NO: 105, and a VL CDR1 comprising SEQ ID NO: 108, a VL CDR2 comprising SEQ ID NO: 110, a VL CDR3 comprising SEQ ID NO: 112; or h. a VH CDR1 comprising SEQ ID NO: 115, a VH CDR2 comprising SEQ ID NO: 117, a VH CDR3 comprising SEQ ID NO: 119, and a VL CDR1 comprising SEQ ID NO: 122, a VL CDR2 comprising SEQ ID NO: 124, a VL CDR3 comprising SEQ ID NO: 126, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0108] In an embodiment the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 16 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 18 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 20 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 23 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 25 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 27 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 30 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 32 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 34 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 37 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 39 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 41 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 44 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 46 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 48 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 51 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 53 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 55 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 58 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 60 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 62 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 65 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 67 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 69 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 72 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 74 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 76 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 79 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 81 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 83 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 86 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 88 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 90 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 93 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 95 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 97 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 100 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 102 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 104 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 107 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 109 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 111 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 114 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 116 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 1 18 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 121 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 123 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 125 or a sequence comprising 1 , 2, or 3 amino acid differences, wherein the CDR sequences are defined according to the Kabat numbering scheme. In an embodiment the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 16, a VH CDR2 comprising SEQ ID NO: 18, a VH CDR3 comprising SEQ ID NO: 20, and a VL CDR1 comprising SEQ ID NO: 23, a VL CDR2 comprising SEQ ID NO: 25, a VL CDR3 comprising SEQ ID NO: 27; b. a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 32, a VH CDR3 comprising SEQ ID NO: 34, and a VL CDR1 comprising SEQ ID NO: 37, a VL CDR2 comprising SEQ ID NO: 39, a VL CDR3 comprising SEQ ID NO: 41 ; c. a VH CDR1 comprising SEQ ID NO: 44, a VH CDR2 comprising SEQ ID NO: 46, a VH CDR3 comprising SEQ ID NO: 48, and a VL CDR1 comprising SEQ ID NO: 51 , a VL CDR2 comprising SEQ ID NO: 53, a VL CDR3 comprising SEQ ID NO: 55; d. a VH CDR1 comprising SEQ ID NO: 58, a VH CDR2 comprising SEQ ID NO: 60, a VH CDR3 comprising SEQ ID NO: 62, and a VL CDR1 comprising SEQ ID NO: 65, a VL CDR2 comprising SEQ ID NO: 67, a VL CDR3 comprising SEQ ID NO: 69; e. a VH CDR1 comprising SEQ ID NO: 72, a VH CDR2 comprising SEQ ID NO: 74, a VH CDR3 comprising SEQ ID NO: 76, and a VL CDR1 comprising SEQ ID NO: 79, a VL CDR2 comprising SEQ ID NO: 81 , a VL CDR3 comprising SEQ ID NO: 83; f. a VH CDR1 comprising SEQ ID NO: 86, a VH CDR2 comprising SEQ ID NO: 88, a VH CDR3 comprising SEQ ID NO: 90, and a VL CDR1 comprising SEQ ID NO: 93, a VL CDR2 comprising SEQ ID NO: 95, a VL CDR3 comprising SEQ ID NO: 97; g. a VH CDR1 comprising SEQ ID NO: 100, a VH CDR2 comprising SEQ ID NO: 102, a VH CDR3 comprising SEQ ID NO: 104, and a VL CDR1 comprising SEQ ID NO: 107, a VL CDR2 comprising SEQ ID NO: 109, a VL CDR3 comprising SEQ ID NO: 111 ; or h. a VH CDR1 comprising SEQ ID NO: 114, a VH CDR2 comprising SEQ ID NO: 116, a VH CDR3 comprising SEQ ID NO: 118, and a VL CDR1 comprising SEQ ID NO: 121 , a VL CDR2 comprising SEQ ID NO: 123, a VL CDR3 comprising SEQ ID NO: 125, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0109] In an embodiment the first antigen binding region comprises a. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; c. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; d. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; e. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; f. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; g. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 112 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; or h. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 1 19 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0110] In an embodiment the first antigen binding region comprises a. a VH CDR1 comprising SEQ ID NO: 16 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 18 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 20 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 23 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 25 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 27 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH CDR1 comprising SEQ ID NO: 30 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 32 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 34 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 37 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 39 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 41 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; c. a VH CDR1 comprising SEQ ID NO: 44 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 46 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 48 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 51 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 53 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 55 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; d. a VH CDR1 comprising SEQ ID NO: 58 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 60 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 62 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 65 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 67 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 69 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; e. a VH CDR1 comprising SEQ ID NO: 72 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 74 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 76 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 79 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 81 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 83 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; f. a VH CDR1 comprising SEQ ID NO: 86 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 88 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 90 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 93 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 95 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 97 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; g. a VH CDR1 comprising SEQ ID NO: 100 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 102 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 104 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 107 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 109 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 111 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; or h. a VH CDR1 comprising SEQ ID NO: 114 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 116 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 1 18 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 121 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 123 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 125 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0111] The first antigen binding region may comprise a VH and a VL domain. In an embodiment the first antigen binding region comprises; a. a VH comprising SEQ ID NO: 15 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 22 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or b. a VH comprising SEQ ID NO: 29 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 36 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or c. a VH comprising SEQ ID NO: 43 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 50 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or d. a VH comprising SEQ ID NO: 57 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 64 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or e. a VH comprising SEQ ID NO: 71 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 78 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or f. a VH comprising SEQ ID NO: 85 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 92 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or g. a VH comprising SEQ ID NO: 99 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 106 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or h. a VH comprising SEQ ID NO: 113 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 120 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0112] The CDR sequences of the antigen binding region are herein provided according to the Kabat numbering scheme and the IMGT numbering scheme. The CDR sequences of the antigen binding regions according to the invention may also comprise the CDR sequences as defined by any antibody numbering scheme known in the art including Kabat, IMGT, Chothia, Martin, AHo. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and was originally derived from observations from sequence alignments of light chain A, K and heavy chain sequences for a number of antibodies and forthe a, p, y, 6 chain sequences for a number of TCRs. Kabat numbering is the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). The ImMunoGeneTics (IMGT) unique numbering scheme (IMGT numbering scheme) is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). The IMGT numbering scheme is based on alignment of germ-line V genes and covers the entire variable region positions 1 to 128 based on the V-gene sequence alignment, with an insertion point between positions 111 and 112 in the CDR3 for lengths exceeding 13 amino acids. The Chothia scheme is a structure-based scheme and refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)) Martin a further structural numbering scheme similar to Chothia which comprises greater sequence variability in the reference dataset, also unconventional CDR lengths and deletions are taken into account in the numbering scheme. The AHo scheme comprises a large number of positions is supposed to account for all possible positions without the need for specifying insertion positions. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

[0113] The first antigen binding region may comprise CDR sequences as provided in the full length VH and VL sequence wherein the CDR is defined according to any known antibody numbering scheme including but not limited to Kabat, IMGT, Chothia, Martin, AHo. In an embodiment the first antigen binding region comprises; a. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 15, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 22, or b. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 29, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 36, or c. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 43 and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 50, or d. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 57, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 64, or e. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 71 , and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 78, or f. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 85 and a VL CDR1 , CDR2, CDR3 of SEQ ID

[0114] NO: 92, or g. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 99, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 106 o, or h. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 113, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 120, wherein the CDR sequences are defined according to one of Kabat, IMGT, Chothia, Martin, AHo numbering schemes.

[0115] The binding agent of the invention comprises a second antigen binding region that binds to human B7H4. In an embodiment the second binding region comprises a VH CDR1 comprising SEQ ID NO: 3 or a sequence comprising 1 , 2 or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 5 or a sequence comprising 1 , 2 or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 7 or a sequence comprising 1 , 2 or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 10 or a sequence comprising 1 , 2 or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 12 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 14 or a sequence comprising 1 , 2 or 3 amino acid differences, wherein the sequences are defined by the IMGT numbering scheme. In an embodiment the second binding region comprises a VH CDR1 comprising SEQ ID NO: 3 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 5 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 7 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 10 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 12 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 14 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the sequences are defined by the IMGT numbering scheme. In an embodiment the second antigen binding region comprises a VH CDR1 comprising SEQ ID NO: 3, a VH CDR2 comprising SEQ ID NO: 5, a VH CDR3 comprising SEQ ID NO: 7, and a VL CDR1 comprising SEQ ID NO: 10, a VL CDR2 comprising SEQ ID NO: 12, a VL CDR3 comprising SEQ ID NO: 14.

[0116] In an embodiment the second binding region comprises a VH CDR1 comprising SEQ ID NO: 2 or a sequence comprising 1 , 2 or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 4 or a sequence comprising 1 , 2 or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 6 or a sequence comprising 1 , 2 or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 9 or a sequence comprising 1 , 2 or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 11 or a sequence comprising 1 , 2 or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 13 or a sequence comprising 1 , 2 or 3 amino acid differences, wherein the sequences are defined by the Kabat numbering scheme. In an embodiment the second binding region comprises a VH CDR1 comprising SEQ ID NO: 2 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 4 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 6 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 9 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 11 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 13 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the sequences are defined by the Kabat numbering scheme. In an embodiment the second antigen binding region comprises a VH CDR1 comprising SEQ ID NO: 2, a VH CDR2 comprising SEQ ID NO: 4, a VH CDR3 comprising SEQ ID NO: 6, and a VL CDR1 comprising SEQ ID NO: 9, a VL CDR2 comprising SEQ ID NO: 11 , a VL CDR3 comprising SEQ ID NO: 13.

[0117] In an embodiment the second antigen binding region comprises a VH comprising SEQ ID NO: 1 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 8 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto. In an embodiment the second antigen binding region comprises a VH comprising SEQ ID NO: 1 , and a VL comprising SEQ ID NO: 8.

[0118] The second antigen binding region may comprise CDR sequences as provided in the full length VH and VL sequence wherein the CDR is defined according to any known antibody numbering scheme including but not limited to Kabat, IMGT, Chothia, Martin, AHo. In an embodiment the first antigen binding region comprises a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 1 , and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 8 wherein the CDR sequences are defined according to one of Kabat, IMGT, Chothia, Martin, AHo numbering schemes.

[0119] The antigen binding region disclosed herein may have therapeutic utility when used in a monospecific or multispecific format, as such an aspect of the invention relates to a binding agent comprising an antigen-binding region that binds human IL1 RAP comprising; a. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 1 12 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 1 19 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0120] In an embodiment the antigen binding region that binds IL1-RAP comprises; a. a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 21 , and a VL CDR1 comprising SEQ ID NO: 24, a VL CDR2 comprising SEQ ID NO: 26, a VL CDR3 comprising SEQ ID NO: 28; b. a VH CDR1 comprising SEQ ID NO: 31 , a VH CDR2 comprising SEQ ID NO: 33, a VH CDR3 comprising SEQ ID NO: 35, and a VL CDR1 comprising SEQ ID NO: 38, a VL CDR2 comprising SEQ ID NO: 40, a VL CDR3 comprising SEQ ID NO: 42; c. a VH CDR1 comprising SEQ ID NO: 45, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 49, and a VL CDR1 comprising SEQ ID NO: 52, a VL CDR2 comprising SEQ ID NO: 54, a VL CDR3 comprising SEQ ID NO: 56; d. a VH CDR1 comprising SEQ ID NO: 59, a VH CDR2 comprising SEQ ID NO: 61 , a VH CDR3 comprising SEQ ID NO: 63, and a VL CDR1 comprising SEQ ID NO: 66, a VL CDR2 comprising SEQ ID NO: 68, a VL CDR3 comprising SEQ ID NO: 70; e. a VH CDR1 comprising SEQ ID NO: 73, a VH CDR2 comprising SEQ ID NO: 75, a VH CDR3 comprising SEQ ID NO: 77, and a VL CDR1 comprising SEQ ID NO: 80, a VL CDR2 comprising SEQ ID NO: 82, a VL CDR3 comprising SEQ ID NO: 84; f. a VH CDR1 comprising SEQ ID NO: 87, a VH CDR2 comprising SEQ ID NO: 89, a VH CDR3 comprising SEQ ID NO: 91 , and a VL CDR1 comprising SEQ ID NO: 94, a VL CDR2 comprising SEQ ID NO: 96, a VL CDR3 comprising SEQ ID NO: 98; g. a VH CDR1 comprising SEQ ID NO: 101 , a VH CDR2 comprising SEQ ID NO: 103, a VH CDR3 comprising SEQ ID NO: 105, and a VL CDR1 comprising SEQ ID NO: 108, a VL CDR2 comprising SEQ ID NO: 110, a VL CDR3 comprising SEQ ID NO: 112; or h. a VH CDR1 comprising SEQ ID NO: 115, a VH CDR2 comprising SEQ ID NO: 117, a VH CDR3 comprising SEQ ID NO: 119, and a VL CDR1 comprising SEQ ID NO: 122, a VL CDR2 comprising SEQ ID NO: 124, a VL CDR3 comprising SEQ ID NO: 126, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0121] In an embodiment the antigen binding region that binds IL1-RAP comprises; a. a VH CDR1 comprising SEQ ID NO: 16 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 18 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 20 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 23 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 25 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 27 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 30 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 32 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 34 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 37 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 39 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 41 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 44 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 46 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 48 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 51 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 53 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 55 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 58 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 60 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 62 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 65 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 67 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 69 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 72 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 74 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 76 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 79 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 81 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 83 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 86 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 88 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 90 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 93 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 95 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 97 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 100 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 102 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 104 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 107 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 109 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 111 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 114 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 116 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 1 18 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 121 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 123 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR3 comprising SEQ ID NO: 125 or a sequence comprising 1 , 2, or 3 amino acid differences, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0122] In an embodiment the antigen binding region that binds IL1-RAP comprises; a. a VH CDR1 comprising SEQ ID NO: 16, a VH CDR2 comprising SEQ ID NO: 18, a VH CDR3 comprising SEQ ID NO: 20, and a VL CDR1 comprising SEQ ID NO: 23, a VL CDR2 comprising SEQ ID NO: 25, a VL CDR3 comprising SEQ ID NO: 27; b. a VH CDR1 comprising SEQ ID NO: 30, a VH CDR2 comprising SEQ ID NO: 32, a VH CDR3 comprising SEQ ID NO: 34, and a VL CDR1 comprising SEQ ID NO: 37, a VL CDR2 comprising SEQ ID NO: 39, a VL CDR3 comprising SEQ ID NO: 41 ; c. a VH CDR1 comprising SEQ ID NO: 44, a VH CDR2 comprising SEQ ID NO: 46, a VH CDR3 comprising SEQ ID NO: 47, and a VL CDR1 comprising SEQ ID NO: 51 , a VL CDR2 comprising SEQ ID NO: 53, a VL CDR3 comprising SEQ ID NO: 55; d. a VH CDR1 comprising SEQ ID NO: 58, a VH CDR2 comprising SEQ ID NO: 60, a VH CDR3 comprising SEQ ID NO: 62, and a VL CDR1 comprising SEQ ID NO: 65, a VL CDR2 comprising SEQ ID NO: 67, a VL CDR3 comprising SEQ ID NO: 69; e. a VH CDR1 comprising SEQ ID NO: 72, a VH CDR2 comprising SEQ ID NO: 74, a VH CDR3 comprising SEQ ID NO: 76, and a VL CDR1 comprising SEQ ID NO: 79, a VL CDR2 comprising SEQ ID NO: 81 , a VL CDR3 comprising SEQ ID NO: 83; f. a VH CDR1 comprising SEQ ID NO: 86, a VH CDR2 comprising SEQ ID NO: 87, a VH CDR3 comprising SEQ ID NO: 90, and a VL CDR1 comprising SEQ ID NO: 93, a VL CDR2 comprising SEQ ID NO: 95, a VL CDR3 comprising SEQ ID NO: 97; g. a VH CDR1 comprising SEQ ID NO: 100, a VH CDR2 comprising SEQ ID NO: 102, a VH CDR3 comprising SEQ ID NO: 104, and a VL CDR1 comprising SEQ ID NO: 107, a VL CDR2 comprising SEQ ID NO: 109, a VL CDR3 comprising SEQ ID NO: 111 ; or h. a VH CDR1 comprising SEQ ID NO: 114, a VH CDR2 comprising SEQ ID NO: 116, a VH CDR3 comprising SEQ ID NO: 118, and a VL CDR1 comprising SEQ ID NO: 121 , a VL CDR2 comprising SEQ ID NO: 123, a VL CDR3 comprising SEQ ID NO: 125, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0123] In an embodiment the antigen binding region that binds IL1-RAP comprises a. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; c. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; d. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; e. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; f. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; g. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 112 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; or h. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 1 19 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the CDR sequences are defined according to the IMGT numbering scheme.

[0124] In an embodiment the antigen binding region that binds IL1-RAP comprises a. a VH CDR1 comprising SEQ ID NO: 16 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 18 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 20 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 23 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 25 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 27 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH CDR1 comprising SEQ ID NO: 30 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 32 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 34 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 37 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 39 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 41 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; c. a VH CDR1 comprising SEQ ID NO: 44 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 46 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 48 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 51 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 53 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 55 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; d. a VH CDR1 comprising SEQ ID NO: 58 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 60 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 62 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 65 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 67 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 69 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; e. a VH CDR1 comprising SEQ ID NO: 72 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 74 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 76 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 79 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 81 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 83 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; f. a VH CDR1 comprising SEQ ID NO: 86 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 88 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 90 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 93 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 95 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 97 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; g. a VH CDR1 comprising SEQ ID NO: 100 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 102 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 104 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 107 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 109 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 111 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; or h. a VH CDR1 comprising SEQ ID NO: 114 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR2 comprising SEQ ID NO: 116 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VH CDR3 comprising SEQ ID NO: 1 18 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL CDR1 comprising SEQ ID NO: 121 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR2 comprising SEQ ID NO: 123 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, a VL CDR3 comprising SEQ ID NO: 125 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0125] The antigen binding region that binds IL1-RAP may comprise a VH and a VL domain. In an embodiment the antigen binding region that binds IL1-RAP comprises; a. a VH comprising SEQ ID NO: 15 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 22 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or b. a VH comprising SEQ ID NO: 29 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 36 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or c. a VH comprising SEQ ID NO: 43 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 50 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or d. a VH comprising SEQ ID NO: 57 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 64 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or e. a VH comprising SEQ ID NO: 71 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 78 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or f. a VH comprising SEQ ID NO: 85 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 92 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or g. a VH comprising SEQ ID NO: 99 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 106 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or h. a VH comprising SEQ ID NO: 113 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 120 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto.

[0126] The antigen binding region that binds IL1-RAP may comprise CDR sequences as provided in the full length VH and VL sequence wherein the CDR is defined according to any known antibody numbering scheme including but not limited to Kabat, IMGT, Chothia, Martin, AHo. In an embodiment the antigen binding region that binds IL1-RAP comprises; a. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 15, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 22, or b. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 29, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 36, or c. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 43 and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 50, or d. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 57, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 64, or e. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 71 , and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 78, or f. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 85 and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 92, or g. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 99, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 106 o, or h. a VH CDR1 , CDR2, CDR3 of SEQ ID NO: 113, and a VL CDR1 , CDR2, CDR3 of SEQ ID NO: 120, wherein the CDR sequences are defined according to one of Kabat, IMGT, Chothia, Martin, AHo numbering schemes.

[0127] The antigen binding region comprises or consists of an antibody or antigen binding fragment thereof. Examples of antigen binding fragments are provided herein. In one example, the fragment is a Fab.

[0128] In an embodiment the binding agent comprising an antigen-binding region that binds human IL1 RAP, comprises one or more further antigen binding region. In an embodiment the one or more further antigen binding regions bind to a tumour associated antigen. In an embodiment the tumour associated antigen is B7H4. In an embodiment the further antigen binding region may comprise any of the features herein described for the second antigen binding region.

[0129] In an embodiment the binding agents of the invention also encompass an antibody or antigen binding fragment thereof which competes for binding to human IL1 RAP with one or more of the molecules described above.

[0130] Human IL1 RAP is also known as Interleukin 1 Receptor Accessory Protein (Uniprot accession number Q9NPH3-1). The amino acid sequence is shown below.

[0131] MTLLWCVVSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEHFLKFNYST AHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTLLNDTGNYTCMLRNTT YCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQRITCPNVDGYFPSSVKPTITWYMG CYKIQNFNNVIPEGMNLSFLIALISNNGNYTCWTYPENGRTFHLTRTLTVKVVGSPKNA VPPVIHSPNDHVVYEKEPGEELLIPCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINE SISHSRTEDETRTQILSIKKVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVEL ACGFGATVLLVVILIVVYHVYWLEMVLFYRAHFGTDETILDGKEYDIYVSYARNAEEEEF VLLTLRGVLENEFGYKLCIFDRDSLPGGIVTDETLSFIQKSRRLLWLSPNYVLQGTQAL LELKAGLENMASRGNINVILVQYKAVKETKVKELKRAKTVLTVIKWKGEKSKYPQGRFWK

[0132] QLQVAMPVKKSPRRSSSDEQGLSYSSLKNV (SEQ ID NO. 127)

[0133] Binding agents as described herein bind to SEQ ID NO. 127 or variants thereof, for example a different isoform variant of SEQ ID NO. 127 produced by alternative splicing. Variants may have at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO. 127.

[0134] Human B7-H4 is also known as B7H4, B7 family member H4, B7 superfamily member, 1 V-set domain containing T cell activation inhibitor 1 , VTCN1 (Uniprot accession number Q7Z7D3-1). The amino acid sequence is shown below. MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEP

[0135] DIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV QLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVV WASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKV TESEIKRRSHLQLLNSKASLCVSSFFAISWALLPLSPYLMLK (SEQ ID NO. 128)

[0136] Binding agents as described herein bind to SEQ ID NO. 127 or variants thereof, for example a different isoform variant of SEQ ID NO. 128 produced by alternative splicing. Variants may have at least 70%, 75%, 80%, 85%, 90% or 95% sequence identity to SEQ ID NO. 128.

[0137] As used herein, the terms "homology" or “identity” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and in some embodiments not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percentage identity between two amino acid sequences can be determined using well known mathematical algorithms. As used herein the term “sequence identity” generally refers to the percentage of amino acid residues in a sequence that are identical and / or comprise conservative amino acid substitutions, where the substituted amino acid has similar physicochemical properties. For example, where a positively charged amino acid has been replaced by a different positively charged amino acid. Percentage identity may be calculated using tools such as “EMBOSS Needle” which implements the Needleman-Wunch algorithm for the pairwise percent identities or similarities, amino acids may be considered similar if they have a positive score in the BLOSUM62 matrix. In an embodiment, where the term “sequence identity” is used herein it may be replaced by the term “sequence similarity” or “sequence homology”.

[0138] 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. Mol. 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. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. For example, the psi-Blast algorithm or Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms may be used. Sequence identity may also be defined using the Bioedit, ClustalW algorithm.

[0139] As used herein where a sequence is defined as comprising 80%, 85%, 90%, 95% or 99% sequence identity, this may include 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity.

[0140] A binding agent of the invention is isolated. An "isolated" binding agent is one that has been identified and separated and / or recovered from a component of its natural environment.

[0141] The terms "IL1 RAP binding molecule / region / protein / polypeptide / agent / moiety / antibody / antibody fragment / binding portion”, "IL1 RAP antigen binding molecule / region / protein / polypeptide / agent / moiety / antibody / antibody fragment / binding portion”, “anti- IL1 RAP antibody”, “anti- IL1 RAP antibody fragment”, “anti- IL1 RAP antibody or antigen binding portion thereof’, “IL1 RAP antibody” or“IL1 RAP antibody fragment” all referto a molecule capable of specifically binding to the human IL1 RAP antigen.

[0142] The terms "B7H4 binding molecule / region / protein / polypeptide / agent / moiety / antibody / antibody fragment / binding portion”, “B7H4 antigen binding molecule / region / protein / polypeptide / agent / moiety / antibody / antibody fragment / binding portion”, “anti- B7H4 antibody”, “anti- B7H4 antibody fragment”, “anti- B7H4 antibody or antigen binding portion thereof’, “B7H4 antibody” or “B7H4 antibody fragment” all refer to a molecule capable of specifically binding to the human B7H4 antigen.

[0143] The present invention relates to a binding agent which target IL1 RAP. In some embodiments the present invention relates to a binding agent which targets at least two antigens, wherein said antigens are IL1 RAP and B7H4. Bispecific refers to a binding agent which binds to two different antigens, i.e. IL1 RAP and B7H4. The bispecific molecule is a heterodimeric molecule that is capable of, e.g. binds both IL1 RAP and B7H4 antigen. Binding is to target epitopes on IL1 RAP and B7H4 antigen respectively. The binding agent is capable of binding to the two antigens expressed on the same cell.

[0144] Unless stated otherwise herein, binding is to human IL1 RAP. In some embodiments binds is to human IL1 RAP and human B7H4 antigen.

[0145] The binding agent of the invention is capable of targeting cells expressing IL1 RAP+ B7H4+, wherein said cells express IL1 RAP at a low level. The binding agent of the invention is also capable of targeting cells expressing IL1 RAP+ B7H4+, wherein said cells express IL1 RAP at a high level.

[0146] The level of expression of a cell surface receptor such as IL1 RAP or B7H4 may be determined using methods known to the skilled person. The level of expression may refer to the number of receptors present. The number of receptors present on a cell can be calculated by determining the specific antigen binding capacity of a cell. In an embodiment, the level of expression refers to the number of receptors present on a cell as determined by specific antigen binding capacity of a cell. The binding agent may target cells expressing IL1 RAP+ B7H4+, wherein said cells express IL1 RAP at a high level. A high level of expression of IL1 RAP may refer to the number of receptors present on a cell as determined by specific antigen binding capacity of a cell. A high level of expression of IL1 RAP may refer to a specific antigen binding capacity, provided in Antibody-Binding Capacity (ABC) units, of a cell in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to

[0147] 45,000, 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to 45,000, 26,000 to

[0148] 45,000, 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to 45,000, 31 ,000 to

[0149] 45,000, 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to 45,000, 15,000 to

[0150] 40,000, 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to

[0151] 40,000, 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to 40,000, 25,000 to

[0152] 40,000, 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to 40,000, 30,000 to

[0153] 40,000, 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to 40,000, 35,000 to

[0154] 40,000.

[0155] A high level of expression of IL1 RAP may refer to a number of receptors in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000, 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to

[0156] 45,000, 26,000 to 45,000, 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to

[0157] 45,000, 31 ,000 to 45,000, 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to

[0158] 45,000, 15,000 to 40,000, 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to

[0159] 40,000, 20,000 to 40,000, 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to

[0160] 40,000, 25,000 to 40,000, 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to

[0161] 40,000, 30,000 to 40,000, 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to

[0162] 40,000, 35,000 to 40,000.

[0163] The binding agent may target cells expressing IL1 RAP+ B7H4+, wherein said cells express IL1 RAP+ at a low level. A low level of expression of IL1 RAP+ may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody-Binding Capacity (ABC) units, of said cell. A low level of expression of IL1 RAP+may refer to a specific antigen binding capacity of a cell in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 11 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0164] A low level of expression of IL1 RAP+ may refer to a number of receptors in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 1 1 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 1 1 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0165] The binding agent may target cells expressing IL1 RAP+ B7H4+, wherein said cells express B7H4+ at a high level. A high level of expression of B7H4+ may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody- Binding Capacity (ABC) units, of a cell. A high level of expression of B7H4+ may refer to a specific antigen binding capacity of a cell in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000,

[0166] 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to 45,000, 26,000 to 45,000,

[0167] 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to 45,000, 31 ,000 to 45,000,

[0168] 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to 45,000, 15,000 to 40,000,

[0169] 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to 40,000,

[0170] 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to 40,000, 25,000 to 40,000,

[0171] 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to 40,000, 30,000 to 40,000,

[0172] 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to 40,000, 35,000 to 40,000.

[0173] A high level of expression of B7H4+ may refer to a number of receptors in the range of 15,000 to 45,000, 16,000 to 45,000, 17,000 to 45,000, 18,000 to 45,000, 19,000 to 45,000, 20,000 to 45,000, 21 ,000 to 45,000, 22,000 to 45,000, 23,000 to 45,000, 24,000 to 45,000, 25,000 to

[0174] 45,000, 26,000 to 45,000, 27,000 to 45,000, 28,000 to 45,000, 29,000 to 45,000, 30,000 to

[0175] 45,000, 31 ,000 to 45,000, 32,000 to 45000, 33,000 to 45,000, 34,000 to 45,000, 35,000 to

[0176] 45,000, 15,000 to 40,000, 16,000 to 40,000, 17,000 to 40,000, 18,000 to 40,000, 19,000 to 40,000, 20,000 to 40,000, 21 ,000 to 40,000, 22,000 to 40,000, 23,000 to 40,000, 24,000 to

[0177] 40,000, 25,000 to 40,000, 26,000 to 40,000, 27,000 to 40,000, 28,000 to 40,000, 29,000 to

[0178] 40,000, 30,000 to 40,000, 31 ,000 to 40,000, 32,000 to 40,000, 33,000 to 40,000, 34,000 to

[0179] 40,000, 35,000 to 40,000.

[0180] The binding agent may target cells expressing IL1 RAP+ B7H4+, and wherein B7H4 is expressed at a low level. A low level of expression of B7H4+ may refer to the number of receptors present on a cell as determined by specific antigen binding capacity, provided in Antibody-Binding Capacity (ABC) units, of a cell. A low level of expression of B7H4+ may refer to a specific antigen binding capacity of a cell in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 11 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0181] A low level of expression of B7H4+ may refers to a number of receptors in the range of 1 ,000 to 15,000, 1 ,000 to 14,000, 1 ,000 to 13,000, 1 ,000 to 12,000, 1 ,000 to 11 ,000, 1 ,000 to 10,000, 1 ,000 to 9,000, 1 ,000 to 8,000, 1 ,000 to 7,000, 1 ,000 to 6,000, 1 ,000 to 5,000, 2,000 to 15,000, 2,000 to 14,000, 2,000 to 13,000, 2,000 to 12,000, 2,000 to 1 1 ,000, 2,000 to 10,000, 2,000 to 9,000, 2,000 to 8,000, 2,000 to 7,000, 2,000 to 6,000, 2,000 to 5,000, 3,000 to 15,000, 3,000 to 14,000, 3,000 to 13,000, 3,000 to 12,000, 3,000 to 11 ,000, 3,000 to 10,000, 3,000 to 9,000, 3,000 to 8,000, 3,000 to 7,000, 3,000 to 6,000, 3,000 to 5,000, 4,000 to 15,000, 4,000 to 14,000, 4,000 to 13,000, 4,000 to 12,000, 4,000 to 11 ,000, 4,000 to 10,000, 4,000 to 9,000, 4,000 to 8,000, 4,000 to 7,000, 4,000 to 6,000, 4,000 to 5,000.

[0182] The binding agent comprises an antibody, antigen binding fragment or antigen binding portion thereof. In some embodiments, the binding agent comprises an antibody, antigen binding fragment or antigen binding portion thereof, wherein the antibody or antigen binding fragment or portion thereof targets at least two antigens, wherein said antigens are IL1 RAP and B7H4. The binding agent comprises a first antibody or antigen binding fragment or portion thereof, and a second antibody or antigen binding fragment or portion thereof wherein the first antibody or antigen binding fragment or portion thereof targets IL1 RAP, and the second antibody or antigen binding fragment or portion thereof targets B7H4. In one embodiment, the binding agents may comprise further antigen binding domains, for example the binding agents may be multispecific e.g., trispecific or tetraspecific. The further antigen binding domain may be provided as an antibody or antigen binding fragment or portion thereof.

[0183] The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g. an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g. fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g. free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[0184] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antigen-binding fragment thereof specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes.

[0185] Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antigen-binding fragment thereof (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides are tested for reactivity with a given antibody or antigen-binding fragment thereof. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. Suitable competition assays are mentioned elsewhere herein and also shown in the examples. In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, mutagenesis mapping (e.g, site-directed mutagenesis mapping), and / or in silico modelling.

[0186] A binding agent of the invention described herein, "which binds" or is “capable of binding” IL1 RAP, is one that binds the antigen IL1 RAP with sufficient affinity such that the antibody or antigen binding portion thereof is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen IL1 RAP as described herein. A binding agent of the invention described herein, "which binds" or is “capable of binding” both IL1 RAP and B7H4, is one that binds both of the antigens IL1 RAP and B7H4 with sufficient affinity such that the antibody or antigen binding portion thereof is useful as a therapeutic agent in targeting a cell or tissue expressing both of the antigens IL1 RAP and B7H4 as described herein.

[0187] The binding agents may target IL1 RAP and B7H4 with different affinity, e.g. some binding agents of the invention may target IL1 RAP with a higher affinity than B7H4, some binding agents of the invention may target IL1 RAP with a lower affinity than B7H4. Binding agents of the invention may target IL1 RAP and B7H4 with approximately the same affinity. The binding agents are capable of selectively targeting cells expressing both IL1 RAP and B7H4, compared to cells expressing IL1 RAP or cells expressing B7H4.

[0188] As the inventors have identified that tumour cells express both IL1 RAP and B7H4, the agents of the invention can be used in treating cancer.

[0189] Therefore, the binding agent can be used to specifically target dual expressing IL1 RAP + B7H4+ malignant cells. Dual expressing IL1 RAP + B7H4+ cells refers to cells which express both IL1 RAP and B7H4. The cells may also express further cell surface markers. In certain embodiments, the bispecific binding agents specifically bind to IL1 RAP and B7H4 antigens that are cell surface expressed. As used herein, the expression “cell surface-expressed” means one or more IL1 RAP and / or B7H4 protein(s) that is / are expressed on the surface of a cell in vitro or in vivo, such that at least a portion of a IL1 RAP and / or a B7H4 protein is exposed to the extracellular side of the cell membrane and is accessible to the bispecific antibody of antigen binding fragments thereof of the invention.

[0190] “Specifically binds", "specific binding" or "selective binding" means that the binding is selective for the antigen, i.e. IL1 RAP and B7H4, and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g., surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays. The binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.

[0191] In an embodiment the binding agents of the invention also encompass an antibody or antigen binding fragment thereof which competes for binding to human IL1 RAP with one or more of the molecules described above for the first antigen binding region or the antigen binding region that binds IL1 RAP. In an embodiment the binding agents of the invention also encompass an antibody or antigen binding fragment thereof which competes for binding to human B7H4 with one or more of the molecules described above for the second antigen binding region.

[0192] The term "antibody" as used herein refers to an immunoglobulin (Ig) protein that is capable of binding an antigen. In particular, the term "antibody" as used herein broadly refers to any polypeptide comprising complementarity determining regions (CDRs) that confer specific binding affinity of the polypeptide for an antigen. The term antibody as used herein encompasses polyclonal and monoclonal antibody preparations.

[0193] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain. The term “antibody” should also be construed as covering antibody mimetics, such as, but not limited to, cyclic peptides, for example bicyclic peptides, cysteine knots and anticalins etc. The antibody may be any immunoglobulin (Ig) molecule, or antigen binding portion / fragment thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such fragments are known in the art for example F(ab')2, Fab, Fab’, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Therefore, an antibody fragment comprises an antigen binding portion. The present invention extends to such antibody fragments. In one embodiment, the antibody fragment is a Fab. In one embodiment, the binding agent comprises a first antigen-binding region that binds IL1 RAP and a second antigen binding region that binds human B7H4 wherein the antigen-binding region that binds IL1 RAP is a Fab antibody fragment and the antigen-binding region that binds B7H4 is a Fab antibody fragment. Thus, in one embodiment, the binding agent comprises a BiFab.

[0194] The term "monoclonal antibody" refers to an antibody obtained from a single close of cells or cell line. The individual antibodies are identical and / or bind the same epitope. Unlike polyclonal antibodies, which include different antibodies directed against different epitopes, each monoclonal antibody of in a preparation is directed against a single epitope.

[0195] The terms “polypeptide(s)” and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art.

[0196] The binding agents of the present invention may comprise a bispecific antibody, or a bispecific antigen binding fragment thereof. The bispecific antibody or antigen binding fragment may comprise two antigen binding domains wherein the antigen binding domains comprise the same format or wherein the antigen binding domains comprise different formats. An antigen binding fragment may only comprise one antigen binding domain, as such a bispecific antigen binding fragment may comprise two antigen binding fragments linked in any suitable manner.

[0197] A full-length antibody comprises two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable region or domain and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0198] The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

[0199] Immunoglobulin molecules can be subdivided into various types class and subclass. In humans the classes (isotype) of immunoglobulin include IgG, IgM, IgA, IgE, and IgD. The immunoglobulin classes are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as y-chains; IgM have p-chains; IgA have a-chains; IgE have e-chains; and IgD have b-chains. The antibody or antigen binding fragment may be IgG type.

[0200] There are two types of light chain that can be present these are kappa(K) and lambda (A). The antibodies or antigen binding fragments of the invention may comprise either K light chain or A light chain. There are fourtypes of heavy chain the a (alpha) (IgA), y (gamma) (lgG1 , lgG2, lgG3, lgG4), 6 (delta) (IgD), £ (epsilon) (IgE) and p (mu) (IgM) heavy chains, or their equivalents in other species (for example rodent, canine etc). Full-length immunoglobulin “light chains” (usually of about 25 kDa or 214 amino acids long) consist of a variable region of approximately 1 10 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin “heavy chains” (usually of about 50 kDa or 446 amino acids long), likewise consist of a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids). Light or heavy chain variable regions are generally composed of a “framework” region (FR) interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and CDRs have been precisely defined. The sequences of the framework regions of different light and heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0201] Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3. Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.

[0202] The antibody may be comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0203] The term "CDR" (complementarity-determining region) refers to the immunoglobulin hypervariable domains within an antibody or antigen binding fragment sequences. It is generally accepted that the CDRs determine the specific antibody binding. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[0204] The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and was originally derived from observations from sequence alignments of light chain A, K and heavy chain sequences for a number of antibodies and for the a, p, y, 6 chain sequences for a number of TCRs. Kabat numbering is the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

[0205] The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one CDR capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo. Methods of producing polyclonal and monoclonal antibodies are known in the art.

[0206] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which generally 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 regions. 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.

[0207] The antibody or antigen-binding fragment thereof may be chimeric, human or humanized. A “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody. Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody therapies, as such molecules would elicit little or no immune response in the human subject. The antibody or antigen-binding fragment thereof is a humanized antibody or antigen-binding fragment thereof. The first and / or second binding domains of the bispecific molecule may be humanized.

[0208] A “humanized antibody” or fragment thereof is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent, canine, feline antibody, are transferred from the heavy and light variable chains of the rodent, canine or feline antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (e.g., rodent) antibody may be substituted into the human antibody framework region sequences. In an embodiment the CDRs disclosed herein may be transferred from the heavy and light variable chains of the antibody disclosed herein into the heavy and light variable domains (e.g., framework region sequences) of a different species. The antibody or antigen-binding fragment may comprise a monoclonal antibody or antigenbinding fragment thereof.

[0209] The binding agents of the present invention may include antibodies or antigen binding fragments thereof. The binding agents of the invention may comprise a first and second antigen binding region, the first antigen binding region may comprise an antibody or an antigen binding fragment thereof, the second antigen binding region may comprise an antibody or an antigen binding fragment thereof. The antigen-binding fragments may be selected from any fragment capable of binding the antigen or antigenic fragment of interest. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, F(ab')3, Fabc, Fd, single chain Fv (scFv), (scFv)2, Fv, scFv- Fc, heavy chain only antibody, diabody, tetrabody, triabody, minibody, antibody mimetic protein, single domain antibody, e.g. a VH. Thus, the antigen-binding fragment may comprise or consist of any of these fragments. The term antigen binding domain as used herein refers to a polypeptide capable of binding an antigen. The term antigen binding region as used herein refers to a polypeptide capable of binding an antigen.

[0210] In one embodiment, the antigen-binding region that binds IL1 RAP is a Fab fragment and the antigen binding region that binds human B7H4 is a Fab fragment. Thus, in one embodiment the binding agent comprises a BiFab. The Fab fragments are linked with a linker. This format is illustrated in the figures.

[0211] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and / or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[0212] The antibody or antigen-binding fragment may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody may be the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g., the CH2 domain of human lgG1 . The sequences of human IgG domains are known in the art.

[0213] The antibody or antigen-binding fragment 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. The hinge region, or part thereof, may be a human IgG 1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, may be an IgG 1 hinge region, or part thereof.

[0214] The sequence of the CH3 domain is not particularly limited. The CH3 domain may be a human immunoglobulin G domain, such as a human lgG1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[0215] The antibody or antigen-binding fragment may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human lgG1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. The antibody or antigen-binding fragment may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0216] The antigen binding fragment according to the present invention include functional fragments of an antibody. A functional fragment of an antibody is a fragment which still retains the ability to bind to a target antigen, as such the functional fragment comprises an antigen binding domain. Examples of functional fragments of an antibody include Fab, Fab’, F(ab’)2, scFv, (scFv), heavy chain only antibody, single domain antibody, nanobody, these terms are known in the art and describe specific formats of antigen binding fragments.

[0217] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and / or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[0218] Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

[0219] The smallest antigen-binding fragment is the single variable fragment, namely the variable heavy (VH) or variable light (VL) chain domain. VH and VL domains respectively are capable of binding to an antigen. They are generally referred to as a “single domain antibody” or “immunoglobulin single variable domain”. A single domain antibody (~12 to 15 kDa) has thus either the VH or VL domain. Antigen-binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341 : 544-546). Ward et al. named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb" or “sdAb” (for single domain antibody) generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. For use in therapy, human single domain antibodies are preferred over camelid derived VHH, primarily because they are not as likely to provoke an immune response when administered to a patient.

[0220] A "Fab molecule" (fragment antigen binding) as such a Fab domain refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin. In certain embodiments the Fab light chain and Fab heavy chain in the Fab construct are linked by a polypeptide sequence to yield a single chain Fab (scFab).

[0221] A “Fab” or “Fab fragment” comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains. For example, a Fab contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen binding. A Fab’ comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains and the thiol group which forms the disulphide bridge between two heavy chains in a full-length antibody. As such Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region. BiFab molecule comprise two Fab fragments as shown in the figures.

[0222] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties, e.g., antigen binding polypeptide construct, is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single- chain Fab molecule. Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linkerto create a single chain Fv (scFv). In certain other embodiments, one of the antigen binding moieties is a single-chain Fv molecule (scFv).

[0223] Single chain fragment variable (scFv) antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In one embodiment the scFv is a fusion protein comprising the heavy chain variable region and the light chain variable region linked via a peptide linker.

[0224] The antigen binding fragment may comprise a Fab, Fab’, F(ab’)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the antigen binding fragment comprises a Fab. In an embodiment the antigen binding fragment comprises a Fab’. In an embodiment the antigen binding fragment comprises an scFv. In an embodiment the bispecific antibody or antigen binding fragment, comprises a first and second antigen binding domain, wherein said first and / or second antigen binding domain comprises a Fab, Fab’, F(ab’)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the first antigen binding domain comprises a Fab, Fab’, F(ab’)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the second antigen binding domain comprises a Fab, Fab’, F(ab’)2, scFv, (scFv), heavy chain only antibody, single domain antibody, or nanobody. In an embodiment the first and second antigen binding domain comprises a Fab or an scFv. In an embodiment the first antigen binding domain comprises a Fab or a scFv and the second antigen binding domain comprises a Fab or an scFv. In an embodiment the first and second antigen binding domain comprises a Fab or a Fab’. In an embodiment the first and second antigen binding domain comprises a Fab. In an embodiment the first and second antigen binding domain comprises or consists of a Fab. In an embodiment the first and second antigen binding domain comprises or consists of a Fab. In an embodiment the first antigen binding domain comprises or consists of a Fab. In an embodiment the second antigen binding domain comprises or consists of a Fab. In an embodiment the first and second antigen binding domain each comprise or consist of a Fab. In an embodiment the first and second antigen binding domain comprises an scFv.

[0225] The binding agent, bispecific binding agent, antibody or antigen binding fragment thereof may comprise an Fc domain. The term "Fc" or "Fc domain" or "Fc region" or "Fc construct" herein is used to define a C-terminal region of an immunoglobulin heavy chain. The term includes native sequence Fc regions and variant Fc regions.

[0226] "Fc region", as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences.

[0227] The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "Fc polypeptide" herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence. The antibody may comprise a CH2 domain. The CH2 domain is for example 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 in one embodiment the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g the CH2 domain of human lgG1. The sequences of human IgG domains are known in the art.

[0228] Thus, binding agent, bispecific binding agent, antibody or antigen binding fragment thereof 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. In one embodiment, the hinge region, or part thereof, is a human IgG 1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, is an lgG1 hinge region, or part thereof.

[0229] The first and / or second antibody or antigen-binding fragment may comprise an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and / or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and / or a single domain antibody and ii) an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and / or a single domain antibody and ii) an Fc domain. The Fc region may be any suitable Fc region for example a human Fc region.

[0230] Thus, binding agents, antibody or antigen binding fragment thereof may comprise a CH3 domain. The sequence of the CH3 domain is not particularly limited. In one embodiment, the CH3 domain is a human immunoglobulin G domain, such as a human IgG 1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[0231] Thus, the binding agents, antibody or antigen binding fragment thereof may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human IgG 1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. A binding agent, antibody or antigen binding fragment of the invention may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0232] The Fc includes two Fc polypeptides each having a CH3 domain for dimerization. The N- terminal end of each Fc polypeptide is linked to the C-terminus of one of the antigen binding polypeptide constructs with or without a linker. An Fc region can mediate downstream effector functions via interaction with Fc-receptors found on immune cells or with C1 q, the recognition molecule of the complement system. The antibody or antigen-binding fragment may comprise an Fc region may be capable of interacting with an Fc receptor. The bispecific molecule may comprise an Fc region may be capable of interacting with an Fc receptor and eliciting a downstream effector function. Effector functions refer to downstream immune effector mechanisms such as but not limited to antibody-dependent cytotoxicity (ADCC), antibody dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), antibody-dependent intracellular neutralization and / or immunomodulatory functions.

[0233] 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 FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRlla, FcyRllb and FcyRllc) and CD16 (FcyRllla and FcyRlllb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRIII are low to intermediate affinity (micromolar range KD).

[0234] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell- mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g. Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell.

[0235] "Complement dependent cytotoxicity" and "CDC" refer to the lysing of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (Clq) to a molecule (e.g. an antibody) complexed with a cognate antigen.

[0236] "Antibody-dependent cellular phagocytosis and "ADCP" refer to the destruction of target cells via monocyte or macrophage-mediated phagocytosis.

[0237] The binding agents may comprise a human IgG Fc with or without effector function.

[0238] In antibody ADCC, FcyRs 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 various for IgG subtypes. Although this is dependent on the allotype and specific FcyRs in simple terms ADCC effector function is high for human IgG 1 and lgG3, and low for lgG2 and lgG4.

[0239] FcyRs 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.

[0240] The binding agent, antibody or antigen binding fragment thereof of the invention may have an Fc region with effector function, with enhanced effector function or with reduced effector function. In particular, the binding agent, antibody or antigen binding fragment thereof may include a modified Fc region.

[0241] The agent may comprise a modified Fc. The modified Fc may be modified in terms of amino acid sequence compared to a wild-type Fc region.

[0242] The Fc region may be modified to modulate the effect of the Fc region. For example the Fc region may be modified to enhance effector function. The bispecific molecule may comprise an Fcwith enhanced effector function. The Fc region may be modified to reduce or abolish effector function.

[0243] The Fc region may be modified to improve certain properties, e.g. to reduce complement mediated effector function, to reduce FcyR- mediated effector functions, to improve half-life. The Fc region may be modified to enhance or reduce interaction of the Fc with one or more of the Fc receptors e.g. FcRn, C1 q, TRIM21 , FcyRI, FcyRlla / b, FcyRllla).

[0244] The potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such ADCC and ADCP. 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, FcyRs 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 FcyRllla.

[0245] Activation of ADCC 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, lgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, lgG4 molecules can exchange half molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous lgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of lgG4 antibodies with a reduced propensity for Fabarm exchange. Fc engineering approaches have been used to determine the key interaction sites for the lgG1 Fc domain with Fey receptors and C1 q 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 Fey receptors, such as FcyRI, FcyRlla, FcyRllb, FcyRIII and / or to complement. CH2 domains of human IgG domains normally bind to Fey receptors and complement, decreased binding to Fey receptors is expected to decrease ADCC and decreased binding to complement is expected to decrease CDC activity of the antibody molecule.

[0246] Mutations to decrease or abrogate binding of the CH2 domain to one or more Fey 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 / P331 S (“TM”), which almost completely abolish FcyR and C1 q 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.

[0247] 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.

[0248] The Fc region may be modified to improve the half-life of the bispecific molecule. The Fc region may be modified to enhance interaction with FcRn receptor and thereby improve half-life of the bispecific molecule. 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, 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. The Fc region may be modified to reduce interaction with FcyR receptor and thereby improve half-life of the bispecific molecule. An example of a modification that may be made to improve half-life is introduction of M252Y / S254T / T256E mutations in lgG1 CH2 domain, this modification is commonly referred to as a “YTE” variant.

[0249] The bispecific binding agent, antibody or antigen binding fragment thereof may comprise an Fc which does not elicit downstream effector function, i.e., where its effector function has been knocked out. In such embodiments, the half-life of the compound may be increased. Methods to modify or modulate the Fc effector function are known in the art and may be achieved via site directed mutagenesis of the Fc region. The modulated Fc refers to a modulated activity compared to that of the wild-type Fc.

[0250] In one embodiment, the Fc is an IgG 1 Fc construct, and lgG2 Fc construct, an lgG3 Fc construct, or an lgG4 Fc construct. In some embodiments, at least one CH3 domain has at least one amino acid modification that promotes the formation of a heterodimeric Fc with stability comparable to a wild-type homodimeric Fc. Exemplary modifications are described below.

[0251] Production of bispecific antibodies of the IgG type by co-expression of the two light and two heavy chains in a single host cell can be highly challenging because of the low yield of desired bispecific IgGs and the difficulty in removing closely related mispaired IgG contaminants. This reflects that heavy chains form homodimers as well as the desired heterodimers - the so-called heavy chain-pairing problem. Additionally, light chains can mispair with non-cognate heavy chains - the so-called light chain pairing problem. Consequently, coexpression of two antibodies can give rise to up to nine unwanted IgG species in addition to the desired bispecific antibody. Various approaches are described in the art in order to promote heterodimerisation, i.e. the formation of a certain bispecific antibody of interest for human therapy, thereby reducing the content of undesired homodimers in the resulting mixture. These approaches have been studied in relation to human or humanised antibodies designed to target disease in humans.

[0252] The homodimerisation of the two heavy chains in an IgG is mediated by the non-covalent interaction between the CH3 domains alone. Thus, CH3-CH3 interaction is the primary driver for Fc dimerisation.

[0253] It is furthermore well-known in the art that when two CH3 domains interact with each other they meet in a protein-protein interface which comprises “contact” residues (also called contact amino acids, interface residues or interface amino acids). Contact amino acids of a first CH3 domain interact with one or more contact amino acids of a second CH3 domain. Contact amino acids are typically within 5.5 A (preferably within 4.5 A) of each other in the three-dimensional structure of an antibody. The interaction between contact residues from one CH3 domain and contact residues from a different CH3 domain may for instance be via Van der Waals forces, hydrogen bonds, water-mediated hydrogen bonds, salt bridges or other electrostatic forces, attractive interactions between aromatic side chains, disulfide bonds, or other forces known to one skilled in the art. The primary drive is thus hydrophobic interaction in the core and electrostatic interactions.

[0254] Approaches to interfere with the dimerisation of antibody heavy chains have been employed in the art to bias production of heterodimeric antibodies. Specific engineering in the CH3 domains was applied in order to favour heterodimerisation over homodimerisation. Examples of such engineering of the CH3-CH3 interface include the introduction of complementary protuberance and cavity mutations, also known as ‘knob-into-hole’ approaches as described for instance in WO9627011 incorporated by reference and J.B. Ridgway et al 'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerisation Protein Eng., 9 (1996), pp. 617-621.

[0255] Generally, the method involves introducing a protuberance at the interface of a first polypeptide and a corresponding cavity in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heteromultimer formation and hinder homomultimer formation. “Protuberances” or “knobs” are constructed by replacing small amino acid side chains from the interface of the first polypeptide with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” or “holes” of identical or similar size to the protuberances are created in the interface of the second polypeptide by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). The protuberance and cavity can be made by synthetic means such as altering the nucleic acid encoding the polypeptides or by peptide synthesis. Starting from a “knob” mutation (T366W) (Ridgway et al., supra) that disfavors CH3 homodimerisation, compensating “hole” mutations (T366S, L368A, and Y407V) (Atwell et al Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library J. Mol. Biol., 270, pp. 26-35, 1997) were identified by phage display providing efficient pairing with the “knob” while disfavoring homodimerisation.

[0256] Several other successful strategies for heavy chain heterodimerisation, including electrostatic steering mutations (W02006 / 106905 and Gunasekaran et al Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG J. Biol. Chem., 285, pp. 19637-19646, 2010). This approach is based on electrostatic engineering of contact residues within the CH3-CH3 interface that are naturally charged. Mutations are introduced in the CH3 domains of heavy chains wherein naturally occurring charged amino acid contact residues are replaced by amino acid residues of opposite charge (i.e. a charge reversal strategy). This creates an altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.

[0257] It has been described that within the human CH3-CH3 interface four unique charges residue pairs are involved in the domain-domain interaction. These are D356 / K439', E357 / K370', K392 / D399' and D399 / K409' (numbering according to Kabat, 1991) where residues in the first chain are separated from residues in the second chain by 7’ and where the prime (') indicates the residue numbering in the second chain). As the CH3-CH3 interface displays a 2-fold symmetry, each unique charge pair is represented twice in intact IgG (i.e., also K439 / D356', K370 / E357', D399 / K392' and K409 / D399' charge interactions are present in the interface). Taking advantage of this two-fold symmetry, it was demonstrated that a single charge reversion, e.g. K409D in the first chain, or D399'K in the second chain resulted in diminished homodimer formation due to repulsion of identical charges. Combining different charge reversions further enhanced this repulsive effect. It was demonstrated that expression of different CH3 domains comprising different, complementary charge reversions, could drive heterodimerisation, resulting in an increased proportion of the bispecific species in the mixture (for review see Kontermann and Brinkmann, supra; Brinkmann and Kontermann: The making of bispecific antibodies, MABS, Vol. 9, No. 2, 2017, pages 182-212, Ha et al, Frontiers in Immunology Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins, Vol 7, article 394, 2016).

[0258] In some embodiments, the dimeric Fc is a heterodimer formed with a purity greater than about 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% when produced; or wherein the Fc is a heterodimer formed with a purity greater than about 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% when expressed or when expressed via a single cell.

[0259] The Fc region may comprise one or more modifications in at least one of the CH3 sequences. The Fc region may comprise one or more modifications in at least one of the CH2 sequences. The Fc region may comprise one or more modifications in the CH2 and in the CH3 sequences.

[0260] In some embodiments, a construct described herein comprises a heterodimeric Fc region comprising a modified CH3 domain that has been asymmetrically modified. The heterodimeric Fc region can comprise two heavy chain constant domain polypeptides: a first heavy chain polypeptide and a second heavy chain polypeptide, which can be used interchangeably provided that Fc comprises one first heavy chain polypeptide and one second heavy chain polypeptide. Generally, the first heavy chain polypeptide comprises a first CH3 sequence and the second heavy chain polypeptide comprises a second CH3 sequence.

[0261] Two CH3 sequences that comprise one or more amino acid modifications introduced in an asymmetric fashion generally results in a heterodimeric Fc region, rather than a homodimer, when the two CH3 sequences dimerize. As used herein, "asymmetric amino acid modifications" refers to any modification where an amino acid at a specific position on a first CH3 sequence is different from the amino acid on a second CH3 sequence at the same position, and the first and second CH3 sequence preferentially pair to form a heterodimer, rather than a homodimer. This heterodimerization can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence; or modification of both amino acids on each sequence at the same respective position on each of the first and second CH3 sequences. The first and second CH3 sequence of a heterodimeric Fc region can comprise one or more than one asymmetric amino acid modification.

[0262] Another suitable method uses a platform based on BEAT® technology (Bispecific Engagement by Antibodies based on the T cell receptor) (Moretti et al, BMC Proc 7, Article Number 09 (2013)).

[0263] Additional methods for modifying monomeric Fc polypeptides to promote heterodimeric Fc formation are as described above. Therefore, the Fc region as the bispecific molecule, e.g. antibody as described herein may comprise one or more modification to promote heterodimerisation, e.g. a knob in hole (KiH) modification.

[0264] The binding agent may comprise an additional moiety. The additional moiety may provide further function to the molecule. For example, the further moiety may be selected from a half-life extending moiety and / or a label.

[0265] In some embodiments, the binding agent may comprise a half-life extension moiety. The inclusion of a half-life extension moiety suitably increases the half-life of the bispecific molecule when compared to the same bispecific molecule that does not include a half-life extension moiety.

[0266] The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and / or clearance or sequestration of the sequence or compound by natural mechanisms. Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding antibodies without such modification. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding antibodies without such modification. The in vivo half-life of the antibody or antigen-binding fragment or compound of the invention a can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Half-life can for example be expressed using parameters such as the t1 / 2-alpha t1 / 2-beta and the area underthe curve (AUC). It will be appreciated by a person skilled in the art that reference to the half-life of an antibody may also refer to the half-life of the compounds of the invention (and may be used interchangeably herein).

[0267] The additional moiety may be selected from one or more half-life extending moieties for example, one or more PEG molecules, a liposome, a serum albumin protein an antibody or antibody fragment that binds serum albumin. The serum albumin may be human serum albumin. The binding agent may be conjugated or linked to the additional moiety in any suitable manner.

[0268] The half-life extension moiety may comprise an antibody and / or antigen-binding fragment, a protein, and or a polypeptide. For example, the half-life extension moiety may comprise a serum albumin protein or an antibody or antigen-binding fragment that binds human serum albumin. For example, the half-life extension moiety may comprise an antibody or antigen-binding fragment that binds human serum albumin, For example an antigen-binding fragment that binds human serum albumin, For example a single domain antibody that binds human serum albumin, such as a VH domain that that binds human serum albumin.

[0269] In some embodiments, the binding agent may comprise one or more further antibodies and / or antigen-binding fragments. For example, the compound may comprise a total of 3, 4, 5, etc., antibodies and / or antigen-binding fragments. In an embodiment the compound may comprise a total of 3 antibodies and / or antigen-binding fragments. The further antibodies and / or antigenbinding fragments may be any suitable antibodies and / or antigen-binding fragments. For example, the further antibodies and / or antigen-binding fragments may bind to human serum albumin or may bind to any other suitable antigen. It will be appreciated by the skilled person that the further antibodies and / or antigen-binding fragments may be attached to the binding agent in any suitable manner for example recombinantly attached or chemically linked.

[0270] For example, production of the multispecific binding agents of the invention may comprise recombinant expression, wherein a nucleic acid is designed which encodes the first and / or second antigen binding domain in combination with the further antibody, antigen binding fragment or domain and the protein is expressed in a host organism such that the antigen binding domains are expressed in a linked manner. Production of the multispecific binding agent of the invention may comprise preparing one or more of the antibodies, antigen binding fragments or antigen binding domains in a manner such that they can be chemically linked, for example by the click chemistry methods described herein. Production of the multispecific binding agent may comprise a mixture of recombinantly expressing some of the binding domains in a linked manner and chemically linking some of the binding domains.

[0271] It will be appreciated by a person skilled in the art that in certain embodiments a further antibody or antigen-binding fragment may also be a half-life extension moiety and vice versa. For example, where the further antibody or antigen-binding fragment is a serum albumin binding protein, the further antibody or antigen binding fragment would also be a half-life extension moiety.

[0272] The additional moiety may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).

[0273] The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).

[0274] The one or more further moieties may be attached to the compound at any suitable position and by any suitable means. For example, when the bispecific molecule comprises a further antibody and / or antigen-binding fragment, protein and / or polypeptide, said antibody and / or antigenbinding fragment, protein and / or polypeptide may be recombinantly attached to the binding agent. Suitable recombinant techniques will be known to a person skilled in the art. For example, nucleic acids encoding the bispecific molecule, and the further antibody and / or antigen-binding fragment, protein and / or polypeptide, suitably separated by a polypeptide spacer / linker sequence, may be inserted into a plasmid and expressed in a suitable expression system. Suitable expression systems include bacterial host cells, such as E. coli, CHO or other host cells expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding the bispecific molecule linked to the additional moiety that is operably linked to a T7 promoter. For example, a bacterial host cell, such as an E. coli, may include a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the bispecific molecule linked to the additional moiety is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). Transformation can be by any known method for introducing polynucleotides into a host cell. Suitable transformation methods are as already described herein.

[0275] As described herein, the binding agent comprises a first and second antibody, antigen binding fragment, or antigen binding domain, in such embodiment the first and second may be attached or linker together. The first and second antibody, antigen binding fragment, or antigen binding domain may be attached via a linker. The linker may be formed when the first and second antibody, antigen binding fragment, or antigen binding domain are conjugated together to form a bispecific molecule. The first and second antibody, antigen binding fragment, or antigen binding domain may be conjugated together via methods known in the art.

[0276] For example, in order to conjugate the first and second antibody, antigen binding fragment, or antigen binding domain together a thiol containing group may be introduced into the first and or second antibody, antigen binding fragment, or antigen binding domain In order to conjugate the first and second antibody, antigen binding fragment, or antigen binding domain together cysteine residues may be introduced into the first and or second antibody, antigen binding fragment, or antigen binding domain. These cysteine residues may be referred to herein a non-naturally occurring cysteine residues i.e., the cysteine residue has been recombinantly introduced, for example the wild-type residue has been replaced with a cysteine using site directed mutagenesis. A “non-naturally occurring cysteine residue” refers to a cysteine residue that has been introduced at a position in the polypeptide wherein a different amino acid was present in the wild-type sequence. Methods for introducing cysteine resides are known in the art, for example site directed mutagenesis.

[0277] The linker may be any suitable linker.

[0278] For example, the linker may comprise a one or more polyethylene glycol (PEG) groups. The linker may comprise a polycyclic group. By “polycyclic”, and like terms as used herein, is meant a group comprising a plurality of ring moieties wherein at least two of said ring moieties share at least one atom, preferably at least two atoms, in common.

[0279] The terms “conjugation” and “conjugate(d)” refer to chemical linkages, either covalent or non- covalent, which proximally associates one molecule of interest with a second molecule of interest.

[0280] The binding agent is capable of binding both IL1 RAP and B7H4, upon binding to the target antigens the bispecific molecule may induce receptor mediate internalisation. In one embodiment, the bispecific molecule is capable of inducing IL1 RAP and / or B7H4 receptor mediated internalization into a IL1 RAP+ and / or B7H4cell. In one embodiment, the bispecific molecule is capable of inducing IL1 RAP receptor mediated internalisation into a IL1 RAP+ cell.

[0281] In one embodiment, the bispecific molecule is capable of inducing B7H4 receptor mediated internalization into a B7H4+ cell. The bispecific molecule is capable of inducing IL1 RAP receptor mediated internalization into a IL1 RAP+ B7H4+ cell. In one embodiment, the bispecific molecule is capable of inducing B7H4 receptor mediated internalization into a IL1 RAP+ B7H4+ cell.

[0282] The inventors show herein that the binding agents are capable of selectively targeting B7H4+ IL1 RAP dual positive cells, providing excellent cell specificity. The bispecific molecule is capable of selectively targeting cells expressing B7H4+ and IL1 RAP+ compared to cells expressing B7H4+ or cells expressing IL1 RAP+.

[0283] Therefore, the binding agent has reduced off-target effects which may occur as a result of targeting non-tumour single positive cells i.e., cells expressing B7H4+ or cells expressing IL1 RAP.

[0284] Dual positive cells expressing both IL1 RAP+B7H4+ may be found in malignant cells. The binding agent is capable of bispecifically binding to a cell expressing IL1 RAP+ and B7H4+ wherein the cell is a malignant cell.

[0285] Immunoconjugates

[0286] In one embodiment, the binding agent of the invention may be conjugated to a payload. A “payload” may be selected from but not limited to a cell killing drug / moiety / agent, an immune- modulating payload, a macrophage class switching agent, a detectable label or a light activatable payload or other suitable payload. Thus, the binding agent of the invention conjugated to a payload can also be described as a cell inhibiting agent.

[0287] In certain embodiments where the binding agent is conjugated to a payload this is referred to as an immunoconjugate or antibody drug conjugate (ADC). Thus, the binding agent may be provided as an immunoconjugate or ADC.

[0288] As shown in the examples, binding agents of the invention conjugated to a payload selectively kill IL1 RAP+B7H4+ cells.

[0289] Therefore, binding agents of the invention provide an advantage over combination therapies as the binding agent has reduced off-target effects which may occur as a result of targeting nontumour single positive cells i.e., cells expressing B7H4+ or cells expressing IL1 RAP.

[0290] In one embodiment, the payload may be a cell killing agent. In one embodiment, the payload may be a macrophage class switching agent. In one embodiment, the payload may be an immune-modulating payload. In one embodiment, the payload may be a light activatable payload. In one embodiment, the payload may be a molecular label.

[0291] Examples of suitable payloads are known to the skilled person and are described in the art, for example Metrangolo et al, Cancers 2024, 16, 447 and Wang et al, Acta Pharmaceutica Sinica B 2023;13(10):4025-4059 incorporated herein by reference.

[0292] By “molecular label”, and like terms as used herein, is meant a group that is operable to aid the detection of the compound. Detection of the compound may be ex vivo and / or in vivo. Examples of suitable molecular labels include, but are not limited to, fluorescent molecules, 0- galactosidase, luciferase molecules, chemical dyes, fluorophores and / or radioisotopes.

[0293] The molecular label may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800). The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA-pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).

[0294] In one embodiment, the payload may be an immune-modulating payload. As used herein, an immune-modulating payload includes any moiety that modulates the immune system, for example which stimulates the immune system and / or kills the target cell. Thus, a moiety that has immuno-activating and / or antineoplastic activities can be used. Such moieties may be synthetic peptides that recognise the specific target and trigger (agonist) or block (antagonist) inflammatory responses. The target may be a pattern recognition receptor (PRR), including Tolllike receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C-type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs).

[0295] Examples of payloads include agonists for the stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173). Such payloads include cyclic dinucleotides and compounds listed in see WO2021113679 incorporated herein by reference). Activation of the STING pathway triggers an immune response that results in generation of specific killer T-cells that shrink tumours and can provide long-lasting immunity so the tumours do not recur. Examples of STING agonists include XMT-2056, TAK-500. Alternatively, payloads that act on toll-like receptors (TLRs) may be used. For example, agonists that bind to TLR7 and / or TLR8 can be used. Examples of TLRs include NJH395, BDC-1001 , SBT6050. Another example is a macrophage class switching agent.

[0296] The payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the payload may be a cytotoxin I cytotoxic moiety.

[0297] In one example, the cytotoxin I cytotoxic moiety is a biologically active cytotoxic material. The cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues, topoisomerase inhibitors and doxorubicin. RNA polymerase II inhibitors such as HDP-101.

[0298] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0299] In some embodiments the payload may be a cytotoxic or cytostatic agent, i.e., a compound that kills or inhibits tumour cells. Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and / or topoisomerase inhibition. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., <211 >At, <131 >l, <125>1 , <90>Y, <186>Re, <188>Re, <153>Sm, <212>Bi, <32>P, <60>C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.

[0300] A "chemotherapeutic agent" and "anticancer agent" are terms that denote a chemical compound useful in the treatment of cancer, and which may be payloads or may be administered in combination therapy with the antibody drug conjugate compounds of the invention. Examples of chemotherapeutic agents include Erlotinib (TARCEVA(R), Genentech / OSI Pharm.), Bortezomib (VELCADE(R), Millenium Pharm.), Fulvestrant (FASLODEX(R), Astrazeneca), Sutent (SUI 1248, Pfizer), Letrozole (FEMARA(R), Novartis), Imatinib mesylate (GLEEVEC(R), Novartis), PTK787 / ZK 222584 (Novartis), Oxaliplatin (Eloxatin(R), Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE(R), Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA(R), Astrazeneca), AG1478, AG1571 (SU 5271 ; Sugen), alkylating agents such as thiotepa and CYTOXAN(R) cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA(TM)); acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL(R)); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN(R)), CPT-II (irinotecan, CAMPTOSAR(R)), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl, 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1 , epirubicin, GPX- 100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN(R) doxorubicin (including morpholino- doxorubicin, cyanomo[phi]holino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti- neoplastic agents such as ALEMTA(R), LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX<1 >A, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISENE(R), FILDESIN (R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL(R) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANE(TM) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE(R) doxetaxel (Rh[delta]ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR(R)); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN(R)); etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(R)); vinca alkaloid; vinorelbine (NAVELBINE(R)); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; topoisomerase inhibitor Dxd, difluorometlhylornithine (DMFO); topoisomerase I inhibitors U3-1402, Ds-7300a, Ds-6157a, Ds- 1062a, IMMU-130, AZD8205, SKB264, PRO1160, PRO1184; topoisomerase II inhibitors SGN- 15, SOT102; retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATESf(TM)) combined with 5-FU and leucovorin.

[0301] Also encompassed by the term “cytotoxic agent” are anti-hormonal agents that act to regulate or inhibit hormone action on tumours such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX(R) tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYI 17018, onapristone, and FARESTON(R) toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE(R) megestrol acetate, AROMAS I N(R) exemestane, formestanie, fadrozole, RIVISOR(R) vorozole, FEMARA(R) letrozole, and ARHVIIDEX(R) anastrozole; and anti-androgens such as fiutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN(R) vaccine, LEUVECTIN(R) vaccine, and VAXID(R) vaccine; PROLEUKIN(R) rlL-2; LURTOTECAN(R) topoisomerase 1 inhibitor; ABARELIX(R) rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0302] The cytotoxic or cytostatic agent may be, for example, a peptide toxin, a small molecule toxin or a radioisotope. This is also referred to herein as drug or cytotoxic payload.

[0303] In one embodiment the cytotoxic or cytostatic agent may be a tubulin inhibitor; or a DNA interacting agent. Tubulin inhibitors modulate tubulin polymerisation. DNA interacting agents target cellular DNA.

[0304] In one embodiment the cytotoxic or cytostatic agent may be a geldanamycin derivative or psymberin.

[0305] In one embodiment the payload may be a proteosome inhibitor.

[0306] In one embodiment the payload may be a protein degrader, for example a PROTAC. PROTACs are heterobifunctional molecules that contain three components: the protein-of-interest (POI) binding moiety, a linker, and E3 ubiquitin ligase binding moiety. Examples of PROTACS may include ORM-5029. In one embodiment, the ADC is a dual payload ADC.

[0307] In an embodiment the cytotoxic or cytostatic agent is a tubulin inhibitor. In an embodiment, the tubulin inhibitor is selected from the group comprising of: (a) an auristatin; and (b) a maytansine derivative. In an embodiment, the cytotoxic or cytostatic agent is an auristatin. Auristatins include synthetic derivatives of the naturally occurring compound Dolastatin-10. Auristatins are a family of antineoplastic I cytostatic pseudopeptides. Dolastatins are structurally unique due to the incorporation of 4 unusual amino acids (Dolavaine, Dolaisoleuine, Dolaproine and Dolaphenine) identified in the natural biosynthetic product. In addition, this class of natural product has numerous asymmetric centres defined by total synthesis studies by Pettit et al (US 4,978,744). It would appear from structure activity relationships that the Dolaisoleuine and Dolaproine residues appear necessary for antineoplastic activity (US 5,635,483 and US 5,780,588). In an embodiment, the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF. In an embodiment, the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine. In an embodiment, the cytotoxic or cytostatic agent is a maytansine. Maytansines include structurally complex antimitotic polypeptides. Maytansines are potent inhibitors of microtubulin assembly which leads towards apoptosis of tumour cells. In an embodiment the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4. Preferably, the drug is MMAE, MMAF or auristatin MMAF. In an embodiment the cytotoxic or cytostatic agent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN- 38.

[0308] In an embodiment, the cytotoxic or cytostatic agent is DNA interacting agent. In an embodiment, the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs). In an embodiment, the cytotoxic or cytostatic agent is a calicheamicin. Calicheamicin is a potent cytotoxic agent that causes doublestrand DNA breaks, resulting in cell death. Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,1 1 , 2103-2117). Calicheamicin was found in the soil microorganism Micromonosporaechinospora. In an embodiment, the calicheamicin is calicheamicin gamma 1 . In an embodiment, the drug is a duocarmycin. Duocarmycins are potent anti-tumour antibiotics that exert their biological effects through binding sequence-selectively in the minor groove of DNA duplex and alkylating the N3 of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, 837-844). In an embodiment, the duocarmycin is selected from the group consisting of: Duocarmycin A; Duocarmycin B1 ; Duocarmycin B2; Duocarmycin C1 ; Duocarmycin C2; Duocarmycin D; Duocarmycin SA; Cyclopropylbenzoindole (CBI) duocarmycin; Centanamycin; Rachelmycin (CC-1065); Adozelesin; Bizelesin; and Carzelesin. In an embodiment, the cytotoxic or cytostatic agent is a pyrrolobenzodiazepine. Pyrrolobenzodiazepines (PBDs) are a class of naturally occurring anti-tumour antibiotics. Pyrrolobenzodiazepines are found in Streptomyces. PBDs exert their anti-tumour activity by covalently binding to the DNA in the minor groove specifically at purine-guanine-purine units. They insert on to the N2 of guanine via an aminal linkage and, due to their shape, they cause minimal disruption to the DNA helix. It is believed that the formation of the DNA-PBD adduct inhibits nucleic acid synthesis and causes excisiondependent single and double stranded breaks in the DNA helix. As synthetic derivatives the joining of two PBD units together via a flexible polymethylene tether allows the PBD dimers to cross-link opposing DNA strands producing highly lethal lesions. In an embodiment, the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether. In an embodiment, the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof); Mazethramycin (and dimers thereof); Tomaymycin (and dimers thereof); Prothracarcin (and dimers thereof); Chicamycin (and dimers thereof); Neothramycin A (and dimers thereof); Neothramycin B (and dimers thereof); DC-81 (and dimers thereof); Sibiromycin (and dimers thereof); Porothramycin A (and dimers thereof); Porothramycin B (and dimers thereof); Sibanomycin (and dimers thereof); Abbeymycin (and dimers thereof); SG3199; SG2000; and SG2285.

[0309] In an embodiment, the cytotoxic or cytostatic agent is a drug that targets DNA interstrand crosslinks through alkylation. A drug that targets DNA interstrand crosslinks through alkylation is selected from: a DNA targeted mustard; a guanine-specific alkylating agent; and a adeninespecific alkylating agent. In an embodiment, the cytotoxic or cytostatic agent is a DNA targeted mustard. For example, the DNA targeted mustard may be selected from the group consisting of: an oligopyrrole; an oligoimidazole; a Bis-(benzimidazole) carrier; a Polybenzamide Carrier; and a 9-Anilinoacridine-4-carboxamide carrier.

[0310] In an embodiment, the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.

[0311] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[0312] A guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions. In an embodiment, the cytotoxic or cytostatic agent is a guaninespecific alkylating agent selected from the group consisting of: a G-N2 alkylators; a A-N3 alkylator; a mitomycin; a carmethizole analogue; a ecteinascidin analogue. In an embodiment, the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW-2149. In an embodiment, the a carmethizole analogue is selected from: Bis-(Hydroxymethyl)pyrrolizidine; and NSC 602668. In an embodiment, the ecteinascidin analogue is Ecteinascidin 743. Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[0313] Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators. In an embodiment, the cytotoxic or cytostatic agent is a cyclopropaindolone analogue. Preferably, the drug is selected from: adozelesin; and carzelesin.

[0314] In an embodiment, the cytotoxic or cytostatic agent is a benz[e]indolone. Preferably, the cytotoxic or cytostatic agent is selected from: CBI-TMI; and iso-CBI.

[0315] In an embodiment, the cytotoxic or cytostatic agent is bizelesin. In an embodiment, the cytotoxic or cytostatic agent is a Marine Antitumour Drug. Marine Antitumour Drugs has been a developing field in the antitumour drug development arena (I. Bhatnagaret al, Mar. Drugs 2010, 8, P2702- 2720 and T. L. Simmons et al, Mol. Cancer Ther. 2005, 4(2), P333-342). Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents.

[0316] In an embodiment, the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate. In an embodiment, the EribulinMesylate is selected from: (E7389); Soblidotin (TZT 1027); Squalamine lactate; CemadotinPlinabulin (NPI-2358); Plitidepsin; Elisidepsin; Zalypsis; Tasidotin, Synthadotin; (ILX-651); Discodermolide; HT1286; LAF389; Kahalalide F; KRN7000; Bryostatin 1 ; Hemiasterlin (E7974); Marizomib; Salinosporamide A; NPI-0052); LY355703; CRYPTO 52; Depsipeptide (NSC630176); Ecteinascidin 743; Synthadotin; Kahalalide F; Squalamine; Dehydrodidemnin B; Didemnin B; Cemadotin; Soblidotin; E7389; NVP-LAQ824; Discodermolide; HTI-286; LAF-389; KRN-7000 (Agelasphin derivative); Curacin A; DMMC; Salinosporamide A; Laulimalide; Vitilevuamide; Diazonamide; Eleutherobin; Sarcodictyin; Peloruside A; Salicylihalimides A and B; Thiocoraline; Ascididemin; Variolins; Lamellarin D; Dictyodendrins; ES-285 (Spisulosine); and Halichondrin B.

[0317] The following cytotoxic or cytostatic agent are also encompassed by the present invention: Amatoxins (a-amanitin)- bicyclic octapeptides produced by basidiomycetes of the genus Amanita, e.g., the Green Deathcap mushroom; Tubulysins; Pseudomonas exotoxin; Cytolysins; dolabellanins; diphtheria toxin; Epothilone A, B, C, D, E, F. Epothilones - constitute a class of non-taxane tubulin polymerisation agents and are obtained by natural fermentation of the myxobacterium Sorangiumcellulosum. These moieties possess potent cytotoxic activity which is linked to the stabilisation of microtubules and results in mitotic arrest at the G2 / M transition. Epothilones have demonstrated potent cytotoxicity across a panel of cancer cell lines and has often exhibited greater potency than paclitaxel (Pivot et al, European Oncology, 2008;4(2), P42- 45). Pseudomonas exotoxin is an exotoxin produced by Pseudomonas aeruginosa which catalyzes the ADP-ribosylation and inactivation of EF2, which leads to protein synthesis inhibition and cell death. In an embodiment, the drug or payload is amatoxin. In an embodiment, the drug or payload is tubulysin. In an embodiment the drug or payload is Pseudomonas exotoxin. In an embodiment, the drug or payload is cytolysin. In an embodiment, the drug or payload is dolabellanin. In an embodiment, the drug or payload is epothilone.

[0318] The following cytotoxic or cytostatic agent are also encompassed by the present invention. In an embodiment, the drug is selected from: Doxorubicin; Epirubicin; Esorubicin; Detorubicin; Morpholino-doxorubicin; Methotrexate; Methopterin; Bleomycin; Dichloromethotrexate; 5- Fluorouracil; Cytosine-p-D-arabinofuranoside; Taxol; Anguidine; Melphalan; Vinblastine; Phomopsin A; Ribosome-inactivating proteins (RIPs); Daunorubicin; Vinca alkaloids; Idarubicin; Melphalan; Cis-platin; Ricin; Saporin; Anthracyclines; Indolino-benzodiazepines; 6- Mercaptopurine; Actinomycin; Leurosine; Leurosideine; Carminomycin; Aminopterin; Tallysomycin; Podophyllotoxin; Etoposide; Hairpin polyamides; Etoposide phosphate; Vinblastine; Vincristine; Vindesine; Taxotere retinoic acid; N8-acetyl spermidine; Camptothecin; Esperamicin; and Ene-diynes.

[0319] In one embodiment, the cell killing portion is a peptide toxin, for example an auristatin such as MMAE or MMAF. In one embodiment, the binding agent comprises a binding portion and a cell killing portion, wherein the binding portion is an anti-IL1 RAP anti-B7H4 bispecific antibody or binding portion thereof and wherein the cell killing portion is a peptide toxin, for example an auristatin such as Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF), vcMMAE, vcMMAF, mcMMAE and mcMMAF.

[0320] In certain embodiments, the binding agent comprises a binding portion that is conjugated to a payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload. Such conjugates may be prepared by in vitro methods known to one of ordinary skill in the art. Techniques for conjugating cytotoxic or cytostatic agent to proteins, and in particular to antibodies, are well-known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.)

[0321] In certain embodiments the payload comprises a small molecule inhibitor with an anti-cancer activity. For example the small molecule inhibitor may be a Bcl-XI inhibitor, Bcl2 inhibitor, Bcl-w inhibitor, Bcr-Abl inhibitor, EGFR inhibitor VEGFR2 inhibitor, RET inhibitor, PDGFR inhibitor, FLT-3 inhibitor, KIT inhibitor, CSF-1 inhibitor, HER2 inhibitor, LCK inhibitor, B-raf inhibitor, mTOR inhibitor, c-KIT inhibitor, FGFR inhibitor, VEGFR inhibitor, HGFR inhibitor, Jak1 inhibitor, Jak2 inhibitor, VEGFR1-3 inhibitor, Src inhibitor, c.MET inhibitor, PDGFR-0 inhibitor, MEK inhibitor, HSP90 inhibitor, MMP inhibitor, proteosome inhibitor, Akt inhibitor, NAMPT inhibitor. In certain embodiments the payload comprises a light-activatable payload, for example an infrared light activatable payload. Immunoconjugates comprising a near-infrared activatable payload enable binding molecule-mediated targeted delivery to achieve a high degree of tumour specificity, while using infrared light to activate the biophysical mechanism of the drug to accurately induce rapid death of cancer cells without harming the surrounding normal tissues. Suitable light-activatable payloads include IRDye700DX. In one embodiment a light activatable payload (IRDye® 700DX, IR700) may also be used. Light activation of the non-toxic payload results in the generation of singlet oxygen species that damage the cell membrane integrity, resulting in necrotic and immunogenic cell death of tumour cells, resulting in minimal damage to surrounding normal tissue.

[0322] The antibodies described and disclosed herein can also be used in ADEPT (Antibody- Directed Enzyme Prodrug Therapy) procedures by conjugating the antibody to a prodrugactivating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent), to an active anticancer drug. The enzyme component of the immunoconjugate useful for ADEPT is an enzyme capable of acting on a prodrug in such a way so as to covert it into its more active, cytotoxic form.

[0323] The binding agent, ADC or the immunoconjugate of the invention may have an IC50 in the range of 0.005 to 5 nM, 0.006 to 5 nM, 0.007 to 5 nM, 0.008 to 5 nM, 0.009 to 5 nM, 0.01 to 5 nM, 0.005 to 4.5 nM, 0.006 to 4.5 nM, 0.007 to 4.5 nM, 0.008 to 4.5 nM, 0.009 to 4.5 nM, 0.01 to 4.5 nM, 0.005 to 4 nM, 0.006 to 4 nM, 0.007 to 4 nM, 0.008 to 4 nM, 0.009 to 4 nM, 0.01 to 4 nM, 0.005 to 3.5 nM, 0.006 to 3.5 nM, 0.007 to 3.5 nM, 0.008 to 3.5 nM, 0.009 to 3.5 nM, 0.01 to 3.5 nM, 0.005 to 3 nM, 0.006 to 3 nM, 0.007 to 3 nM, 0.008 to 3 nM, 0.009 to 3 nM, 0.01 to 3 nM, 0.005 to 2.5 nM, 0.006 to 2.5 nM, 0.007 to 2.5 nM, 0.008 to 2.5 nM, 0.009 to 2.5 nM, 0.01 to 2.5 nM, 0.005 to 2 nM, 0.006 to 2 nM, 0.007 to 2 nM, 0.008 to 2 nM, 0.009 to 2 nM, 0.01 to 2 nM, 0.005 to 1 .5 nM, 0.006 to 1 .5 nM, 0.007 to 1 .5 nM, 0.008 to 1 .5 nM, 0.009 to 1 .5 nM, 0.01 to 1 .5 nM, or 0.02 to 1 .2 nM

[0324] Techniques for conjugating cytotoxic or cytostatic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.). In certain embodiments, a linking group is used to conjugate the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload, to the first and / or second antibody or antigen-binding fragment, as appropriate. The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment. The cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A-expressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).

[0325] In certain embodiments, a linking group is used to conjugate the binding portion and the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

[0326] The payload can be linked to an antibody by a linker. Attachment of a linker to a mAb can be accomplished in a variety of ways, such as through surface lysines, reductive- coupling to oxidized carbohydrates, and through cysteine residues liberated by reducing interchain disulfide linkages. A variety of linkage systems are known in the art, including hydrazone-, disulfide- and peptide-based linkages.

[0327] Suitable linkers include, for example, cleavable and non-cleavable linkers. A cleavable linker is typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, a peptide linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. In exemplary embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (mc-Val-Cit- PABA) linker. Another linker is Sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l- carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while its Sulfo-NHS ester is reactive toward primary amines (as found in Lysine and the protein or peptide N-terminus). Yet another linker is maleimidocaproyl (me). Other suitable linkers include linkers hydrolyzable at a specific pH or a pH range, such as a hydrazone linker. Additional suitable cleavable linkers include disulfide linkers. The linker may be covalently bound to the antibody to such an extent that the antibody must be degraded intracellularly in order for the drug to be released e.g. the me linker and the like.

[0328] Thus, a linker can include a group for linkage to the antibody or antibody fragment. For example, a linker can include an amino, hydroxyl, carboxyl or sulfhydryl reactive groups (e.g., malemide, haloacetamides (e.g., iodo, bromo or chloro), haloesters (e.g., iodo, bromo or chloro), halomethyl ketones (e.g., iodo, bromo or chloro), benzylic halides (e.g., iodide, bromide or chloride), vinyl sulfone and pyridylthio). See generally Wong, Chemistry of Protein.

[0329] The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment. The cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A- expressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).

[0330] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH- sensitive linker is hydrolysable under acidic conditions. For example, an acid- labile linker that is hydrolysable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.

[0331] Other linkers are cleavable under reducing conditions (e.g., a disulfide linker). The cleavable linker can also be a malonate linker (Johnson et al, Anticancer Res. 15 :1387-93, 1995), a maleimidobenzoyl linker (Lau et al, Bioorg-Med-Chem. 3: 1299-1304, 1995), or a 3' -N-amide analogue (Lau et al, Bioorg-Med-Chem. 3: 1305-12, 1995).

[0332] In some embodiments the linker can be a protease cleavable linker, for example a valinecitrulline, which may be cleaved by cathepsin B in the lysosome.

[0333] The linker also can be a non-cleavable linker, such as a maleimidoca-proyl (me) linker or maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the binding portion.

[0334] The terms “conjugation” and “conjugate(d)” refer to chemical linkages, either covalent or non- covalent, which proximally associates one molecule of interest with a second molecule of interest. The drug-to- antibody ratio (DAR) can be, e.g., 1 .0 to 6.0, e.g. 4 or 6.

[0335] The conjugate may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group or an electrophilic group of an antibody with a bivalent linker reagent, to form antibodylinker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody. Conjugation methods (1) and (2) may be employed with a variety of antibodies, drug moieties, and linkers to prepare the antibodydrug conjugates described here. The bispecific agents of the present invention may be formed by any suitable method, for example a bispecific antibody / antibody frgament may be recombinantly expressed using standard techniques known in the art, or a bispecific molecule may be formed by linking the first and second antigen binding domain together. Methods of producing bispecific molecule are known in the art.

[0336] Several specific examples of methods of preparing bispecific antibodies ADCs are known in the art. Traditional methods such as the hybrid hybridoma and chemical conjugation methods can be used in the preparation of the bispecific antibodies of the invention. Co-expression in a host cell of two antibodies, consisting of different heavy and light chains, leads to a mixture of possible antibody products in addition to the desired bispecific antibody, which can then be isolated by, e.g., affinity chromatography or similar methods.

[0337] Strategies favoring the formation of a functional bispecific, product, upon co-expression of different antibody constructs can also be used. Strategies for promoting heterodimerization are known in the art. One strategy to promote formation of heterodimers over homodimers is a "knob-into-hole" strategy in which a protuberance is introduced on a first heavy-chain polypeptide and a corresponding cavity in a second heavy-chain polypeptide, such that the protuberance can be positioned in the cavity at the interface of these two heavy chains so as to promote heterodimer formation and hinder homodimer formation.

[0338] Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent.

[0339] Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.

[0340] Antibody-drug conjugates may also be produced by modification of the antibody to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g. with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g. by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated antibody with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug. In another embodiment, proteins containing N-terminal serine or threonine residues can react with sodium metaperiodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[0341] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[0342] Methods for conjugating the payload to the binding agent may utilise the presence of one or more non-naturally occurring amino acids i.e. a non-wild-type residue. The payload and / or linker may be attached to the binding agent via a non-naturally occurring amino acid residue present in the bispecific antibody or antigen binding fragment. Methods to introduce non-naturally occurring amino acid residues are known in the art.

[0343] The non-naturally occurring amino acid residue to which the payload and / or linker is attached is may be selected from but not limited to a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.

[0344] Where a canonical amino acid is used to attach the payload and / or linker, site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding agent. In an embodiment a non-canonical amino acid is used to attach the payload linker. A “non-canonical” amino acid refers to one of the non-proteinogenic (unnatural) amino acids i.e., an amino acid which is not introduced via the cell’s natural translation machinery. There are many examples of non-canonical amino acids in the art many of which provide a bio- orthogonal handle on which to attach a payload. Where a non-canonical amino acid is used to attach the payload linker, suitable techniques are known in the art to introduce such non- canonical amino acid residues such as chemical modification, tRNA suppressor technology, engineered tRNA / tRNA synthetase pairs.

[0345] The payload may be attached at various positions within the bispecific molecule. One or more copies of the payload may be attached to the molecule, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the payload may be attached. The position at which the payload is attached to the binding agent may directed by the position at which the non-naturally occurring amino acid is introduced or the position at which the electrophilic moiety is introduced. As such site-specific attachments of the payload can be achieved.

[0346] In another embodiment, the second moity is a label, for example a fluorescent molecule, 0- galactosidase, luciferase molecules, chemical dyes, fluorophores or a radioisotope.

[0347] Where the binding agents of the invention are provided as an immunoconjugate or an ADC they demonstrates low payload deconjugation e.g. <10%. The immunoconjugate or ADC may comprise a payload deconjugation of <15%, <14%, <13%, <12%, <11 %, <10%, <9%, <8%, <7%, <6%, <5%, <4%, <3%, <2%, when assessed over 5 days.

[0348] In one embodiment of the binding agent, the antigen-binding region that binds IL1 RAP is a Fab fragment and the antigen binding region that binds human B7H4 is a Fab fragment and the binding agent further comprises a payload. Thus, in one embodiment the binding agent comprises or consists of a BiFab conjugated to a payload. This format is illustrated in the figures.

[0349] Nucleic Acid, Vectors, Host Cells

[0350] An aspect of the invention relates to an isolated nucleic acid molecule encoding the binding agent of the invention. The isolated nucleic acid may be encoded in a construct for example a plasmid, vector, transcription or expression cassette. In an embodiment the nucleic acid is provided in a vector. Any suitable vector may be used.

[0351] The isolated nucleic acid or the construct comprising said nucleic acid may be provided in a host cell. The host cell may be a bacterial, viral, plant, mammalian or another suitable host cell.

[0352] The host cell comprising said vector or isolated nucleic acid may be used to produce the binding agent. There are several methods by which to produce recombinant antibodies which are known in the art. One of these is production in an E. coll expression system. In this embodiment, nucleic acids encoding the antibody or antigen-binding fragment thereof as described in previous aspects of the invention may be inserted into a plasmid and expressed in a suitable expression system. For example, the present invention includes methods for expressing a binding agent in a host cell (e.g., bacterial host cell such as E. coll, CHO, HEK or other host cell).

[0353] Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, inter alia, Chinese hamster ovary (CHO) cells, such as ExpiCHO cells, NSO, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK- 293 cells and a number of other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, bovine, horse and hamster cells. Other cell lines that may be used are insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells and fungal cells. Fungal cells include yeast and filamentous fungus cells including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa.

[0354] Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene- mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.

[0355] Engineered cells

[0356] In one embodiment, the binding agent comprises an immune effector cell. The immune effector cell may comprise a T cell and / or a NK cell.

[0357] Adoptive cellular therapy (ACT) has received much attention as a technique for cancer treatment. One therapeutic approach of ACT involves genetic engineering of T cells to express chimeric antigen receptors (CARs) on the surface of T cells to enable targeting of specific tumours. Once the CAR is expressed in T cells, the CAR modified T cell (CAR-T or CAR- T cell) acquires properties that include antigen-specific recognition, activation and proliferation and the cells thus act as “living drugs”. The purpose of expressing a CAR in a T cell is therefore to redirect immune reactivity of the cell to a chosen target. Furthermore, CARs with different strength and signalling can also modulate T cell expansion as well as alter the strength of T cell activation. CARs are synthetic receptors typically consisting of a targeting / binding moiety that is associated with one or more signaling domains in a single fusion molecule. CAR molecules are recombinant fusion proteins and are distinguished by their ability to both bind antigen and transduce activation signals via immunoreceptor activation motifs (ITAMs) present in their cytoplasmic tails in order to activate genetically modified immune effector cells for killing, proliferation, and cytokine production.

[0358] The binding moiety of the CAR of the invention comprises a first antigen-binding region that binds IL1 RAP and a second antigen binding region that binds human B7H4. The antigen binding portion, may for example be and antibody fragment, e.g. a single-chain antibody (scFv) comprising paired antibody light chain and heavy chain variable domains (VL and VH) that are fused into a single polypeptide chain via a short flexible linker. The scFv retains the same specificity and a similar affinity as the full antibody from which it was derived and is capable of binding to the specific target of interest. The term antibody fragment tis defined elsewhere and includes Fab fragment. In one embodiment, the first antigen-binding region that binds IL1 RAP is a Fab and the second antigen binding region that binds human B7H4 is a Fab.

[0359] In addition to an extracellular antigen-binding domain CARs also comprise a transmembrane domain and signaling molecules such as costimulatory endodomains and CD3 chain.

[0360] CARs combine antigen-specificity and T cell activating properties in a single fusion molecule. First generation CARs typically included the cytoplasmic region of the CD3zeta or Fc receptor y chain as their signalling domain. First generation CARs have been tested in phase I clinical studies in patients with ovarian cancer, renal cancer, lymphoma, and neuroblastoma, where they have induced modest responses (reviewed in Sadelain et al., Curr Opin Immunol, 21 (2): 215- 223, 2009). Second generation CARs, which contain the signalling domains of both CD28 and CD3zeta, provide dual signalling to direct combined activating and co-stimulatory signals. Third generation CARs are more complex with three or more signalling domains.

[0361] For example, cells such as T cells (e.g., CTLs), stem cells (e.g., induced pluripotent stem cells), or NK cells can be engineered to express one or more CARs having the ability to bind to an IL1 RAP polypeptide and a B7H4 polypeptide, thus being capable of targeting dual positive IL1 RAP+ B7H4+ cells.

[0362] Thus, the invention also provides an immune effector cell comprising a T cell and / or a NK cell comprising a first antigen-binding region that binds IL1 RAP and a second antigen binding region that binds human B7H4 as described herein. Such cells (e.g., IL1 RAP and B7H4-specific CAR-T cells or NK cells) can also be used to treat cancer.

[0363] In some embodiments, the immune effector cell is autologous to the subject. In another embodiment, the immune effector cell is not autologous to the subject.

[0364] Embodiments of the CARs described herein include nucleic acids encoding a bispecific antigenspecific CAR polypeptide comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen-binding domain. Optionally, a CAR can comprise a hinge domain positioned between the transmembrane domain and the antigen binding domain. A CAR may further comprise a signal peptide that directs expression of the CAR to the cell surface.

[0365] A chimeric antigen receptor can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. A nucleic acid sequence encoding the several regions of the chimeric antigen receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning (genomic library screening, PCR, primer- assisted ligation, libraries from yeast and bacteria, site-directed mutagenesis, etc.). The resulting coding region can be inserted into an expression vector and used to transform a suitable expression host allogeneic or autologous immune effector cells, such as a T cell.

[0366] The chimeric construct may be introduced into immune effector cells as naked DNA or in a suitable vector. Methods of stably transfecting cells by electroporation using naked DNA are known in the art. See, e.g., U.S. Pat. No. 6,410,319. Naked DNA generally refers to the DNA encoding a chimeric receptor contained in a plasmid expression vector in proper orientation for expression. Alternatively, a viral vector (e.g., a retroviral vector, adenoviral vector, adeno- associated viral vector, or lentiviral vector) can be used to introduce the chimeric construct into immune effector cells. Suitable vectors for use in accordance with the method of the present invention are non-replicating in the immune effector cells. A large number of vectors are known that are based on viruses, where the copy number of the virus maintained in the cell is low enough to maintain the viability of the cell, such as, for example, vectors based on HIV, SV40, EBV, HSV, or BPV.

[0367] A skilled person would also know that such elements of a CAR (other than antigen-specific targeting domain described herein) are well known in the art. Thus, the invention is not limited to specific domains of the CAR in addition to the antigen-specific targeting domain described herein. As mentioned above, the first generation CARs have been tested in various phase I clinical studies in patients with cancer. Second generation CARs and third generation CARs have also been described are more complex with three or more signalling domains (reviewed references 4 and 5 and in Sadelain et al., Curr Opin Immunol, 21 (2): 215-223, 2009, Sterner, R.C., Sterner, R.M. CAR-T cell therapy: current limitations and potential strategies. Blood Cancer J. 11 , 69, 2021). CARs are also described in US2004043401 , WO2019200007 and WO2021108613, all incorporated herein by reference.

[0368] For example, the CAR of the invention may comprise a molecule of the general formula: MSLN binding human VH sdAb- transmembrane domain- Intracellular signaling domain. Exemplary domains are listed below. As will also be apparent, the CAR may comprise additional domains as explained below.

[0369] In one embodiment, the CAR may comprise a mesothelin binding VH single domain antibody as described herein, an extracellular domain (which may comprise a “hinge” domain), a transmembrane domain, and an intracellular signaling domain.

[0370] The Intracellular (Cytoplasmic) Domain

[0371] The intracellular (cytoplasmic) domain of the CAR can provide activation of at least one of the normal effector functions of the immune cell. The CAR of the invention may thus further comprise an intracellular signaling domain. An "intracellular signaling domain", "cytoplasmic domain" or “endodomain” is the domain that transmits activation signals to T cells and directs the cell to perform its specialized function.

[0372] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CART cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines.

[0373] In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0374] The intracellular domain may comprise at least in part an activating domain, preferably comprised of a CD3 family member such as CD3 zeta, CD3 epsilon, CD3 gamma, or portions thereof. The antigen binding molecule, i.e. the mesothelin binding VH single domain antibody may be engineered such that it is located in the extracellular portion of the molecule / construct, such that it is capable of recognizing and binding to its target or targets.

[0375] Examples of domains that transduce the effector function signal and can be used according to the invention include but are not limited to the chain of the T-cell receptor complex or any of its homologs (e.g., q chain, FcsRIy and p chains, MB1 (Igalpha) chain, B29 (Igbeta) chain, human CD3zeta chain, CD3 gamma or other CD3 polypeptides (A, 6 and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5, 0X40 and CD28.

[0376] It will be appreciated that suitable intracellular molecules may also include but are not limited to, 4-1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function- associated antigen-1 (LFA-I, CDI-la / CDI8), CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-I, B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, IT GAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0377] In some embodiment, the cytoplasmic domain of the CAR can be designed to comprise the CD3 zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and a costimulatory signaling region. The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order.

[0378] The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein provided as GenBan Acc. No. BAG36664.1 , or the equivalent residues from a non- human species, e.g., mouse, rodent, monkey, ape and the like, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1.

[0379] The extracellular signaling domain

[0380] The CAR may also comprise an extracellular signaling domain. The extracellular domain is beneficial for signaling and for an efficient response of lymphocytes to an antigen. Extracellular domains may be derived from (i.e., comprise) CD28, CD28T, OX-40, 4-1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 , CDI-la / CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, IT GAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRAN CE / R ANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.

[0381] Hinge region

[0382] In one embodiment, the CAR of the invention further comprises a hinge or spacer region which connects the extracellular antigen binding domain and the transmembrane domain. In particular, extracellular domains often comprise a hinge portion. This hinge or spacer region can be used to achieve different lengths and flexibility of the resulting CAR. Examples of the hinge or spacer region that can be used according to the invention include, but are not limited to, Fc fragments of antibodies or fragments or derivatives thereof, hinge regions of antibodies, or fragments or derivatives thereof, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacer sequences, for example peptide sequences, or combinations thereof. Other hinge or spacer region will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In one embodiment, the hinge is an lgG4 hinge or a CD8A hinge, an immunoglobulin (Ig) sequence or other suitable molecule to achieve the desired special distance from the target cell. In some embodiments, the entire extracellular region comprises a hinge region. In some embodiments, the hinge region comprises CD28T, or the EC domain of CD28.

[0383] The transmembrane domain

[0384] The CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR.

[0385] A "transmembrane domain" (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane and is connected to the endoplasmic signaling domain and the antigen binding domain, in case of the latter optionally via a hinge. In one embodiment, the transmembrane domain of the CAR of the invention is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins), an artificial hydrophobic sequence or a combination thereof. In one embodiment, the transmembrane domain comprises the CD3zeta domain or CD28 transmembrane domain.

[0386] In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0387] Transmembrane regions of particular use in this invention may be derived from (i.e. comprise) CD28, CD28T, OX-40, 4- 1 BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-I, CDI-la / CDI8), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHO class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM- 1 , B7-H3, CDS, ICAM-I, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-I, ITGAM, CD1 lb, ITGAX, CD1 Ic, ITGB1 , CD29, ITGB2, CD 18, LFA-I, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1 , CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1 , CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG / Cbp, CDI9a, a ligand that specifically binds with CD83, or any combination thereof. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0388] Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0389] Pharmaceutical Composition

[0390] The binding agent, cell, ADC, antibody or antibody fragment of the invention may be provided as a pharmaceutical composition. The pharmaceutical composition may optionally comprise a pharmaceutically acceptable excipient, and / or adjuvant.

[0391] The pharmaceutical compositions described herein may comprise the binding agent. The pharmaceutical compositions described herein may comprise the immunoconjugate or the ADC comprising said binding agent. The pharmaceutical compositions described herein can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. In another embodiment, delivery is of the nucleic acid encoding the drug, e.g. a nucleic acid encoding the molecule of the invention is delivered.

[0392] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration. In an embodiment, the compositions are administered parenterally.

[0393] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a binding agent of the present invention is administered. Suitable diluents, adjuvants and excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the bispecific antibody or antigen binding portion thereof of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0394] The pharmaceutical composition can be in the form of a liquid, e.g., a solution, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneous.

[0395] When intended for oral administration, the composition can be in solid or liquid form, where semisolid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[0396] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[0397] When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavour enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Compositions can take the form of one or more dosage units.

[0398] In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

[0399] The amount of the binding agent or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0400] Therapy

[0401] The binding agents of the present invention are capable of targeting specific cell populations found within malignant cell populations and therefore have utility in therapy.

[0402] An aspect of the invention therefore relates to a binding agent, immunoconjugate, antibody drug conjugate, cell or antibody or antibody fragment thereof described herein for use in the treatment or prevention of a disease.

[0403] An aspect of the invention relates to a method of treating or preventing a disease comprising administering a therapeutically effective amount of a binding agent, immunoconjugate, antibody drug conjugate, cell or antibody or antibody fragment thereof described herein.

[0404] An aspect of the invention relates to the use of a binding agent, immunoconjugate, antibody drug conjugate, cell or antibody or antibody fragment thereof described herein for the manufacture of a medicament for the treatment or prevention of a disease.

[0405] In one embodiment, the disease is associated with cells expressing both IL1 RAP and B7H4.

[0406] The terms malignancy or disease as used herein includes any disease state comprising the uncontrolled growth and division of abnormal cells, for example cancer. The binding molecules of the invention are for use in the treatment of disease state comprising malignant cellular proliferation including but not limited to neoplasms and tumours. The terms "cancer", "cancerous" or “tumour” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.

[0407] In one embodiment, the cancer is a solid tumour.

[0408] In one embodiment, the solid tumour is selected from tumour is selected from prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain / CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head / neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, endometrial cancer or sarcomas.

[0409] In one embodiment, the cancer is selected from ovarian cancer, breast cancer, liver cancer, bile duct cancer, pancreatic cancer and lung cancer.

[0410] In one embodiment, the lung cancer is non-small cell lung cancer (NSCLC).

[0411] In one embodiment, the cancer is ovarian cancer. The ovarian cancer may be epithelial ovarian carcinoma, germ cell tumour (teratomas and dysgerminomas) or stromal cell tumour.

[0412] In some embodiments, the ovarian cancer is early stage (e.g., stage I or II).

[0413] In some embodiments, the ovarian cancer is advanced (e.g., stage III or IV).

[0414] In one embodiment, the ovarian cancer is ovarian adenocarcinoma.

[0415] In one embodiment, the ovarian cancer is HGSOC (high-grade serous ovarian cancer).

[0416] In one embodiment, the ovarian cancer is a recurrent, platinum- sensitive ovarian cancer.

[0417] In one embodiment, the disease is a haematologic disorder. In one embodiment, the haematologic disorder is selected from chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML).

[0418] The inventors have also surprisingly shown that bispecific binding agents of the invention comprising a payload have a greater cell killing effect of IL1 RAP+ B7H4+ cells compared to anti- IL1 RAP antibody or B7H4 antibody combined as a mixture or alone. Thus, in one embodiment, a binding agent of the invention comprising a payload has a greater cell killing effect of IL1 RAP+ B7H4+ cells compared to anti-IL1 RAP antibody or B7H4 antibody combined as a mixture or alone. In particular, the cell killing effect may be synergistic.

[0419] A dual targeting therapy using a bispecific binding agent described herein is defined as affording an “additive effect”, “synergistic effect” or a “synergistic treatment” if the effect is therapeutically superior, as measured by, for example, the extent of the response (e.g. apoptosis or cell viability), the response rate, the time to disease progression or the survival period, to that achievable on dosing one or other of the components of the dual targeting therapy at its conventional dose. For example, the effect of the dual targeting therapy is additive if the effect is therapeutically superior to the effect achievable with an antibody or antigen binding fragments thereof that specifically binds to IL1 RAP or B7H4 alone. For example, the effect of the bispecific treatment may be synergistic if the effect of the bispecific treatment supersedes the effect of the individual treatments added together and / or the mixture of the individual components.

[0420] Thus, the effect of the bispecific binding agent is beneficial (e.g. additive or synergistic) if a beneficial effect is obtained in a group of subjects that does not respond (or responds poorly) to an agent that specifically binds to IL1 RAP alone or an agent that specifically binds to B7H4 alone. In addition, the effect of the bispecific binding agent treatment is defined as affording a benefit (e.g. additive or synergistic effect) if one of the components is dosed at its conventional dose and the other component is dosed at a reduced dose and the therapeutic effect, as measured by, for example, the extent of the response, the response rate, the time to disease progression or the survival period, is equivalent to or better than that achievable on dosing conventional amounts of either one of the components that specifically binds to IL1 RAP alone or an agent that specifically binds to B7H4 alone and / or their combination. An agent that specifically binds to IL1 RAP may be selected from an antibody that binds to IL1 RAP or a fragment thereof and agent that specifically binds to B7H4 may be selected from an antibody that binds to IL1 RAP or a fragment thereof.

[0421] As used herein, "killing of a target cell" relates for example to an inhibition of protein synthesis, for example such that cell viability is reduced, or an induction of apoptosis resulting in elimination or death of target cells. Assays to determine cell killing and apoptosis are well known in the art. Cytotoxicity assays assess the number of live and dead cells in a population after treatment with a pharmacological substance (e.g. an LDH cytotoxicity assay, or a live-dead cell assay). Apoptosis assays assess how cells are dying by measuring markers that are activated upon cell death (e.g. a Phosphatidylserine (PS) expos ure / ann exin V binding assay, a caspase activation assay, a DNA fragmentation assay, a GSH / GSSG determination, a LDH cytotoxicity assay, a live-dead cell assay, or a non-caspase protease activation assay). As used herein "inhibit the cell growth” (e.g., referring to target cells) refers to any measurable decrease in the growth or proliferation of a target cell when contacted with the antibody or antigen binding fragments thereof according to the present invention as compared to the growth of the same cell not in contact with the antibody or antigen binding fragments thereof according to the present disclosure, e.g., the inhibition of growth of a cell by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. Assays to determine cell viability or proliferation are well known in the art. Cell viability assays assess how healthy the cells are by measuring markers of cellular activity (e.g. an ATP and ADP determination assay, a cell cycle assay, a cell proliferation assay, a cell viability assay, an LHD cytotoxicity assay, or a live-dead cell assay). Cell proliferation assays assess the growth rate of a cell population or to detect daughter cells in a growing population (e.g. a cell cycle assay, a cell proliferation assay, a cell viability assay, or a senescence assay).

[0422] Administration regimens

[0423] The skilled person will be able to determine appropriate dosage regimens of the binding agent or conjugates described herein. The amount of the binding agent, conjugate or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0424] As an example, the binding agent, conjugate or pharmaceutical composition may be provided at a dose of 0.1 mg / kg to 100mg / kg, 0.5 mg / kg to 100mg / kg, 1 mg / kg to 100mg / kg, 2 mg / kg to 100mg / kg, 5 mg / kg to l OOmg / kg, 10 mg / kg to 100mg / kg, 20 mg / kg to 100mg / kg, 0.1 mg / kg to 80mg / kg, 0.5 mg / kg to 80mg / kg, 1 mg / kg to 80mg / kg, 2 mg / kg to 80mg / kg, 5 mg / kg to 80mg / kg, 10 mg / kg to 80mg / kg, 20 mg / kg to 80mg / kg, 0.1 mg / kg to 60mg / kg, 0.5 mg / kg to 60mg / kg, 1 mg / kg to 60mg / kg, 2 mg / kg to 60mg / kg, 5 mg / kg to 60mg / kg, 10 mg / kg to 60mg / kg, 20 mg / kg to 60mg / kg, 0.1 mg / kg to 40mg / kg, 0.5 mg / kg to 40mg / kg, 1 mg / kg to 40mg / kg, 2 mg / kg to 40mg / kg, 5 mg / kg to 40mg / kg, 10 mg / kg to 40mg / kg, 20 mg / kg to 40mg / kg, 0.1 mg / kg to 30mg / kg, 0.5 mg / kg to 30mg / kg, 1 mg / kg to 30mg / kg, 2 mg / kg to 30mg / kg, 5 mg / kg to 30mg / kg, 10 mg / kg to 30mg / kg, 15 mg / kg to 30mg / kg, 20 mg / kg to 30mg / kg, 0.1 mg / kg to 20mg / kg, 0.5 mg / kg to 20mg / kg, 1 mg / kg to 20mg / kg, 2 mg / kg to 20mg / kg, 5 mg / kg to 20mg / kg, 10 mg / kg to 20mg / kg, or 15 mg / kg to 20mg / kg. As an example, the binding agent, conjugate or pharmaceutical composition may be administered with a specific treatment schedule. The treatment cycle may be 1 day to 42 days 1 day to 35 days, 1 day to 28 days, 1 day to 21 days, 1 day to 14 days, 1 day to 7 days. Within the treatment cycle the binding agent, conjugate or pharmaceutical composition may be administered to the subject. Th treatment cycle may comprise 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses. The treatment cycle may comprise 1 dose every 7 days, 2 doses every 7 days, 3 doses every 7 days. The treatment cycle may comprise 1 dose every 14 days, 2 doses every 14 days, 3 doses every 14 days, 4 doses every 14 days, 5 doses every 14 days, 6 doses every 14 days.

[0425] As will be appreciated by the skilled person, the terms “treating”, “treats” and “treatment” include both preventative and curative treatment of a condition, disease or disorder. These terms also include slowing, interrupting, controlling or stopping the progression of a condition, disease or disorder and preventing, curing, slowing, interrupting, controlling or stopping the symptoms of a condition, disease or disorder. As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disease. For example, treatment can include a postponement of development of the symptoms associated with a disease or disease, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., canine patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0426] The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline.

[0427] As used herein, a "patient" is typically a human who is undergoing treatment for, or has been diagnosed as having a malignancy associated with preferably a IL1 RAP+B7H4+ expression. In some embodiments, the binding agent is administered to a patient in remission from IL1 RAP+B7H4+ malignancy, whereby the recurrence of the haematological malignancy is prevented or delayed. In some embodiments, the patient lacks detectable cells of the haematological malignancy.

[0428] Wherein the binding agent, immunoconjugate or antibody drug conjugate is used in therapy it will be administered to the subject at a therapeutically effective dose. The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumour size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumour metastasis; inhibit, to some extent, tumour growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can, for example, be measured by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0429] The therapeutically effective amount of the binding agent or a pharmaceutical composition comprising the binding agent may be administered orally, topically, by inhalation, insufflation or parenterally. As the skilled person will appreciate the binding agent may be formulated, for example in a pharmaceutical composition as described herein. The binding agent may be formulated for a particular administration route. For example, formulations suitable for oral administration include tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs. Suitable formulations for topical use include, for example, creams, ointments, gels, or aqueous or oily solutions or suspensions. Suitable formulations for inhalation include, for example, as a fine powder or a liquid aerosol. Suitable formulations for administration by insufflation include, for example, a fine powder. Suitable formulations for parenteral administration include, for example, a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing.

[0430] As will be appreciated by the skilled person, the therapeutically effective amount of the binding agent or pharmaceutical composition as described herein, will necessarily vary depending on the subject to be treated, the route of administration and the nature and severity of the disease to be treated.

[0431] As used herein, the term “effective amount” means an amount of the binding agent or pharmaceutical composition of the invention, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration. The term “effective amount” of a composition, as used herein, is intended to denote a non-lethal but sufficient amount of the composition to provide the desired effect. For example, in order to elicit a favourable response in a subject when treating a disorder or infection, the effective amount is the one which eliminates or diminishes the symptoms associated with the disorder. An effective amount may be determined by one of ordinary skill in the art, using routine experimentation. The term “administering” as used herein refers to any action that results in exposing or contacting a composition containing the therapeutic monoclonal antibodies of the present invention in combination with the disclosed chemotherapy regimen. As used herein, administering may be conducted in vivo, in vitro, or ex vivo. Particularly, administering is to an ovarian cancer patient, for example a stage lll-IV ovarian cancer patient, and more specifically, stage lll-IV ovarian cancer patient that are BRCA-wild type that are platinum sensitive. For example, a composition may be administered by injection or through an endoscope. Administering also includes the direct application to cells of a composition according to the present invention. For example, during the course of surgery, tumour cells may be exposed. In accordance with an embodiment of the invention, these exposed cells (or tumours) may be exposed directly to a composition of the present invention, e.g., by washing or irrigating the surgical site and / or the cells, or by direct intra-tumoural injection of the binding agent.

[0432] In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease ordisorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

[0433] Typically, the amount is at least about 0.01 % of the binding agent or pharmaceutical composition of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the binding agent or pharmaceutical composition of the present invention by weight of the composition.

[0434] Compositions of the present invention can be prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the binding agent or pharmaceutical composition of the present invention.

[0435] For administration by injection, the binding agent or pharmaceutical composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, for example, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, for example about 1 mg / kg to about 10 mg / kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

[0436] Combination therapies The binding agent, immunoconjugate, antibody drug conjugate may be used in combination with any other anti-cancer therapy. A binding agent, immunoconjugate, antibody drug conjugate may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy, immunotherapy, surgery and / or radiation therapy. Such therapy or therapeutic agent includes for example an anticancer compound, such as a chemotherapy agent, biologic, cytokine, small molecule, immune cell, e.g. CAR-T therapy or radiotherapy treatment.

[0437] Chemotherapy agents include alkylating agents, plant alkaloids, antimetabolites, anthracyclines, topoisomerase inhibitors and corticosteroids. For example, the chemotherapy can include vinorelbine, cisplatin, carboplatin, gemcitabine, paclitaxel, topotecan, docetaxel, irinotecan, pemetrexed, etoposide, or any combination thereof.

[0438] A biologic may be an antibody therapy, for example an antibody that targets a checkpoint inhibitor, such as PD-1 (e.g. Pembrolizumab, Nivolumab or Cemiplimab), PD-L1 (e.g. Atezolizumab, Avelumab or Durvalumab), PD-L2, LAG-3 (e.g. Relatlimab), Tim-3 or CTLA4 (e.g. Ipilimumab).

[0439] The small molecule therapy may be tyrosine kinase inhibitor. In one embodiment, the small molecule inhibitor is selected from sorafenib, sunitinib, pazopanib, tivantinib, and erlotinib. In one embodiment, the small molecule inhibitor is selected from a PARP inhibitor.

[0440] Examples of chemotherapeutics for ovarian cancer include paclitaxel (e.g. albumin bound paclitaxel or nab-paclitaxel, trade name Abraxane®), altretamine (Hexalen®), capecitabine (Xeloda®), cyclophosphamide (Cytoxan®), etoposide (VP-16), gemcitabine (Gemzar®), ifosfamide (Ifex®), irinotecan (CPT-11 , Camptosar®), liposomal irinotecan (Onivyde®), liposomal doxorubicin (Doxil®), melphalan, pemetrexed (Alimta®), topotecan, and vinorelbine (Navelbine®); as well as combination regimens of chemotherapy including cisplatin + paclitaxel, TIP (paclitaxel / Taxol, ifosfamide, and cisplatin / Platinol), VelP (vinblastine, ifosfamide, and cisplatin / Platinol), VIP (etoposide / VP-16, ifosfamide, and cisplatin / Platinol), VAC (vincristine, dactinomycin, and cyclophosphamide), and PEB (cisplatin / Platinol, etoposide, and bleomycin). Examples of polyamine inhibitors that have been or are being explored in clinical trials for anticancer treatment include (but are not limited to) eflomithine (Vaniqa®) and AMXT-1501 dicaprate. Examples of hormone therapies for ovarian cancer include luteinizing-hormone- releasing hormone (LHRH) agonists (such as goserelin (Zoladex®) and leuprolide (Lupron®)), tamoxifen, and aromatase inhibitors (such as letrozole (Femara®), anastrozole (Arimidex®), and exemestane (Aromasin®)). Examples of targeted therapies for ovarian cancer include angiogenesis inhibitors such as bevacizumab (Avastin) as well as (poly(ADP)-ribose polymerase) (PARP) inhibitors such as Olaparib (Lynparza), rucaparib (Rubraca), and niraparib (Zejula).

[0441] Administration may be concurrently or sequentially.

[0442] In one embodiment, the subject has received prior treatment with an anti cancer therapy.

[0443] The binding agent, immunoconjugate, antibody drug conjugate of the invention may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to a binding agent, immunoconjugate, antibody drug conjugate of the combination such that they do not adversely affect each other.

[0444] Methods

[0445] The binding agents may also be used in various methods, for example in non-therapeutic methods.

[0446] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of detecting IL1 RAP+B7H4+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a binding agent as described herein.

[0447] The bispecific molecule may be used in a variety of diagnostic tests that comprise the detection of IL1 RAP+B7H4+ cells, for example immunoassays for the detection of cancer. The immunoassays per se are well-known and any of the well-known immunoassays may be employed. That is, classifying the known immunoassays according to the reaction type, known immunoassays include sandwich immunoassays, competition immunoassays, agglutination immunoassays, Western blot and the like. Classifying the known immunoassays according to the label employed, known immunoassays include fluorescence immunoassays, enzyme immunoassays, radio immunoassays, biotin immunoassays and the like. Any of these immunoassays may be employed. Further, diagnosis may be attained by immunohistostaining. In cases where a labelled antibody antigen-binding fragment thereof or binding agent is used in the immunoassay, the methods per se for labelling an antibody are well-known, and any of the well-known methods may be employed. 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.

[0448] The binding agent, for use in the method of detecting IL1 RAP+B7H4+ cells, may further comprise an additional moiety that allows identification of the IL1 RAP+B7H4+ cells. The binding agent may comprise a label, for example a fluorescent molecule, p-galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope. The method may the comprise a further step of detecting the fluorescent molecule, p-galactosidase, luciferase molecules, secondary antibody, chemical dyes, fluorophores or a radioisotope

[0449] An aspect of the invention relates to an in vitro, ex vivo or in vivo method of delivering a payload to IL1 RAP+B7H4+ cells within a biological sample, comprising obtaining a biological sample contacting said biological sample with a binding agent as described herein, wherein the bispecific molecule is conjugated to a payload.

[0450] The payload may be any payload as described herein, including but not limited to a cell killing agent, an immune-modulating payload, a macrophage class switching agent, a light activatable payload, or a detectable label.

[0451] An aspect of the invention relates to a method for reducing off target toxicity of a cancer treatment comprising administering to a subject a binding agent, an immunoconjugate or an antibody drug conjugate as described herein.

[0452] An aspect of the invention relates to a method for identifying a tumour that responds to therapy with a binding agent of the invention comprising analysing tumour cells for co-expression of IL1 RAP and B7H4.

[0453] The tumour cells may be obtained from a patient or a cell line.

[0454] The biological sample may be a biological tissue sample, a biological fluid. A biological fluid may be, for example, blood, serum, lymph, urine, inflammatory exudate, cerebrospinal fluid, amniotic fluid, a tissue extract or homogenate, and the like.

[0455] Kits

[0456] An aspect of the invention relates to a kit comprising the binding agent, cell, ADC, antibody or antibody fragment of the invention and optionally instructions for use. The kit may contain materials useful for the treatment of the disorders described above is provided. The kit comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds the binding agent, immunoconjugate or antibody drug conjugate of the invention which is effective for treating the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0457] The label or package insert indicates that the composition is used for treating the condition of choice, such as cancer. The kit may further contain a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0458] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present disclosure, including methods, as well as the best mode thereof, of making and using this disclosure, the following examples are provided to further enable those skilled in the art to practice this disclosure. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present disclosure will be apparent to those skilled in the art in view of the present disclosure.

[0459] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers, scientific publications and references to patent publications. Citation of publications and patent documents is not intended as an admission that any is pertinent prior art, nor does it constitute any admission as to the contents or date of the same.

[0460] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of’ or “consists essentially of’ means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like.

[0461] The term “consisting of’ or “consists of’ means including the components specified but excluding other components.

[0462] Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of’ or “consisting essentially of’, and also may also be taken to include the meaning “consists of’ or “consisting of’.

[0463] The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments.

[0464] It should be understood that while the use of words such as “preferable”, “preferably”, “preferred” or “more preferred” in the description suggest that a feature so described may be desirable, it may nevertheless not be necessary and embodiments lacking such a feature may be contemplated as within the scope of the invention as defined in the appended claims. In relation to the claims, it is intended that when words such as “a,” “an,” or “at least one,” are used to preface a feature there is no intention to limit the claim to only one such feature unless specifically stated to the contrary in the claim.

[0465] The invention is further illustrated in the following non-limiting examples.

[0466] Features of the use of the present invention are described in further detail in relation to the abovementioned aspects of the invention, in the examples below.

[0467] The described and illustrated examples are to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the scope of the inventions as defined in the claims are desired to be protected.

[0468] The invention is also described in the following aspects.

[0469] 1. A binding agent comprising a first antigen-binding region that binds human interleukin-1 receptor accessory protein (IL1 RAP) and a second antigen binding region that binds human B7H4, wherein the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 112 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 1 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 119 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences.

[0470] 2. The binding agent of embodiment 1 , wherein the first antigen binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 21 , and a VL CDR1 comprising SEQ ID NO: 24, a VL CDR2 comprising SEQ ID NO: 26, a VL CDR3 comprising SEQ ID NO: 28; b. a VH CDR1 comprising SEQ ID NO: 31 , a VH CDR2 comprising SEQ ID NO: 33, a VH CDR3 comprising SEQ ID NO: 35, and a VL CDR1 comprising SEQ ID NO: 38, a VL CDR2 comprising SEQ ID NO: 40, a VL CDR3 comprising SEQ ID NO: 42; c. a VH CDR1 comprising SEQ ID NO: 45, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 49, and a VL CDR1 comprising SEQ ID NO: 52, a VL CDR2 comprising SEQ ID NO: 54, a VL CDR3 comprising SEQ ID NO: 56; d. a VH CDR1 comprising SEQ ID NO: 59, a VH CDR2 comprising SEQ ID NO: 61 , a VH CDR3 comprising SEQ ID NO: 63, and a VL CDR1 comprising SEQ ID NO: 66, a VL CDR2 comprising SEQ ID NO: 68, a VL CDR3 comprising SEQ ID NO: 70; e. a VH CDR1 comprising SEQ ID NO: 73, a VH CDR2 comprising SEQ ID NO: 75, a VH CDR3 comprising SEQ ID NO: 77, and a VL CDR1 comprising SEQ ID NO: 80, a VL CDR2 comprising SEQ ID NO: 82, a VL CDR3 comprising SEQ ID NO: 84; f. a VH CDR1 comprising SEQ ID NO: 87, a VH CDR2 comprising SEQ ID NO: 89, a VH CDR3 comprising SEQ ID NO: 91 , and a VL CDR1 comprising SEQ ID NO: 94, a VL CDR2 comprising SEQ ID NO: 96, a VL CDR3 comprising SEQ ID NO: 98; g. a VH CDR1 comprising SEQ ID NO: 101 , a VH CDR2 comprising SEQ ID NO: 103, a VH CDR3 comprising SEQ ID NO: 105, and a VL CDR1 comprising SEQ ID NO: 108, a VL CDR2 comprising SEQ ID NO: 1 10, a VL CDR3 comprising SEQ ID NO: 112; or h. a VH CDR1 comprising SEQ ID NO: 115, a VH CDR2 comprising SEQ ID NO: 1 17, a VH CDR3 comprising SEQ ID NO: 119, and a VL CDR1 comprising SEQ ID NO: 122, a VL CDR2 comprising SEQ ID NO: 124, a VL CDR3 comprising SEQ ID NO: 126.

[0471] 3. The binding agent of embodiment 1 or 2 wherein the first antigen binding region comprises; a. a VH comprising SEQ ID NO: 15 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 22 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or b. a VH comprising SEQ ID NO: 29 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 36 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or c. a VH comprising SEQ ID NO: 43 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 50 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or d. a VH comprising SEQ ID NO: 57 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 64 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or e. a VH comprising SEQ ID NO: 71 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 78 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or f. a VH comprising SEQ ID NO: 85 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 92 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or g. a VH comprising SEQ ID NO: 99 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 106 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or h. a VH comprising SEQ ID NO: 113 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 120 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto. 4. The binding agent of any preceding embodiment where...

Claims

CLAIMS1 . A binding agent comprising a first antigen-binding region that binds human interleukin-1 receptor accessory protein (IL1 RAP) and a second antigen binding region that binds human B7H4, wherein the first antigen-binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 119 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequencecomprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; or h. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 1 12 or a sequence comprising 1 , 2, or 3 amino acid differences.

2. The binding agent of claim 1 , wherein the first antigen binding region comprises; a. a VH CDR1 comprising SEQ ID NO: 115, a VH CDR2 comprising SEQ ID NO: 1 17, a VH CDR3 comprising SEQ ID NO: 1 19, and a VL CDR1 comprising SEQ ID NO: 122, a VL CDR2 comprising SEQ ID NO: 124, a VL CDR3 comprising SEQ ID NO: 126; b. a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 21 , and a VL CDR1 comprising SEQ ID NO: 24, a VL CDR2 comprising SEQ ID NO: 26, a VL CDR3 comprising SEQ ID NO: 28; c. a VH CDR1 comprising SEQ ID NO: 31 , a VH CDR2 comprising SEQ ID NO: 33, a VH CDR3 comprising SEQ ID NO: 35, and a VL CDR1 comprising SEQ ID NO: 38, a VL CDR2 comprising SEQ ID NO: 40, a VL CDR3 comprising SEQ ID NO: 42; d. a VH CDR1 comprising SEQ ID NO: 45, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 49, and a VL CDR1 comprising SEQ ID NO: 52, a VL CDR2 comprising SEQ ID NO: 54, a VL CDR3 comprising SEQ ID NO: 56; e. a VH CDR1 comprising SEQ ID NO: 59, a VH CDR2 comprising SEQ ID NO: 61 , a VH CDR3 comprising SEQ ID NO: 63, and a VL CDR1 comprising SEQ ID NO: 66, a VL CDR2 comprising SEQ ID NO: 68, a VL CDR3 comprising SEQ ID NO: 70;f. a VH CDR1 comprising SEQ ID NO: 73, a VH CDR2 comprising SEQ ID NO: 75, a VH CDR3 comprising SEQ ID NO: 77, and a VL CDR1 comprising SEQ ID NO: 80, a VL CDR2 comprising SEQ ID NO: 82, a VL CDR3 comprising SEQ ID NO: 84; g. a VH CDR1 comprising SEQ ID NO: 87, a VH CDR2 comprising SEQ ID NO: 89, a VH CDR3 comprising SEQ ID NO: 91 , and a VL CDR1 comprising SEQ ID NO: 94, a VL CDR2 comprising SEQ ID NO: 96, a VL CDR3 comprising SEQ ID NO: 98; h. a VH CDR1 comprising SEQ ID NO: 101 , a VH CDR2 comprising SEQ ID NO: 103, a VH CDR3 comprising SEQ ID NO: 105, and a VL CDR1 comprising SEQ ID NO: 108, a VL CDR2 comprising SEQ ID NO: 110, a VL CDR3 comprising SEQ ID NO: 112.

3. The binding agent of any preceding claim wherein the first antigen binding region comprises; a. a VH comprising SEQ ID NO: 113 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 120 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH comprising SEQ ID NO: 15 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 22 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or c. a VH comprising SEQ ID NO: 29 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 36 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or d. a VH comprising SEQ ID NO: 43 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 50 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or e. a VH comprising SEQ ID NO: 57 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 64 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or f. a VH comprising SEQ ID NO: 71 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 78 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or g. a VH comprising SEQ ID NO: 85 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 92 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or h. a VH comprising SEQ ID NO: 99 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 106 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto.

4. The binding agent of any preceding claim wherein the first antigen binding region comprises; a. a VH comprising SEQ ID NO: 113, and a VL comprising SEQ ID NO: 120, or b. a VH comprising SEQ ID NO: 15, and a VL comprising SEQ ID NO: 22, or c. a VH comprising SEQ ID NO: 29, and a VL comprising SEQ ID NO: 36, or d. a VH comprising SEQ ID NO: 43, and a VL comprising SEQ ID NO: 50, ore. a VH comprising SEQ ID NO: 57, and a VL comprising SEQ ID NO: 64, or f. a VH comprising SEQ ID NO: 71 , and a VL comprising SEQ ID NO: 78, or g. a VH comprising SEQ ID NO: 85, and a VL comprising SEQ ID NO: 92, or h. a VH comprising SEQ ID NO: 99, and a VL comprising SEQ ID NO: 106.

5. The binding agent of any preceding claim wherein the second antigen binding region comprises a VH CDR1 comprising SEQ ID NO: 3 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 5 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 7 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 10 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 12 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 14 or a sequence comprising 1 , 2, or 3 amino acid differences.

6. The binding agent of any preceding claim wherein the second antigen binding region comprises a VH comprising SEQ ID NO: 1 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 8 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto.

7. The binding agent of any preceding claim wherein the first and / or second antigen binding region comprises an antibody or antigen binding fragment thereof.

8. The binding agent of claim 7 wherein the antigen binding fragment is selected from a Fab, scFv, F(ab')2, single domain antibody or single chain antibody.

9. The binding agent of any preceding claim wherein the antigen-binding region that binds IL1 RAP is a Fab and the antigen binding region that binds B7H4 is a Fab.

10. The binding agent of any preceding claim wherein the binding agent comprises an Fc region.

11. The binding agent of any preceding claim wherein the binding agent is capable of selectively targeting cells expressing IL1 RAP+ and B7H4+, compared to cells expressing IL1 RAP- / B7H4+ or cells expressing IL1 RAP+ / B7H4-.

12. The binding agent of any preceding claim wherein the antigen-binding region that binds IL1 RAP and the antigen binding region that binds B7H4 are linked by a linking portion.

13. The binding agent of any preceding claim wherein the binding agent comprises a payload.

14. The binding agent of claim 13 wherein the antibody or antigen binding fragment is conjugated to a pay load.

15. The binding agent of claim 14 wherein the payload is a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

16. The binding agent of claim 15, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

17. The binding agent of claim 15 wherein the cell killing agent comprises a cytotoxic moiety.

18. The binding agent of claim 17 wherein the cytotoxic moiety is selected from a radioisotope, peptide toxin or chemical toxin.

19. The binding agent of claim 17 or 18 wherein the cytotoxic moiety is selected from an auristatin, maytansinoid, tubulysin, RNA polymerase II inhibitor, transcription inhibitor, calicheamicin, duocarmycin, pyrrolobenzodiazepine, camptothecin analogue, topoisomerase inhibitor or doxorubicin.

20. The binding agent of any of claims 14 to 19 wherein the binding agent has an IC50 of 0.1 nM to 5 nM.

21. The binding agent of any of claims 14 to 20 wherein the first and / or second antigen binding region is linked to the payload with a linker.

22. The binding agent of any claim 21 wherein the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a pH sensitive linker, a redox sensitive linker.

23. The binding agent of any preceding claim wherein the binding agent is capable of being internalised.

24. The binding agent of any preceding claim comprising an additional moiety selected from a halflife extending moiety and / or a label.

25. The binding agent of any of claims 1 to 13 wherein said binding agent comprises a T cell and / or a NK cell.

26. A nucleic acid encoding the binding agent of any of claims 1 to 25.

27. A nucleic encoding a chimeric antigen receptor (CAR) comprising a) an extracellular antigenbinding domain comprising first antigen-binding region that binds human IL1 RAP and a second antigen binding region that binds human B7H4; b) a co- stimulatory signalling domain and c) a cytoplasmic signalling domain.

28. A vector comprising the nucleic acid of claim 26 or 27.

29. A host cell comprising the nucleic acid of claim 26 or 27 or the vector of claim 28.

30. The host cell of claim 29 wherein the host cell is a bacterial cell, viral cell, plant cell or mammalian cell.31 . A CAR-T or NK cell comprising the binding agent of any of claims 1 to 13.

32. An antibody drug conjugate comprising the binding agent of any of claims 1 to 24.

33. A pharmaceutical composition comprising the binding agent of any of claims 1 to 25 and a pharmaceutical excipient.

34. The binding agent of any of claims 1 to 25 or cell of claim 31 or the antibody drug conjugate of claim 32 for use in treating a disease.

35. The binding agent of any of claims 1 to 25 or cell of claim 31 or the antibody drug conjugate of claim 32 for use in a method of treating a disease associated with cells expressing IL1 RAP and B7H4.

36. The binding agent of any of claims 1 to 25 or cell of claim 31 or the antibody drug conjugate of claim 32 for use in a method for reducing or preventing progression of a tumour or treating cancer in a subject.

37. A method of treating a disease associated with cells expressing IL1 RAP and B7H4 in a subject comprising administering a binding agent of any of claims 1 to 25 or cell of claim 31 or the antibody drug conjugate of claim 32 to said subject.

38. A method of reducing or preventing progression of a tumour or treating cancer in a subject comprising administering a binding agent of any of claims 1 to 25 or cell of claim 31 or the antibody drug conjugate of claim 32 to said subject.

39. The binding agent or cell or antibody drug conjugate for use according to claim 36 or the method of claim 38 wherein said cancer is a solid tumour.

40. The binding agent, antibody drug conjugate or cell for use according to claim 39 or the method of claim 39 wherein the solid tumour is selected from tumour is selected from the group consisting of prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain / CNS cancer, cervical cancer, oesophageal cancer, gastric cancer, head / neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas.41 . The binding agent, antibody drug conjugate or cell for use according to claim 40 or the method of claim 40 wherein the cancer is selected from ovarian cancer, breast cancer, liver cancer, bile duct cancer, pancreatic cancer and lung cancer.

42. The binding agent, antibody drug conjugate or cell for use according to claim 41 or the method of claim 41 wherein the ovarian cancer is selected from ovarian adenocarcinoma.

43. The binding agent or cell for use according to claim 35 or the method of claim 37 wherein the disease associated with cells expressing IL1 RAP and B7H4 is a haematological disorder.

44. The binding agent, antibody drug conjugate or cell of claim 43 or the method of claim 43 wherein the haematologic disorder is selected from the group consisting of chronic myeloid leukemia (CML), myeloproliferative disorders (MPD), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML).

45. The use of the binding agent of any one of claims 1 to 25 or of a cell of claim 31 for the manufacture of a medicament for the treatment of a disease associated with cells expressing IL1 RAP and B7H4.

46. The binding agent of any of claims 1 to 25 or the pharmaceutical composition of claim 33, cell of claim 31 or the antibody drug conjugate wherein said binding agent, pharmaceutical composition, cell or antibody drug conjugate is administered together with another therapy.

47. The binding agent of any of claim 46 or the pharmaceutical composition of claim 46 wherein said administration is sequentially or concurrently.

48. The binding agent or cell or antibody drug conjugate of any of claim 40 or 41 or the pharmaceutical composition of claims 40 or 41 wherein said other therapy is selected from chemotherapy, radiotherapy or therapy with a checkpoint inhibitor49. A method of producing the binding agent of any of claims comprising culturing the host cell of claim 29 or 30.

50. A method for targeting tumour cells that express both IL1 RAP and B7H4 comprising administering the binding agent of any of claims 1 to 25, the cell of claim 31 , the antibody drug conjugate of claim 32 or the pharmaceutical composition of claim 33.

51. An in vitro, ex vivo or in vivo method of delivering a payload to IL1 RAP+ / B7H4+ cells within a biological sample, comprising obtaining or providing a biological sample contacting said biological sample with a binding agent according to any one of claims 1 to 25.

52. A method for reducing off target toxicity of a cancer treatment comprising administering to a subject a binding agent according to any one of claims 1 to 25 the cell of claim 31 , the antibody drug conjugate of claim 32 or the pharmaceutical composition of claim 33.

53. A kit comprising the pharmaceutical composition of claim 33 and instructions for use.

54. A method for identifying a patient that responds to therapy with a binding agent of any of claims 1 to 25 comprising analysing tumour cells for co-expression of IL1 RAP and B7H4.

55. The method of claim 54 further comprising (a) providing a tumour sample of cells from a patient to be tested; (b) optionally, extracting and / or purifying the cells present in the sample; (c) analysing tumour cells for co-expression of IL1 RAP and B7H4.

56. A method for identifying a tumour that responds to therapy with a binding agent of any of claims 1 to 25 comprising analysing tumour cells for co-expression of IL1 RAP and B7H4.

57. The method of claim 56 wherein the tumour cells are obtained from a patient or a cell line.

58. An in vitro, ex vivo or in vivo method of detecting IL1 RAP+ B7H4+ cells in a biological sample, comprising obtaining a biological sample contacting said biological sample with a binding molecule according to any one of claims 1 to 25.

59. A binding agent comprising an antigen-binding region that binds human interleukin-1 receptor accessory protein (IL1 RAP) comprising a. a VH CDR1 comprising SEQ ID NO: 115 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 117 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 119 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 122 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 124 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 126 or a sequence comprising 1 , 2, or 3 amino acid differences; b. a VH CDR1 comprising SEQ ID NO: 17 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 19 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 21 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 24 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 26 or a sequencecomprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 28 or a sequence comprising 1 , 2, or 3 amino acid differences; c. a VH CDR1 comprising SEQ ID NO: 31 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 33 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 35 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 38 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 40 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 42 or a sequence comprising 1 , 2, or 3 amino acid differences; d. a VH CDR1 comprising SEQ ID NO: 45 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 47 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 49 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 52 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 54 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 56 or a sequence comprising 1 , 2, or 3 amino acid differences; e. a VH CDR1 comprising SEQ ID NO: 59 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 61 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 63 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 66 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 68 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 70 or a sequence comprising 1 , 2, or 3 amino acid differences; f. a VH CDR1 comprising SEQ ID NO: 73 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 75 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 77 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 80 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 82 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 84 or a sequence comprising 1 , 2, or 3 amino acid differences; g. a VH CDR1 comprising SEQ ID NO: 87 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 89 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR3 comprising SEQ ID NO: 91 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 94 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 96 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 98 or a sequence comprising 1 , 2, or 3 amino acid differences; h. a VH CDR1 comprising SEQ ID NO: 101 or a sequence comprising 1 , 2, or 3 amino acid differences, a VH CDR2 comprising SEQ ID NO: 103 or a sequence comprising 1 , 2, or 3 aminoacid differences, a VH CDR3 comprising SEQ ID NO: 105 or a sequence comprising 1 , 2, or 3 amino acid differences, and a VL CDR1 comprising SEQ ID NO: 108 or a sequence comprising 1 , 2, or 3 amino acid differences, a VL CDR2 comprising SEQ ID NO: 110 or a sequence comprising 1 amino acid difference, a VL CDR3 comprising SEQ ID NO: 1 12 or a sequence comprising 1 , 2, or 3 amino acid differences.

60. The binding agent of claim 59 wherein the antigen binding region that binds IL1 RAP comprises; a. a VH CDR1 comprising SEQ ID NO: 115, a VH CDR2 comprising SEQ ID NO: 1 17, a VH CDR3 comprising SEQ ID NO: 1 19, and a VL CDR1 comprising SEQ ID NO: 122, a VL CDR2 comprising SEQ ID NO: 124, a VL CDR3 comprising SEQ ID NO: 126; b. a VH CDR1 comprising SEQ ID NO: 17, a VH CDR2 comprising SEQ ID NO: 19, a VH CDR3 comprising SEQ ID NO: 21 , and a VL CDR1 comprising SEQ ID NO: 24, a VL CDR2 comprising SEQ ID NO: 26, a VL CDR3 comprising SEQ ID NO: 28; c. a VH CDR1 comprising SEQ ID NO: 31 , a VH CDR2 comprising SEQ ID NO: 33, a VH CDR3 comprising SEQ ID NO: 35, and a VL CDR1 comprising SEQ ID NO: 38, a VL CDR2 comprising SEQ ID NO: 40, a VL CDR3 comprising SEQ ID NO: 42; d. a VH CDR1 comprising SEQ ID NO: 45, a VH CDR2 comprising SEQ ID NO: 47, a VH CDR3 comprising SEQ ID NO: 49, and a VL CDR1 comprising SEQ ID NO: 52, a VL CDR2 comprising SEQ ID NO: 54, a VL CDR3 comprising SEQ ID NO: 56; e. a VH CDR1 comprising SEQ ID NO: 59, a VH CDR2 comprising SEQ ID NO: 61 , a VH CDR3 comprising SEQ ID NO: 63, and a VL CDR1 comprising SEQ ID NO: 66, a VL CDR2 comprising SEQ ID NO: 68, a VL CDR3 comprising SEQ ID NO: 70; f. a VH CDR1 comprising SEQ ID NO: 73, a VH CDR2 comprising SEQ ID NO: 75, a VH CDR3 comprising SEQ ID NO: 77, and a VL CDR1 comprising SEQ ID NO: 80, a VL CDR2 comprising SEQ ID NO: 82, a VL CDR3 comprising SEQ ID NO: 84; g. a VH CDR1 comprising SEQ ID NO: 87, a VH CDR2 comprising SEQ ID NO: 89, a VH CDR3 comprising SEQ ID NO: 91 , and a VL CDR1 comprising SEQ ID NO: 94, a VL CDR2 comprising SEQ ID NO: 96, a VL CDR3 comprising SEQ ID NO: 98; or h. a VH CDR1 comprising SEQ ID NO: 101 , a VH CDR2 comprising SEQ ID NO: 103, a VH CDR3 comprising SEQ ID NO: 105, and a VL CDR1 comprising SEQ ID NO: 108, a VL CDR2 comprising SEQ ID NO: 110, a VL CDR3 comprising SEQ ID NO: 112.

61. The binding agent of claim 59 or 60 wherein the antigen binding region that binds IL1 RAP comprises; a. a VH comprising SEQ ID NO: 113 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 120 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto; b. a VH comprising SEQ ID NO: 15 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 22 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, orc. a VH comprising SEQ ID NO: 29 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 36 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or d. a VH comprising SEQ ID NO: 43 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 50 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or e. a VH comprising SEQ ID NO: 57 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 64 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or f. a VH comprising SEQ ID NO: 71 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 78 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or g. a VH comprising SEQ ID NO: 85 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 92 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, or h. a VH comprising SEQ ID NO: 99 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto, and a VL comprising SEQ ID NO: 106 or a sequence comprising 80%, 85%, 90%, 95% or 99% sequence identity thereto.

62. The binding agent of any one of claims 59 to 61 wherein the antigen binding region that binds IL1 RAP comprises; a. a VH comprising SEQ ID NO: 113, and a VL comprising SEQ ID NO: 120, or b. a VH comprising SEQ ID NO: 15, and a VL comprising SEQ ID NO: 22, or c. a VH comprising SEQ ID NO: 29, and a VL comprising SEQ ID NO: 36, or d. a VH comprising SEQ ID NO: 43, and a VL comprising SEQ ID NO: 50, or e. a VH comprising SEQ ID NO: 57, and a VL comprising SEQ ID NO: 64, or f. a VH comprising SEQ ID NO: 71 , and a VL comprising SEQ ID NO: 78, or g. a VH comprising SEQ ID NO: 85, and a VL comprising SEQ ID NO: 92, or h. a VH comprising SEQ ID NO: 99, and a VL comprising SEQ ID NO: 106.

63. The binding agent of any one of claims 59 to 62 comprising one or more further antigen binding region.

64. The binding agent of any one of claims 59 to 63 comprising a second antigen binding region that binds to a tumour associated antigen.

65. The binding agent of claim 64 wherein the antigen binding region and / or the second antigen binding region comprises an antibody or antigen binding fragment thereof.

66. The binding agent of claim 65 wherein the antigen binding fragment is selected from a Fab, scFv, F(ab')2, single domain antibody or single chain antibody.

67. The binding agent of any one of claims 59 to 66 wherein the binding agent comprises a payload.

68. The binding agent of claim 67 wherein the payload is a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload.

69. The binding agent of claim 68, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

70. The binding agent of claim 68 wherein the cell killing agent comprises a cytotoxic moiety.

71. The binding agent of claim 70 wherein the cytotoxic moiety is selected from a radioisotope, peptide toxin or chemical toxin.

72. The binding agent of claim 71 wherein the cytotoxic moiety is selected from an auristatin, maytansinoid, tubulysin, RNA polymerase II inhibitor, transcription inhibitor, calicheamicin, duocarmycin, pyrrolobenzodiazepine, camptothecin analogue, topoisomerase inhibitor or doxorubicin.

73. The binding agent of any one of claims 59 to 72 wherein the antigen binding region and / or second antigen binding region is linked to the payload with a linker.

74. The binding agent of claim 73 wherein the linker is selected from one or more of a cleavable linker, a non-cleavable linker, a pH sensitive linker, a redox sensitive linker.

75. The binding agent of any one of claims 59 to 74 wherein the binding agent is capable of being internalised.

76. The binding agent of any one of claims 59 to 75 comprising an additional moiety selected from a half-life extending moiety and / or a label.

77. The binding agent of any one of claims 59 to 67 wherein said binding agent comprises a T cell and / or a NK cell.

78. An isolated nucleic acid encoding a binding agent according to any one of claims 59 to 77.

79. A vector comprising a nucleic acid according to claim 78.

80. A host cell comprising a nucleic acid according to claim 78 or a vector according to claim 79.81 . A CAR-T or NK cell comprising the binding agent of any one of claims 59 to 67.

82. An antibody drug conjugate comprising the binding agent of any of claims 59 to 76.

83. A pharmaceutical composition comprising the binding agent of any of claims 59 to 77 and a pharmaceutical excipient.

84. The binding agent of any of claims 59 to 77 or cell of claim 81 for use in treating a disease.

85. The binding agent of any of claims 59 to 77 or cell of claim 81 for use in a method of treating a disease associated with cells expressing IL1 RAP.

86. The binding agent of any of claims 59 to 77 or cell of claim 81 for use in a method for reducing or preventing progression of a tumour or treating cancer in a subject.

87. A method of treating a disease associated with cells expressing IL1 RAP in a subject comprising administering a binding agent of any of claims 59 to 77 or cell of claim 81 to said subject.

88. A method of reducing or preventing progression of a tumour or treating cancer in a subject comprising administering a binding agent of any of claims 59 to 77 or cell of claim 81 to said subject.

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