Multispecific antigen binding proteins for tumor-targeting of ΓΔ1 t cells and use thereof

Abstract

The present invention relates to multispecific antigen binding proteins that comprise an antigen-binding regions specific for a tumor-associated antigen (TAA), an antigen-binding region that specifically binds an epitope of a γδ T cell receptor (TCR), a γδ T cell-activating cytokine, and optionally, a γδ T cell co-stimulatory agonist. The γδ T cell-activating cytokine preferably is at least 5 one of an interleukin 21 receptor (IL21R) agonist and an interleukin 15 receptor (IL15R) agonist. The γδ T cell co-stimulatory agonist cytokine preferably is at least one of a 4-1BB agonist, a CD27 agonist and a GITR agonist. The multispecific antigen binding proteins of the invention specifically redirect and activate γδ T cell to lyse targeted tumor cells. The invention further relates to the use of such multispecific antigen binding proteins in the treatment of cancer, preferably a cancer 10 expressing the TAA.

Description

[0001] Multispecific antigen binding proteins for tumor-targeting of y61 T cells

[0002] and use thereof

[0003] Field of the invention

[0004] The present invention relates to the field of medicine, in particular to the fields of oncology, immunology and immunotherapy of tumors, infections and / or autoimmune disease. Specifically, the invention relates to multispecific antigen binding proteins that specifically redirect and activate y6 T cells to lyse targeted tumor cells. The invention further relates to the use of such multispecific antigen binding proteins in the treatment of cancer.

[0005] Background of the invention

[0006] What today we consider a medical milestone in immune oncology was the application of redirecting a patient’s immune system, predominantly through manipulating alpha (a) beta (p) T cells to overcome tolerance to consequently attack tumor cells. This can be achieved by multiple means such as (i) antibodies directing such T cell responses called engagers as well as antibodies that overcome various ways of immune suppression by blocking immune checkpoint interactions, (ii) Alternatively, cellular immunotherapy has proven highly effective against certain types of leukemias using chimeric antigen receptors (CAR), genetically introduced into a patient’s own T cells which are subsequently grown in commercial manufacturing settings into large numbers and administrated intra venous to an autologous receiver.

[0007] Despite initial successes using both technologies, a myriad of problems remain to be overcome. For example, checkpoint inhibitors as well as engagers induce a high occurrence rate of severe toxicities, additionally, predictions on why some but not all patients respond to specific antibodies cannot yet be made. Cellular therapies are also facing struggles on multiple levels, poor manufacturability due to the source of cells coming from very ill patients, lengthy and very costly manufacturing per se and the lack of predictive strategies to know which patients will benefit from the expensive therapy. Finally, cellular immunotherapy is yet limited to a short list of malignant indications.

[0008] Advancements in the field to overcome these adversities focus on enhancing existing technologies to modulate ap T-cells, but increasingly, efforts are expanded to include members of the innate immune system which are capable of orchestrating complete and natural immune responses, involving cells and mechanisms that go beyond the direct mode of action of a therapeutic. Furthermore, innate lymphocytes are often Major Histocompatibility Complex (MHC) un-restricted, allowing for their potential allogeneic use in multiple recipients without causing graft versus host disease (GvHD). Both, antibody and adoptive cell therapies targeting innate cells also show a much lower prevalence for therapy associated toxicities such as cytokine release syndrome (CRS) and neurotoxicity. Candidates for such therapeutics are Natural Killer (NK) cells, induced NK (iNK) cells, macrophages and gamma (y) delta (6) T cells.

[0009] Although promising, innate immuno-oncology still comes with its own challenges. Cellular therapies show great potential especially when genetically modified with CARs, but difficulties in manufacturing and the allogeneic nature of these therapies cast questions regarding their durability and persistence, facing early rejection by a patient after infusion. Antibody based engagers and checkpoint blockade molecules targeted against innate cells are in development, but yet need to show benefit beyond pre-clinical results. Innate cells in general have different growth kinetics and signaling requirements from adaptive ap T-cells and may need more optimized engagement using multiple receptor interactions rather than one or two as is the case for the ap TCR. Such multispecific antigen binding proteins are already in development for NK cells (WO2024 / 056862; WO2024 / 056861).

[0010] yb T-cells are a highly conserved, third type of lymphocyte expressing a VDJ recombined antigen receptor after ap T cells and B cells. Whilst the receptor, through gene rearrangement, can be 2-3 logs more diverse than ap TCRs, the cells use a limited repertoire of TCR genes to achieve this. Ignoring pseudogenes, the yb TCR combines one y chain (TRGV2, TRGV3, TRGV 4, TRGV5, TRGV8, TRGV9 and TRGV11) with one 6 chain (TRDV1 , TRDV2, TRDV3, TRAV14 / DV4, TRAV29 / DV5, TRAV23 / DV6, TRAV36 / DV7, TRAV38-2 / DV8). The y uses one of two constant region genes for its TCR chain, the 6 chain uses one gene. The functional TCR then forms a complex with several CD3 molecules.

[0011] Evolutionary, yb T-cells play a significant role in immune surveillance, recognizing cells that show early signs of malignancy through a combination of the expression of various stress-ligand sensing natural cytotoxicity (NCRs) and natural killer receptors (NKRs), as well as a TCR that can recognize metabolic intermediates and proteins linked to hyper proliferation or tumor antigens directly. This self-directed stress surveillance lies in stark contrast to the recognition of foreign antigen mediated by ap T cells and B cells. Representing the interface between innate and adaptive immunity, yb T-cells cells are MHC unrestricted and are not inhibited by KIR expression. Initiating potent cytotoxicity against stressed or malignant cells, yb T cells orchestrate wider immune responses by attracting and modulating macrophages, monocytes, dendritic cells, B cells and ultimately ap T-cells. Multiple mouse models have shown that a total or selective knock out of yb T-cells leads to increased tumor occurrence and severity, a phenotype that is unique to these cells. In humans, clinical studies have shown the presence, number and polyclonality of yb T cells to be the most significant correlate of overall survival (OS) and progression free survival (PFS) in 39 tumor types spanning both hematological and solid malignancies. New therapeutics that would manage to activate and increase the numbers of yb T cells are thus strongly desirable and clinically indicated.

[0012] In human biology and clinical translation, yb T cells are largely classified into two groups based on functionality, (i) variable (V) 62 T-cells that almost exclusively pair with a TRGV9 chain, and (ii) Vb1 T-cells that can pair with any y chain. Vb2 T-cells have been studied and manipulated for clinical application for decades using amino-bisphosphonates, phospho-antigen, antibodies and engagers. Predominantly, Vb2 T-cells have been tested as cellular immunotherapies. Despite a great safety profile, induction of growth and / or targeting / redirection in-situ via the Vb2 TCR with antibodies or drugs has been shown to lead to early exhaustion. Additionally, their canonical role in immunology of predominantly responding to mycobacterial infections makes them poor cellular therapies in the absence of engineered stimuli. To trigger anti-cancer effects, it is very likely that the introduction of CARs and engineering strategies to enhance migration are required. On top of these difficulties, allogeneic V62 T-cells will most likely suffer from the same rejection challenges as other allogeneic cell therapies.

[0013] The second functional group of y6 T cells are V61 T-cells, the dominant cell type in almost all pre-clinical models that have proven their evolutionary significance as well as in clinical papers showing positive correlations with OS and PFS in patients with numbers and TOR clonality. Whilst V62 T cells circulate in the blood, V61 T cells show a strong tropism to travel to and reside within tissues. A lack of understanding on how these cells and their TOR specifically function has been holding back the clinical application of V51 T-cells. In terms of V51 targeting antibodies or TOR agonists, only recently have breakthroughs been made (W02021 / 032960; WO2022 / 034562A1 ; WO2021 / 113558; WO2012 / 156958) allowing for clinical-scale ex-vivo manufacturing of V51 T-cells. These advantages have translated into multiple V51 -based allogeneic therapies, nonengineered or enhanced with CARs, which remain to show benefits in clinical studies. In theory at least, such therapies will have the same persistence challenges as other allogeneic therapies.

[0014] A big clinical need which would furthermore allow to bypass all challenges and costs ofV51 T cell therapy would be the development of technologies to activate and expand V51 T cells within humans. Regarding in-situ manipulation of V51 T-cells, ligands, drugs or antibodies that induce proliferation of V51 T-cells in-situ which lead to a measurable increase in systemic V51 numbers have not yet been discovered. Also, targeting V51 T-cells with checkpoint inhibitors is in its very infancy due to the lack of good descriptive studies on how these cells are modulated or what qualifies as exhausted y5 T-cells in the first place. First V51 -targeting therapeutic applications in development (WO2022 / 034562) propose that V51 T-cells can be directed against tumor cells using binders against the V51 TCR and tumor antigen. It remains to be seen if this TCR focused approach will lead to other results than with V52 cells. Other approaches already postulate (WO2019 / 147735) that V51 T cells are deleted when using V51-TCR targeting antibodies which would be counterintuitive in almost all cancer settings.

[0015] Thus, whilst the proposal of V51 T cell based immuno-therapy is intriguing, there remains a significant need in the art for improved multispecific antigen binding proteins that provide therapeutic advantages over existing immune cell and y5 T cell engagers and even y5 T-cell based cellular therapies. It is thus an object of the present invention to provide for improved multispecific antigen binding proteins for the targeted engagement of V51 T cells, and for methods for their therapeutic use in treating cancers, as well as infectious diseases and autoimmune diseases.

[0016] Summary of the invention

[0017] In a first aspect, there is provided a multispecific antigen binding protein comprising: a) a first antigen-binding region and an optional second antigen-binding region that specifically bind a tumor or pathogen associated antigen (TAA); b) a third antigen-binding region that specifically binds an epitope of a y5 T cell receptor (TCR); c) a y5 T cell-activating agonist that induces at least one of STAT5 and STAT3 activation; and optionally, d) a y6 T cell co-stimulatory agonist that is at least one of: i) a 4-1 BB agonist; ii) a CD27 agonist; and, iii) a GITR agonist.

[0018] In one embodiment of the multispecific antigen binding protein, at least one of the first, optional second and third antigen-binding region comprise an immunoglobulin-derived antigenbinding region, wherein preferably the immunoglobulin-derived antigen-binding region comprises or consists of a Fab or an immunoglobulin single variable domain (ISVD).

[0019] In one embodiment of the multispecific antigen binding protein, the TAA is selected from the group consisting of: Her2 (ErbB2 / Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171 , CTLA-4 (CD152), PD1 , PSCA, L1-CAM, EpCAM, PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1 , CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), B7.1 , B7.2, B7-H3, B7-H4, B7-H6, PD-L1 , IL-6 receptor, IL1 accessory Protein, MAGE, MART-1 / Melan-A, gp100, MICA, MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1A / GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1 , nectin-4, a UL16-binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1 , prostate specific antigen (PSA), T-cell receptor / CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Mullerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1 , TRAILR2, BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC1 , MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), HER-3 / ERBB3, HER-4 / ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, integrin receptors, a5p3 integrins, a5p1 integrins, allbp3-integrins, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, CSF1R, a-fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18)), folate receptor alpha (FRa, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, a-catenin, p-catenin and y-catenin, Plexin-A1 , TNFRSF10B, AXL, EDNRB, OLR1 , ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1 , cdc27, CDCP1 , adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp-1 , P1A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, muc16, muc17, mmp9, FAP, Lewis-Y, EGFRvlll, GPC3, gpA33, 5T4, SSTR2, CD73, CD25, CD45, CD133, FGFR2b, CD79B, BTLA, Fibronectin extra-domain B, GM3, LAG-3, 0X40, PDGFRa, TIGIT, VEGF-a, GPCR5D and scatter factor receptor kinase.

[0020] In one embodiment of the multispecific antigen binding protein, the third antigen-binding region specifically binds an epitope in the V region of a V61 chain of a yb TCR.

[0021] In one embodiment of the multispecific antigen binding protein, the y5 T cell-activating agonist is selected from the group consisting of: a) an IL-2 polypeptide that is an IL-2 mutein that is modified to reduce (or enhance) affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide; b) an IL-6 polypeptide that is an IL-6 mutein that is modified to reduce (or enhance) affinity for IL-6R, relative to a corresponding wild type IL-6 polypeptide; c) an IL-7 polypeptide that is an IL-7 mutein that is modified to reduce (or enhance) affinity for IL-7R, relative to a corresponding wild type IL-7 polypeptide; d) an IL-9 polypeptide that is an IL-9 mutein that is modified to reduce (or enhance) affinity for IL-9R, relative to a corresponding wild type IL-9 polypeptide; e) an IL-12 polypeptide that is an IL-12 mutein that is modified to reduce (or enhance) affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide; f) an IL-15 polypeptide that is an IL-15 mutein that is modified to reduce (or enhance) affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide; g) an IL-18 polypeptide that is an IL-18 mutein that is modified to reduce (or enhance) affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide; h) an IL-21 polypeptide that is an IL-21 mutein that is modified to reduce (or enhance) affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide; i) an IL-23 polypeptide that is an IL-23 mutein that is modified to reduce (or enhance) affinity for IL-23R, relative to a corresponding wild type IL-23 polypeptide; and, j) an IL-27 polypeptide that is an IL-27 mutein that is modified to reduce (or enhance) affinity for IL-27R, relative to a corresponding wild type IL-27 polypeptide.

[0022] In one embodiment of the multispecific antigen binding protein, the IL-15 mutein has reduced affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide, and wherein the IL-15 mutein has a mutation in at least one amino acid position selected from the group consisting of: N1 , D8, D30, H32 L45, E46, I50, V49, S51 , L52, V63, E64, N65, I68, L69, Q108, M109 and N112, of which D8, E64, N65, I68, and L69 are preferred.

[0023] In one embodiment of the multispecific antigen binding protein, the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has at least one of: a) a mutation in one or more amino acid positions selected from the group consisting of: D4, R5, I8, R9, R11 , Q12, L13, 114, D15, 116, D18 Q19, L20, K21 , Y23, R65, I66, I67, N68, V69, S70, K72, K73, L74, K75, R76, K77, P78, P79, S80, E100, E109, R110, K112, S113, Q116, K117, 1119, H120, and L123; b) a deletion of at least one, two, three, four, five, six, seven or eight amino acids in the region of N59 to I66 or a deletion of amino acids N63 to I66; and / or a deletion of amino acids K75 and R76 or a deletion of amino acids K75, R76 and R77; and, c) an insertion of at least two, three or four amino acids immediately C-terminal to G84 and / or an insertion K75 insX, wherein X is one, two or three amino acids selected from the group consisting of: G, S and D.

[0024] In one embodiment of the multispecific antigen binding protein, at least one of: i) the 4-1 BB agonist comprises or consists of at least one 4-1 BB ligand (4-1 BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds 4-1 BB; ii) the CD27 agonist comprises or consists of at least one CD70 extracellular domain (ECD) or agonistic antigen-binding region that specifically binds CD27 and, iii) the GITR agonist comprises or consists of at least one GITR ligand (GITRL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds GITR. In one embodiment of the multispecific antigen binding protein, at least one of: i) the 4-1 BBL ECD is a mutein with reduced affinity for 4-1 BB, relative to a corresponding wild type 4-1 BBL ECD, and wherein the 4-1 BBL ECD mutein comprises the amino acid sequence:

[0025] REGPELSPDD PAGLLDLRQG MFAQLVAQNX XLIDGPLSWX SDPXXXGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVXXGEGSGS VSLALHLQPL XSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWXLTX GATVLGLFRV TPEIPAGLPS PRSE

[0026] ( SEQ ID NO : 362 ) ,

[0027] wherein X represents any amino acid, and wherein the 4-1 BBL ECD mutein amino acid sequence differs from the amino acid sequence of the wild type human 4-1 BBL ECD (SEQ ID NO: 359) by at least 1 amino acid, and wherein preferably, the 4-1 BBL ECD comprises one or more amino acid substitutions selected from the group consisting of: V100T, V100Q, L101 N, Y110Q, G114K, L115R, A116D, V153Q, A154D, R171D, Q227E, Q227R, Q230S, Q230K, V100T, Q227R, Q230S and Q230K; ii) the CD70 ECD is a mutein with reduced affinity for CD27 relative to a corresponding wild type CD70 ECD, and wherein CD70 ECD mutein comprises at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 365 selected from the group consisting of: Q61 , A80, S137, S146 and H148, wherein preferably, the CD70 ECD mutein comprises at least one amino acid substitution selected from the group consisting of: Q61A, A80F, A80R, S137A, S137E, S137K, S146A, H148A, H148E and H148D; and iii) the GITRL ECD is a mutein with reduced affinity for GITR, relative to a corresponding wild type GITRL ECD.

[0028] In one embodiment of the multispecific antigen binding protein, at least one of: i) the 4-1 BB agonist comprises or consists of a fusion protein comprising three 4-1 BBL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three 4-1 BBL ECD monomers are connected by polypeptide linkers; and, ii) the CD27 agonist comprises or consists of a fusion protein comprising three CD70 ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three CD70 ECD monomers are connected by polypeptide linkersl and, iii) the GITR agonist comprises or consists of a fusion protein comprising three GITRL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three GITRL ECD monomers are connected by polypeptide linkers.

[0029] In one embodiment of the multispecific antigen binding protein, at least one of the first, second and third antigen-binding region is a Fab, wherein the Fab is operably linked to a polypeptide chain of the dimeric Fc region, wherein preferably, the Fc region has reduced affinity for at least one of the human Fc y receptor (FcyR) and human C1q, relative to a corresponding wild-type Fc region, wherein more preferably, the Fc region is a variant human lgG1 Fc region comprising at least one modification selected from the group consisting of: L234A, L235A and G237A.

[0030] In one embodiment of the multispecific antigen binding protein, the multispecific antigen binding protein has at least one biological activity selected from: a) the multispecific antigen binding protein causes an increase in at least one y5 T cell activity selected from y5 T cell proliferation, cytokine production, y5 T cell cytotoxicity, y5 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y5 T cells and target cells that are not brought into contact with the multispecific antigen binding protein; and, b) the multispecific antigen binding protein causes an increase in at least one y6 T cell activity selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are brought into contact with a conventional human lgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the multispecific antigen binding protein.

[0031] In a second aspect, there is provided a pharmaceutical composition comprising a multispecific antigen binding protein according to the first aspect, and a pharmaceutically acceptable carrier.

[0032] In a third aspect, there is provided a multispecific antigen binding protein according to the first aspect, or a composition according to the second aspect, for use as a medicament.

[0033] In a fourth aspect, there is provided a multispecific antigen binding protein according to the first aspect, or a composition according to the second aspect, for use in the prevention or treatment of a cancer, an infectious disease or an inflammatory disease, wherein preferably, the cancer is a cancer comprising tumor cells expressing the TAA.

[0034] In one embodiment of the use in the treatment of a cancer, at least one of: a) the multispecific antigen binding protein is administered as a neoadjuvant therapy before a primary therapy comprising at least one of surgery and radiation therapy of the cancer; and, b) the multispecific antigen binding protein is administered as an adjuvant therapy after a primary therapy comprising at least one of surgery and radiation therapy of the cancer.

[0035] Description of the invention

[0036] Definitions

[0037] Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice fortesting of the present invention, the preferred materials and methods are described herein.

[0038] “A,” “an,” and “the”: these singular form terms include plural referents unless the content clearly dictates otherwise. The indefinite article “a” or “an” thus usually means “at least one”. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0039] “About” and “approximately”: these terms, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a ratio in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual ratios such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0040] “And / or”: The term “and / or” refers to a situation wherein one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

[0041] “Comprising”: this term is construed as being inclusive and open ended, and not exclusive. Specifically, the term and variations thereof mean the specified features, steps or components are included. These terms are not to be interpreted to exclude the presence of other features, steps or components.

[0042] “Exemplary”: this term means “serving as an example, instance, or illustration,” and should not be construed as excluding other configurations disclosed herein.

[0043] As used herein “cancer” and “cancerous”, refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer is also referred to as malignant neoplasm.

[0044] As used herein, “in combination with” is intended to refer to all forms of administration that provide a first drug together with a further (second, third) drug. The drugs may be administered simultaneous, separate or sequential and in any order. Drugs administered in combination have biological activity in the subject to which the drugs are delivered.

[0045] As used herein “simultaneous” administration refers to administration of more than one drug at the same time, but not necessarily via the same route of administration or in the form of one combined formulation. For example, one drug may be provided orally whereas the other drug may be provided intravenously during a patient’s visit to a hospital. “Separate” includes the administration of the drugs in separate form and / or at separate moments in time, but again, not necessarily via the same route of administration. “Sequential(ly)” indicates that the administration of a first drug is followed, immediately or in time, by the administration of the second drug.

[0046] A used herein "compositions", "products" or "combinations" useful in the methods of the present disclosure include those suitable for various routes of administration, including, but not limited to, intravenous, subcutaneous, intradermal, subdermal, intranodal, intratumoral, intramuscular, intraperitoneal, oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and / or parenteral or mucosal application. The compositions, formulations, and products according to the disclosure invention normally comprise the drugs (alone or in combination) and one or more suitable pharmaceutically acceptable excipients.

[0047] As used herein, “an effective amount” is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of a cancer varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is “effective against” a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

[0048] “Sequence identity” is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. “Identity” and “similarity” can be readily calculated by known methods. The terms “sequence identity” or “sequence similarity” means that two (poly)peptide or two nucleotide sequences, when optimally aligned, preferably over the entire length (of at least the shortest sequence in the comparison) and maximizing the number of matches and minimizes the number of gaps such as by the programs ClustalW (1.83), GAP or BESTFIT using default parameters, share at least a certain percentage of sequence identity as defined elsewhere herein. GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimizes the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is BLOSUM62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). A preferred multiple alignment program for aligning protein sequences disclosed herein is ClustalW (1.83) using a BLOSUM matrix and default settings (Gap opening penalty:10; Gap extension penalty: 0.05). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are BLOSUM62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called “conservative” amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. Examples of classes of amino acid residues for conservative substitutions are given in the Tables below.

[0049] Acidic Residues Asp (D) and Glu (E)

[0050] Basic Residues Lys (K), Arg (R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn (N), and

[0051] Gin (Q)

[0052] Aliphatic Uncharged Residues Gly (G), Ala (A), Vai (V), Leu (L),

[0053] and lie (I)

[0054] Non-polar Uncharged Residues Cys (C), Met (M), and Pro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

[0055]

[0056] Alternative conservative amino acid residue substitution classes.

[0057] 1 A S T

[0058] 2 D E

[0059] 3 N Q

[0060] 4 R K

[0061] 5 I L M

[0062] 6 F Y W

[0063]

[0064] Alternative physical and functional classifications of amino acid residues.

[0065] Alcohol group-containing residues S and T

[0066] Aliphatic residues I, L, V, and M

[0067] Cycloalkenyl-associated residues F, H, W, and Y

[0068] Hydrophobic residues A, C, F, H, I, L, M, R, T, V, W, and Y

[0069] Negatively charged residues D and E

[0070] Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R

[0071] Small residues A, C, D, G, N, P, S, T, and V

[0072] Very small residues A, G, and S

[0073] Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0074]

[0075] The term "agent" refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be a compound or a composition. An agent can e.g. be selected from the group consisting of: polynucleotides, polypeptides, small molecules, (multispecific) antigen binding proteins, such as antibodies and functional fragments thereof.

[0076] The term "antigen-binding domain" or "antigen-binding region" refers to the portion of an antigen-binding protein that is capable of specifically binding to an antigen or epitope. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region, e.g. comprising both an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Examples of such antigen-binding regions include single-chain Fv (scFv), single-chain antibody, Fv, single-chain Fv2 (scFv2), Fab, and Fab'. In one embodiment, the antigen-binding region is an immunoglobulin-derived antigen-binding region from a single domain antibody consisting only of heavy chains and devoid of light chains as are known e.g. from camelids, wherein the antigen-binding site is present on, and formed by, the single variable domain (also referred to as an "immunoglobulin single variable domain" or "ISVD"). Examples of such ISVDs include the single variable domains of camelid heavy chain antibodies (VHHS), also referred to as nanobodies, domain antibodies (dAbs), and single domains derived from shark antibodies (IgNAR domains). In other embodiments, an antigen-binding region comprises a non-immunoglobulin-derived domain capable of specifically binding to an antigen or epitope, such as DARPpins; Affilins; anticalins, etc.

[0077] The term "antibody" herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies), and antibody fragments and derivatives, so long as they exhibit the desired biological and / or immunological activity. Various techniques relevant to the production of antibodies are provided in, e.g., Harlow, et al.. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988). An antibody can be human and / or humanized. "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.

[0078] The terms "full length antibody", "intact antibody", and "whole antibody" are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure. "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1 , CH2, and CH3), also called a heavy chain constant region. Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a light chain constant domain (CL), also called a light chain constant region. The heavy chain of an antibody may be assigned to one of five types, called a (IgA), 6 (IgD), e (IgE), y (IgG), or m (IgM), some of which may be further divided into subtypes, e.g. y1 (lgG1), y2 (lgG2), y3 (lgG3), y4 (lgG4), a1 (lgA1) and a2 (lgA2). The light chain of an antibody may be assigned to one of two types, called kappa (K) and lambda (A), based on the amino acid sequence of its constant domain.

[0079] An "antibody fragment" comprises a portion of a full-length antibody, e.g. the antigen-binding or variable regions thereof. Examples of antibody fragments include Fab, Fab', F(ab)2, F(ab’)2, F(ab)s, Fv (typically the VH and VL domains of a single arm of an antibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VH and CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VHH, and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, and kappa bodies (see, e.g.. Ill et al.. Protein Eng 1997;10: 949-57); camel IgG; IgNAR; and multispecific antibody fragments formed from antibody fragments, and one or more isolated CDRs or a functional paratope, where isolated CDRs or antigen-binding residues or polypeptides can be associated or linked together so as to form a functional antibody fragment. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003). For a review of scFv fragments, see e.g. Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, N.Y., pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571 ,894 and 5,587,458. For discussion of Fab and F(ab’)2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific, see, for example, EP 404,097; WO 1993 / 01161 ; Hudson et al., Nat Med 9, 129-134 (2003); and Hollinger et al., Proc Natl Acad Sci USA 90, 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat Med 9, 129-134 (2003). Various types of antibody fragments have been described or reviewed in, e.g.. Heiliger and Hudson, Nat Biotechnol 2005; 23, 1126-1136; W02005 / 040219, US20050238646 and US20020161201. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. CHO, E. coli or phage), as described herein.

[0080] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. The term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, N.Y. (1988); Hammerling et al., in: "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, N.Y. (1981), pp. 563-681 (both of which are incorporated herein by reference in their entireties).

[0081] The term "monospecific" antibody as used herein denotes that the antibody-part of a multispecific antigen binding protein comprising antigen-binding regions as described herein, has one or more antigen-binding sites each of which bind to the same epitope of the same antigen. The term "bispecific" means that the antibody-part of a multispecific antigen binding protein as described herein, has at least two antigen-binding sites that are able to specifically bind to at least two distinct antigenic determinants. Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenic determinant. In certain embodiments the bispecific antigen binding molecule is capable of simultaneously binding two antigenic determinants, particularly two antigenic determinants expressed on two distinct cells. The term "valent" or "valency" as used within the current application denotes the presence of a specified number of binding sites or number of ligands in an antigen binding molecule or multispecific antigen binding protein described herein. As such, the terms "bivalent", "tetravalent", and "hexavalent" denote the presence of two, four and six binding sites or ligands, respectively, in an antigen binding molecule or multispecific antigen binding protein.

[0082] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, see, e.g., Huse et al., Science 246:1275-1281 (1989); Ward et al., Nature 341 :544-546 (1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. In a non-limiting example, mice can be immunized with an antigen of interest or a cell expressing such an antigen. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells. Hybridomas are selected and cloned by limiting dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding the antigen. Ascites fluid, which generally contains high levels of antibodies, can be generated by inoculating mice intraperitoneally with positive hybridoma clones.

[0083] Typically, an immunoglobulin has a heavy and light chain. Each heavy and light chain contains a constant region and a variable region, (the regions are also known as "domains"). Light and heavy chain variable regions contain four "framework" regions interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs". The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

[0084] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable region generally comprises amino acid residues from a "complementarity-determining region" or "CDR" (e.g. residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain; Kabat et al. 1991 , Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, USA) and / or those residues from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chain variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as “Kabat position”, "variable domain residue numbering as in Kabat" and "according to Kabat" herein refer to this numbering system for heavy chain variable domains or light chain variable domains. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, an FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of CDR H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.

[0085] The term "framework" or "FR" residues as used herein refers to the region of an antibody variable domain exclusive of those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into the contiguous regions separated by the CDRs (FR1 , FR2, FR3 and FR4).

[0086] The term "constant region" as defined herein refers to an antibody-derived constant region that is encoded by one of the light or heavy chain immunoglobulin constant region genes. By "constant light chain" or "light chain constant region" as used herein is meant the region of an antibody encoded by the kappa (Ck) or lambda (CA) light chains. The constant light chain typically comprises a single domain, and as defined herein refers to positions 108-214 of CK or CA, wherein numbering is according to the EU index (Kabat et al., 1991 , supra).

[0087] The term "constant heavy chain" or "heavy chain constant region" as used herein refers to the region of an antibody encoded by the mu, delta, gamma, alpha, or epsilon genes to define the antibody's isotype as IgM, IgD, IgG, IgA, or IgE, respectively. For full length IgG antibodies, the constant heavy chain, as defined herein, refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, thus comprising positions 118-447, wherein numbering is according to the EU index.

[0088] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments containing each the heavy- and light-chain variable domains and also the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. "Fab" fragments can also be recombinantly produced by methods known in the art. As used herein, Thus, the term "Fab fragment" " or "Fab region" refers to an antibody fragment comprising a light chain fragment comprising a VL domain and a constant domain of a light chain (CL), and a VH domain and a first constant domain (CH1) of a heavy chain. Fab may refer to this region in isolation, or this region in the context of a polypeptide, multispecific antigen binding protein or antigen-binding region, or any other embodiments as outlined herein. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab’-SH are Fab’ fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-combining sites (two Fab fragments) and a part of the Fc region.

[0089] The term "single-chain Fv" or "scFv" as used herein refers to antibody fragments comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, 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. Methods for producing scFvs are well known in the art. For a review of methods for producing scFvs see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, N.Y., pp. 269-315 (1994).

[0090] "Scaffold antigen-binding proteins" are known in the art, for example, fibronectin and designed ankyrin repeat proteins (DARPins) have been used as alternative scaffolds for antigenbinding domains, see, e.g., Gebauer and Skerra, Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol 13:245-255 (2009) and Stumpp et al., Darpins: A new generation of protein therapeutics. Drug Discovery Today 13: 695-701 (2008). In one embodiment, a scaffold antigen-binding protein is selected from the group consisting of: CTLA-4 (Evibody), Lipocalins (Anticalin), monobodies, centyrins, kunitz domains, knottins, fynomers, lipocalins, a Protein A-derived molecule such as Z-domain of Protein A (Affibody), an A-domain (Avimer / Maxibody), a serum transferrin (frans-body); a designed ankyrin repeat protein (DARPin), a variable domain of antibody light chain or heavy chain (single-domain antibody, sdAb), a variable domain of antibody heavy chain (nanobody, aVH), VNAR fragments, a fibronectin (AdNectin), a C-type lectin domain (Tetranectin); a variable domain of a new antigen receptor beta-lactamase (VNAR fragments), a human gamma-crystallin or ubiquitin (Affilin molecules); a Kunitz type domain of human protease inhibitors, microbodies such as the proteins from the knottin family, peptide aptamers and fibronectin (adnectin).

[0091] CTLA-4 (Cytotoxic T Lymphocyte-associated Antigen 4) is a CD28-family receptor expressed on mainly CD4+T-cells. Its extracellular domain has a variable domain- like Ig fold. Loops corresponding to CDRs of antibodies can be substituted with heterologous sequence to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Evibodies (e.g. US7166697B1). Evibodies are around the same size as the isolated variable region of an antibody (e.g. a domain antibody). For further details see Journal of Immunological Methods 248 (1-2), 31-45 (2001).

[0092] Lipocalins are a family of extracellular proteins which transport small hydrophobic molecules such as steroids, bilins, retinoids and lipids. They have a rigid beta-sheet secondary structure with a number of loops at the open end of the conical structure which can be engineered to bind to different target antigens. Anticalins are between 160-180 amino acids in size and are derived from lipocalins. For further details see Biochim Biophys Acta 1482: 337-350 (2000), US7250297B1 and US20070224633.

[0093] An affibody is a scaffold derived from Protein A of Staphylococcus aureus which can be engineered to bind to antigen. The domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details see Protein Eng. Des. Sei. 17, 455-462 (2004) and EP1641818A1.

[0094] Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007). A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999).

[0095] Designed Ankyrin Repeat Proteins (DARPins) are derived from Ankyrin which is a family of proteins that mediate attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two alpha-helices and a beta-turn. They can be engineered to bind different target antigens by randomizing residues in the first alpha-helix and a beta-turn of each repeat. Their binding interface can be increased by increasing the number of modules (a method of affinity maturation). For further details see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003) and J. Mol. Biol. 369, 1015-1028 (2007) and US20040132028A1.

[0096] A single-domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. The first single variable domains were derived from the variable domain of the antibody heavy chain from camelids (nanobodies or Vi-iH fragments). Furthermore, the term single variable domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.

[0097] Fibronectin is a scaffold which can be engineered to bind to antigen. Adnectins consists of a backbone of the natural amino acid sequence of the 10th domain of the 15 repeating units of human fibronectin type III (FN3). Three loops at one end of the p-sandwich can be engineered to enable an Adnectin to specifically recognize a therapeutic target of interest. For further details see Protein Eng. Des. Sei. 18, 435- 444 (2005), US20080139791 , W02005056764 and US6818418B1.

[0098] Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein, typically thioredoxin (TrxA) which contains a constrained variable peptide loop inserted at the active site. For further details see Expert Opin. Biol. Ther. 5, 783-797 (2005).

[0099] Microbodies are derived from naturally occurring microproteins of 25-50 amino acids in length which contain 3-4 cysteine bridges - examples of microproteins include KalataBI and conotoxin and knottins. The microproteins have a loop which can be engineered to include up to 25 amino acids without affecting the overall fold of the microprotein. For further details of engineered knottin domains, see W02008098796.

[0100] The term "Fv" or "Fv fragment" or "Fv region" as used herein refers to a polypeptide that comprises the VH and VL domains of a single antibody.

[0101] The term "Fc" or "Fc region", as used herein refers to the polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Fc may refer to this region in isolation, or this region in the context of an Fc polypeptide, as described below. By "Fc polypeptide" or “Fc-derived polypeptide” as used herein is meant a polypeptide that comprises all or part of an Fc region. Fc polypeptides herein include but are not limited to antibodies, Fc fusions and Fc fragments. Also, Fc regions provided herein include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function. Also, Fc regions provided herein include chimeric Fc regions comprising different portions or domains of different Fc regions, e.g., derived from antibodies of different isotype or species. Fc thus refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge N-terminal to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc comprises immunoglobulin domains Cy2 (CH2) and Cy 3 (CH3) and the hinge between Cy 1 and Cy 2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to comprise residues C226, P230 or A231 to its carboxyl-terminus, wherein the numbering is according to the EU index. The "CH2 domain" of a human IgG Fc region usually extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. In one embodiment, a carbohydrate chain is attached to the CH2 domain. The CH2 domain herein may be a native sequence CH2 domain or variant CH2 domain. The "CH3 domain" comprises the stretch of residues C-terminal to a CH2 domain in an Fc region (i.e. from an amino acid residue at about position 341 to an amino acid residue at about position 447 of an IgG). The CH3 region herein may be a native sequence CH3 domain or a variant CH3 domain (e.g. a CH3 domain with an introduced "protuberance" ("knob") in one chain thereof and a corresponding introduced "cavity" ("hole") in the other chain thereof; see US Patent No. 5,821 ,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical antibody heavy chains as herein described. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0102] The "knob-into-hole" technology is described e.g. in US 5,731 ,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Generally, the method involves introducing a protuberance ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protuberance can be positioned in the cavity so as to promote heterodimer formation and hinder homodimer formation. Protuberances 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 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 altering the nucleic acid encoding the polypeptides, e.g. by site-specific mutagenesis, or by peptide synthesis. In a specific embodiment a knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc region, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other one of the two subunits of the Fc domain. In a further specific embodiment, the subunit of the Fc region comprising the knob modification additionally comprises the amino acid substitution S354C, and the subunit of the Fc region comprising the hole modification additionally comprises the amino acid substitution Y349C. Introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thus further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering is according to EU index of Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0103] A "region equivalent to the Fc region of an immunoglobulin" is intended to include naturally occurring allelic variants of the Fc region of an immunoglobulin as well as variants having alterations which produce substitutions, additions, or deletions but which do not decrease substantially the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without substantial loss of biological function. Such variants can be selected according to general rules known in the art so as to have minimal effect on activity (see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

[0104] The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibodydependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen presenting cells, down regulation of cell surface receptors (e.g. B cell receptor), and B cell activation.

[0105] An "activating Fc receptor" is an Fc receptor that following engagement by an Fc region of an antibody elicits signaling events that stimulate the receptor-bearing cell to perform effector functions. Activating Fc receptors include FcyRllla (CD16a), FcyRI (CD64), FcyRlla (CD32), and FcaRI (CD89). A particular activating Fc receptor is human FcyRllla (see UniProt accession no. P08637, version 141), also referred to as CD16 or CD16A. In humans, CD16 consists of two isoforms, CD16A and CD16B, encoded by two highly homologous genes. CD16A is a transmembrane protein expressed by lymphocytes and some monocytes, whereas CD16B is linked to the plasma membrane via a GPI anchor and primarily expressed by neutrophils. Therefore, when reference is made herein to CD16 in the context of expression on NK cells herein, usually CD16A is meant unless otherwise indicated.

[0106] By "variable region" as used herein is meant the region of an antibody that comprises one or more Ig domains substantially encoded by any of the VL (including VK and VA) and / or VH genes that make up the light chain (including K and A) and heavy chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL or VH) comprise four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity.

[0107] The term "hypervariable region" or "HVR," as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops ("hypervariable loops"). Generally, native four-chain antibodies comprise six HVRs; three in the VH (H1 , H2, H3), and three in the VL (L1 , L2, L3). HVRs generally comprise amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs), the latter being of highest sequence variability and / or involved in antigen recognition. Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (HI), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987).) Exemplary CDRs (CDR-L1 , CDR-L2, CDR-L3, CDR-H1 , CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1 , 50-56 of L2, 89-97 of L3, 31-35B of H1 , 50-65 of H2, and 95-102 of H3. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).) Hypervariable regions (HVRs) are also referred to as complementarity determining regions (CDRs), and these terms are used herein interchangeably in reference to portions of the variable region that form the antigen-binding regions. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), where the definitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table A as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody.

[0108] Table A. CDR defintions1

[0109] CDR Kabat Chotia AbM2

[0110] VH CDR1 31-35 26-32 26-35

[0111] VH CDR2 50-65 52-58 50-58

[0112] VH CDR3 95-102 95-102 95-102 VL CDRI 24-34 26-32 24-34

[0113] VL CDR2 50-56 50-52 50-56

[0114] VL CDR3 89-97 91-96 89-97

[0115] 1Numbering of all CDR definitions in Table A is according to the numbering conventions set forth by Kabat et al. (see below).

[0116] 2"AbM" with a lowercase "b" as used in Table A refers to the CDRs as defined by Oxford Molecular’s "AbM" antibody modeling software.

[0117]

[0118] Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system. With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1 , a-CDRL2, a-CDR-L3, a-CDR-H1 , a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31 -34 of L1 , 50-55 of L2, 89-96 of L3, 31 -35B of H1 , 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.

[0119] As used herein, the term "affinity matured" in the context of antigen binding molecules (e.g., antibodies) refers to an antigen-binding molecule that is derived from a reference antigen-binding molecule, e.g., by mutation, binds to the same antigen, preferably binds to the same epitope, as the reference antibody; and has a higher affinity for the antigen than that of the reference antigenbinding molecule. Affinity maturation generally involves modification of one or more amino acid residues in one or more CDRs of the antigen-binding molecule. Typically, the affinity matured antigen-binding molecule binds to the same epitope as the initial reference antigen-binding molecule.

[0120] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 , and lgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, 5, e, y, and m respectively.

[0121] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. An "agonist antibody", as used herein, is an antibody which mimics at least one of the functional activities of a polypeptide of interest.

[0122] The term "specifically binds" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigenbinding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 / KD. Affinity can be determined in a manner known per se, depending on the specific combination of antigen-binding protein and antigen of interest. Avidity is herein understood to refer to the strength of binding of a target molecule with multiple binding sites by a larger complex of binding agents, i.e. the strength of binding of multivalent binding. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding protein and the valency, i.e. the number of binding sites present on the antigen-binding protein. Affinity, on the other hand refers to simple monovalent receptor ligand systems. Typically, an antigen-binding region of a multispecific antigen binding protein as described herein will specifically bind its target molecule (antigen) with a dissociation constant (KD) of about 106to IO12M or less, and preferably 108to 1012M or less, and / or with a binding affinity of at least IO6M or 107M, preferably at least 108M, more preferably at least 109M, such as at least 10’10, IO11, IO12M or less. Any KD value greater than 104M (i.e. less than 100 pM) is generally considered to indicate non-specific binding. Thus, an antigen-binding region that “specifically binds” an antigen, is an antigen-binding domain that binds the antigen with a KD value of no more than 1 O’4M, as may be determined as herein described below. Preferably, an antigen-binding region of a multispecific antigen binding protein as described herein will specifically bind to the target molecule with an affinity less than 800, 400, 200, 100 50, 10 or 5 nM, more preferably less than 1 nM, such as less than 500, 200, 100, 50, 10 or 5 pM. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure (see e.g. Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988), Coligan et al., eds.. Current Protocols in Immunology, Greene Publishing Assoc, and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983)). Specific illustrative embodiments are described in the following.

[0123] A "KD" or "KD value" can be measured by using an ELISA as described in the Examples herein or by using surface plasmon resonance assays using a BIAcore™-2000 or a BIAcore ™-3000 (BIAcore, Inc., Piscataway, NJ) In an exemplary method, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N’-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier’s instructions. Antigen is diluted with 10mM sodium acetate, pH 4.8, into 5 pg / ml (~0.2 pM) before injection at a flow rate of 5pl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of the antibody or Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25pl / min. Association rates (kon) and dissociation rates (kOff) are calculated using a simple one-to-one Langmuir binding model (BIAcore Evaluation Software version 3.2) by simultaneous fitting the association and dissociation sensogram. The equilibrium dissociation constant (KD) is calculated as the ratio koff / kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds 106M’1S’1by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25°C of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophotometer (Aviv Instruments) or a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir red cuvette.

[0124] The term "humanized antibody" or "humanized immunoglobulin" refers to an immunoglobulin comprising a human framework, at least one and preferably all complementarity determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85%, at least 90%, and at least 95% identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. See, e.g., Queen et al., U.S. Pat. Nos. 5,530,101 ; 5,585,089; 5,693,761 ; 5,693,762; 6,180,370 (each of which is incorporated by reference in its entirety). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Mol. Immunol., 28:489 498 (1991); Studnicka et al., Prot. Eng. 7:805 814 (1994); Roguska et al., Proc. Natl. Acad. Sci. 91 :969 973 (1994), and chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby incorporated by reference in their entireties.

[0125] One class of antigen-binding regions for use in the multispecific antigen binding protein described herein comprises immunoglobulin single variable domains (ISVDs) with an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring single variable domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring single variable domain sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being. This can be performed in a manner known per se, which will be clear to the skilled person, for example on the basis of the prior art on humanization including e.g. Jones et al. (Nature 321 :522-525, 1986); Riechmann et al., (Nature 332:323-329, 1988); Presta (Curr. Op. Struct. Biol. 2:593-596, 1992), Vaswani and Hamilton (Ann. Allergy, Asthma and Immunol., 1 :105-1151998); Harris (Biochem. Soc. Transactions, 23:1035-1038, 1995); Hurle and Gross (Curr. Op. Biotech., 5:428-433, 1994), and specific prior art relating to humanization of VHHS such as e.g. Vincke et al. (2009, J. Biol. Chem. 284:3273-3284). Again, it should be noted that such humanized single variable domains can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring single variable domain as a starting material.

[0126] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1 , FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

[0127] An "acceptor human framework" for the purposes herein is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or a human consensus framework, as defined below. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain amino acid sequence changes. In some embodiments, the number of amino acid changes are 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

[0128] As an alternative to humanization, human antibodies can be generated. By “human antibody” is meant an antibody containing entirely human light and heavy chains as well as constant regions, produced by any of the known standard methods. For example, transgenic animals (e.g., mice) are available that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region PH gene in chimeric and germline mutant mice results in the complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ line mutant mice will result in the production of human antibodies after immunization. See, e.g., Jakobovits et al., Proc. Nat. Acad. Sci. USA, 90:255 1 (1993); Jakobovits et al., Nature, 362:255-258 (1993). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for their review see, e.g., Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-57 1 (1993). Human antibodies may also be generated by in vitro activated B cells or SCID mice with its immune system reconstituted with human cells. Once a human antibody is obtained, its coding DNA sequences can be isolated, cloned and introduced into an appropriate expression system i.e. a cell line, preferably from a mammal, which subsequently express and liberate it into a culture media from which the antibody can be isolated.

[0129] Amino acid substitutions are herein indicated as AOXAS, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence, and As indicates the substitute amino acid as present in that position in the modified amino acid sequence. For example, I8H denotes that the original amino acid isoleucine (I) in position 8 is changed to a histidine (H).

[0130] Amino acid deletions are herein indicated as A0X-, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence and the dash indicates that the original amino acid Aois no longer present in the modified amino acid sequence. For example N59- indicates that the asparagine (N) in position 59 is deleted in the modified amino acid sequence.

[0131] Amino acid insertion are herein indicated as A0X insAi-n, wherein Aoindicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence and insAi-n indicates that amino acids 1 - n replace the original amino acid Ao. For example G84 insGGGGG indicates that the original glycine in position 84 is replaced by a sequence of 5 glycines in the modified amino acid sequence.

[0132] As used herein, the term gamma delta (yb) T cells refers to a small subset of T cells that express on their surface a distinct, defining T Cell Receptor (TCR). This TCR is made up of one gamma (y) and one delta (6) chain. Each chain contains a variable (V) region, a constant (C) region, a transmembrane region and a cytoplasmic tail. The V region contains an antigen binding site. There are two major sub-types of human yb T cells: one that is dominant in the peripheral blood and one that is dominant in non-haematopoietic tissues. The two sub-types may be defined by the type of 5 and / or y present on the cells. For example, yb T cells that are dominant in peripheral blood primarily express the delta variable 2 chain (Vb2). yb T cells that are dominant in non-haematopoietic tissues (i.e. are tissue-resident) primarily express the delta variable 1 chain (Vb1).

[0133] References to “Vb1 T cells” or “Vb1+ T cells” refer to yb T cells with a Vb1 chain, i.e. Vb1 + cells. References to “delta variable 1” may also referred to asVbl orVdl , and a nucleotide encoding a TCR chain containing this region or the TCR protein complex comprising this region may be referred to as “TRDV1”. Antibodies or fragments thereof which interact with the Vb1 chain of a yb TCR, are all effectively antibodies or fragments thereof which bind to Vb1 and may referred to as “anti-TCR delta variable 1 antibodies or fragments thereof’ or “anti-Vb1 antibodies or fragments thereof’ or “anti-TRDV1 antibodies or fragments thereof’ or “anti-TRDV1 antibodies or fragments thereof’.

[0134] Additional references are made herein to other delta chains such as the “delta variable 2” chain. These can be referred to in a similar manner. For example, delta variable 2 chains can be referred to as Vb2, while a nucleotide encoding a TCR chain containing this region or the TCR protein complex comprising this region may be referred to as “TRDV2”.

[0135] References to “gamma variable chains” are also made herein. These may be referred to as y-chains or Vy, while a nucleotide encoding a TCR chain containing this region or the TCR protein complex comprising this region may be referred to as TRGV. For example, TRGV4 refers to Vy4 chain.

[0136] The term “T-cell receptor complex” is the complex of proteins comprising the “T-cell receptor” (or “TCR”) found on the surface of T-cells responsible for recognising a variety of antigens. The T-cell receptor complex comprises either the alpha and beta chains of the T-cell receptor, or in the case of gamma delta T cells, the gamma and delta chains of the T-cell receptor, and up to 6 additional chains or more, such as CD3b, CD3y, CD3e and CD3 , although the precise makeup of T-cell receptor complexes can vary. The T-cell receptor complex mediates intracellular signalling in the T-cell, which may lead to T-cell activation. The term “tumor associated antigen” (TAA) as used herein means any antigen including but not limited to a protein, glycoprotein, ganglioside, carbohydrate, lipid that is associated with cancer. Such antigen can be expressed on malignant cells or in the tumor microenvironment such as on tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltrates. Expressly included in the term TAA are homologues of a wild-type TAA that differs therefrom as a result of tumor-specific mutations (which can be patient-specific or shared) and that result in altered amino acid sequences, i.e. so-called neoantigens.

[0137] A “nucleic acid construct” or “nucleic acid vector” is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term “nucleic acid construct” therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. The terms “expression vector” or expression construct" refer to nucleic acid molecules that are capable of effecting expression of a nucleotide sequence or gene in host cells or host organisms compatible with such expression vectors or constructs. These expression vectors typically include regulatory sequence elements that are operably linked to the nucleotide sequence to be expressed to effect its expression. Such regulatory elements usually at least include suitable transcription regulatory sequences and optionally, 3’ transcription termination signals. Additional elements necessary or helpful in effecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector will be suitable for replication in the host cell or organism whereas an expression construct will usually integrate in the host cell’s genome for it to be maintained. Techniques for the introduction of nucleic acid into cells are well established in the art and any suitable technique may be employed, in accordance with the particular circumstances. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. adenovirus, AAV, lentivirus or vaccinia. For microbial, e.g. bacterial, cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage. The introduced nucleic acid may be on an extra-chromosomal vector within the cell or the nucleic acid may be integrated into the genome of the host cell. Integration may be promoted by inclusion of sequences within the nucleic acid or vector which promote recombination with the genome, in accordance with standard techniques. The introduction may be followed by expression of the nucleic acid to produce the encoded fusion protein. In some embodiments, host cells (which may include cells actually transformed although more likely the cells will be descendants of the transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, so that the encoded fusion protein polypeptide is produced, when an inducible promoter is used, expression may require the activation of the inducible promoter.

[0138] As used herein, the term “promoter” or “transcription regulatory sequence” refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A “constitutive” promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An “inducible” promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer.

[0139] The term “selectable marker” is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. The term “reporter” may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP). Selectable markers may be dominant or recessive or bidirectional.

[0140] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

[0141] The terms “protein” or “polypeptide” are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin.

[0142] The term “signal peptide” (sometimes referred to as signal sequence) is a short peptide (usually 16-30 amino acids long) present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. At the end of the signal peptide there is usually a stretch of amino acids that is recognized and cleaved by signal peptidase either during or after completion of translocation (from the cytosol into the secretory pathway, i.e. ER) to generate a free signal peptide and a mature protein. Signal peptides are extremely heterogeneous, and many prokaryotic and eukaryotic signal peptides are functionally interchangeable even between different species however the efficiency of protein secretion may depend on the signal peptide. Suitable signal peptides are generally known in the art e.g. from Kall et al. (2004 J. Mol. Biol. 338: 1027-1036) and von Heijne (1985, J Mol Biol. 184 (1): 99-105).

[0143] The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site. “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide. The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and / or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

[0144] The term "heterologous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced but has been obtained from another cell or synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is herein encompassed by the term heterologous nucleic acid or protein. The term heterologous also applies to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

[0145] Detailed description of the invention

[0146] A multispecific antigen binding protein

[0147] y6 T cells have been tested as cellular immunotherapies in humans. Despite a great safety profile, cellular therapies have thus far lacked efficacy or persistence. True T cell receptor agonist antibodies or antibody conjugates do not exist, as binding of an antibody or fragment to the TCR alone does not induce T cell activation. TCR activation using antibodies or engaging molecules requires TCR and CD3 clustering and cross linking, ideally causing the formation of a T cell receptor synapse. Whilst achievable with current technology T cell engagers, attempts of inducing growth and / or targeting / redirecting y6 T cells in-situ via their TCR with antibodies or drugs has been shown to lead to early exhaustion, in the case of V62 T cells even to the disappearance of the cells from human blood entirely. The present inventors realized that this may, at least in part, be due to a lack of any co-stimulation or cytokine delivery in these settings. Indeed, they have surprisingly found that a targeted engagement via the V6 TCR with multispecific antigen binding proteins that include a local supply of a signal 2 (co-stimulation) and / or signal 3 (cytokine), leads to the strong induction of proliferation, differentiation into hyper functional, activated innate T cells and redirection of y6 T cells in-situ, without causing terminal differentiation, anergy or even depletion of these highly desirable cells.

[0148] Therefore, in a first aspect, there is provided pertains to a multispecific antigen binding protein. In one embodiment, the multispecific antigen binding protein comprises at least: a) at least one antigen-binding region that specifically binds a tumor- or pathogen-associated antigen; b) an antigen-binding region that specifically binds an epitope of a y6 T cell receptor (TCR); and, c) a y6 T cell-activating agonist. In one embodiment, the multispecific antigen binding protein thus comprises at least: a) a first antigen-binding region and an optional second antigen-binding region that specifically bind a tumor or pathogen associated antigen; b) a third antigen-binding region that specifically binds an epitope of a y6 T cell receptor (TCR); and, c) a y6 T cell-activating agonist.

[0149] Antigen binding regions binding to tumor- or pathogen-associated antigens

[0150] In one embodiment, a multispecific antigen binding protein described herein comprises a first antigen-binding region that specifically binds a tumor or pathogen associated antigen. The multispecific antigen binding protein can further comprise another i.e. second antigen-binding region that also specifically binds a tumor or pathogen associated antigen.

[0151] A multispecific antigen binding protein as provide herein this comprises at least one antigenbinding region that specifically binds a tumor associated antigen (TAA) or a pathogen associated antigen. For practical purposes we shall herein refer to a tumor or pathogen associated antigen as a “tumor associated antigen” or “TAA”, which term is thus to be understood that it can also refer to a pathogen associated antigen.

[0152] An antigen-binding region as used in a multispecific antigen binding protein described herein can be derived from any of a variety of immunoglobulin or non-immunoglobulin scaffolds, for example affibodies based on the Z-domain of staphylococcal protein A, engineered Kunitz domains, monobodies or adnectins based on the 10th extracellular domain of human fibronectin III, anticalins derived from lipocalins, DARPins (designed ankyrin repeat domains), Affilins, multimerized LDLR-A module, avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra (2009) Current Opinion in Chemical Biology 13:245-255, the disclosure of which is incorporated herein by reference.

[0153] In a preferred embodiment, an antigen-binding region as used in a multispecific antigen binding protein described herein comprises or consists of an immunoglobulin variable region. Such immunoglobulin variable regions can comprise or consist of variable domains that are commonly derived from antibodies (immunoglobulin chains), e.g. in the form of associated VL and VH domains found on two polypeptide chains, such as present in a Fab. Alternatively, immunoglobulin variable domains can comprise or consist of a single chain antigen-binding domain such as a scFv, a VH domain, a VL domain, or an immunoglobulin single variable domain (ISVD) such as a dAb, a V-NAR domain or a VHH domain. An immunoglobulin variable region to be used in a multispecific antigen binding protein described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as herein defined above.

[0154] In one embodiment, the antigen-binding region that specifically binds a TAA is an antigenbinding region derived from immunoglobulin or non-immunoglobulin scaffolds as defined above. Preferably, the antigen-binding region that specifically binds a TAA comprises or consists of at least one immunoglobulin variable domain. More preferably, the antigen-binding region that specifically binds a TAA comprises or consists of a Fab that specifically binds a TAA or an immunoglobulin single variable domain (ISVD) that specifically binds a TAA. In one embodiment, the antigen-binding region that specifically binds a TAA is an antigen-binding region that binds the TAA with a KD value of no more than 1CM M, as may be determined as herein described above.

[0155] In one embodiment, the antigen-binding region that specifically binds a TAA comprises or consists of a human or humanized immunoglobulin variable region or immunoglobulin single variable region as herein defined above.

[0156] In one embodiment, a multispecific antigen binding protein as described herein comprises two antigen-binding regions that specifically bind a TAA, i.e. a first and a second antigen-binding region. In a multispecific antigen binding protein that comprises two antigen-binding regions that specifically bind a TAA, the two antigen-binding regions can bind one and the same TAA or they can bind at least two different TAAs. In one embodiment of a multispecific antigen binding protein that comprises two antigen-binding regions that specifically bind a TAA, the two antigen-binding regions are identical. Thus, as regards the two antigen-binding regions that specifically bind a TAA, a multispecific antigen binding protein as described herein can be a homodimeric or a heterodimeric antigen binding protein.

[0157] As used herein, the term tumor-associated antigen (TAA) refers to an antigen that is differentially expressed by cancer / tumor cells as compared to normal, i.e. non-tumoral cells. Alternatively, a TAA can be an antigen that is expressed by non-tumoral cells (e.g. immune cells) having a pro-tumoral effect (e.g. an immunosuppressive effect), and can thereby be exploited in order to target cancer cells. A TAA can thus be any antigen that potentially stimulates apparently tumor-specific immune responses. Some of these antigens are encoded, although not necessarily expressed, or expressed at lower levels or less frequently, by normal cells. These antigens can be characterized as those which are normally silent (i.e., not expressed) in normal cells, those that are expressed only at certain stages of differentiation and those that are temporally expressed such as embryonic and fetal antigens. Other TAAs, also referred to as tumor-specific antigens (TSAs), are encoded by mutant cellular genes, such as oncogenes (e.g., activated ras oncogene), suppressor genes (e.g., mutant p53), fusion proteins resulting from internal deletions or chromosomal translocations, including neo-antigens. Still other TAAs antigens can be encoded by viral genes such as those carried on RNA and DNA tumor viruses. Still other TAAs can be expressed on immune cells capable of contributing to or mediating a pro-tumoral effect, e.g. cell that contributes to immune evasion, a monocyte or a macrophage, optionally a suppressor T cell, regulatory T cell, or myeloid-derived suppressor cell. The TAAs are usually normal cell surface antigens which are either overexpressed or expressed at abnormal times or are expressed by a targeted population of cells. Ideally the target TAA is expressed only on proliferative cells (e.g., tumor cells) or pro-tumoral cells (e.g. immune cells having an immunosuppressive effect), however this is rarely observed in practice. As a result, target antigens are in many cases selected on the basis of differential expression between proliferative / disease tissue and healthy tissue.

[0158] As regards a pathogen associated antigen that is specifically bound by a first and / or second antigen-binding regions of a multispecific antigen binding protein as described herein, the antigen can be of a microbial or parasitic pathogen such a virus, a bacterium, a fungus or a protozoan.

[0159] In one embodiment, the first and / or second antigen-binding regions can specifically bind specific classes of immune cells so as to deplete them for treating autoimmune diseases. In one embodiment, the first and / or second antigen-binding regions can specifically bind CD19 to target B cells in the treatment of an autoimmune disease such as Lupus.

[0160] Thus, in one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA selected from the group consisting of: Her2 (ErbB2 / Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171 , CTLA-4 (CD152), PD1 , PSCA, L1-CAM, EpCAM, PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1 , CD52, CD56, CD80, CD70, CRIPTO-1 , E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), B7.1 , B7.2, B7-H3, B7-H4, B7-H6, CA125, PD-L1 , IL-6 receptor, IL-8 receptor, IL1 accessory Protein, MAGE, MART-1 / Melan-A, gp100, MICA, MICB, adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017-1 A / GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1 , nectin-4, a UL16-binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1 , prostate specific antigen (PSA), T-cell receptor / CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Mullerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1 , TRAILR2, BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC1 , MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), HER-3 / ERBB3, HER-4 / ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, integrin receptors, a5p3 integrins, a5p1 integrins, allbp3-integrins, PDGF alpha receptor, PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, CSF1R, a-fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18)), folate receptor alpha (FRa, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, a-catenin, p-catenin and y-catenin, Plexin-A1 , TNFRSF10B, AXL, EDNRB, OLR1 , ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1 , cdc27, CDCP1 , adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp-1 , P1 A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, muc16, mud 7, mmp9, FAP, Lewis-Y, EGFRvlll, GPC3, gpA33, 5T4, SSTR2, CD73, CD25, CD45, CD133, FGFR2b, CD79B, BTLA, Fibronectin extra-domain B, GM3, LAG-3, 0X40, PDGFRa, TIGIT, VEGF-a, GPCR5D and scatter factor receptor kinase, although this is not intended to be exhaustive.

[0161] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA that is expressed in a solid tumor, which TAA is selected from the group consisting of: B7-H3, BAGE, CD52, CD70, CRIPTO-1 , CTLA-4 (CD152), EGF, EGF receptor (EGFR / ERBB1), EpCAM, E-selectin, folate receptor alpha (FRa, FOLR1), hCG, Ig-idiotype, IL-8 receptor, integrin receptors, Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18), L1-CAM, LAGE-1 , PDGF, receptor protein tyrosine kinase 3 (TYRO-3), STEAP1 , tissue factor (TF, CD142), TNFRSF10B, allbp3-integrins, a GAGE-tumor antigen, a GD2 ganglioside, a GM2 ganglioside, a heterodimeric receptor comprised of at least one HER subunit, a RAET1 protein, carcinoembryonic antigen (CEA), a UL16-binding protein (ULBP), a5p1 integrins, a5p3 integrins, ADAM12, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), aml1 , Angiopoietin-2, anti-Mullerian hormone Type II receptor, AXL, B7.1 , B7.2, B7-H4, , B7-H6, BCMA, brain glycogen phosphorylase, CA125, CanAg, CCR4, CCR6, CD133, CD138, CD171 , CD19, CD2, CD20, CD25, CD30, CD33 (Siglec3), CD38, CD4, CD40, CD45, CD47, CD56, CD73, CD79, CD80, cdc27, CDCP1 , CDK4, CEACAM5, c-erbB-2, cMET, colorectal associated antigen (CRC)-C017-1A / GA733, Connexin 37, CSF1R, CT-7, cyclophilin b, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, EBV-encoded nuclear antigen (EBNA)-I, E-cadherin, EDNRB, EGFRvlll, EPHA4, EphB2, etv6, FAP, Flt3, fodrin, gastrin releasing peptide receptor antigen, Glycoprotein NMB, GnT-V, gp100, gp75, gpA33, GPC3, GPRC5D, Her2 (ErbB2 / Neu), HER-3 / ERBB3, HER-4 / ERBB4, human papillomavirus protein, IL-1 accessory protein, IL-1 accessory Protein, IL-6 receptor, IL-6 receptor, imp-1 , Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), Lewis-Y, MAGE, MAGE-A3, MART-1 ZMelan-A, Melanotransferrin, mesothelin (MSLN), MICA, MICB, mmp9, MUC1 , muc16, muc17, MUC1-C, Muc 6, MUM-1 , NAG, NaPi2b, nectin-4, NTRKR1 (EC 2.7.10.1), NY-ESO-1 , OLR1 , p120ctn, p15, P-cadherin, PD1 , PDGF alpha receptor, PDGF beta receptor, PD-L1 , PLAUR, Plexin-A1 , PRAME, prostate specific antigen (PSA), protein tyrosine kinase 7(PTK7), PSCA, PSMA (prostate specific membrane antigen), RAGE, Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), SCP-1 , SLAMF7, SSTR2, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, sVE-cadherin, T-cell receptor / CD3-zeta chain, TGF-alpha, TMEFF2, TRAILR1 , TRAILR2, TROP2, TYRP1 , VEGF, a-catenin, a-fetoprotein, p-catenin, y-catenin, and 5T4.

[0162] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA that is expressed in a haem (liquid) tumor, which TAA is selected from the group consisting of: B7-H3, BAGE, CD52, CD70, CRIPTO-1 , CTLA-4 (CD152), EGF receptor (EGFR / ERBB1), E-selectin, Ig-idiotype, IL-8 receptor, integrin receptors, Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18), LI-CAM, LAGE-1 , PDGF, receptor protein tyrosine kinase 3 (TYRO-3), STEAP1 , tissue factor (TF, CD142), TNFRSF10B, allbp3-integri ns, a GAGE-tumor antigen, a GD2 ganglioside, a GM2 ganglioside, a heterodimeric receptor comprised of at least one HER subunit, a RAET1 protein, carcinoembryonic antigen (CEA), a UL16-binding protein (ULBP), a5p1 integrins, a5p3 integrins, ADAM12, aml1 , Angiopoietin-2, AXL, B7.1 , B7.2, B7-H4, , B7-H6, BCMA, CA125, CCR4, CCR6, CD123, CD133, CD138, CD171 , CD19, CD2, CD20, CD22 (Siglec2), CD25, CD30, CD33 (Siglec3), CD38, CD4, CD40, CD45, CD47, CD56, CD73, CD79, CD80, cdc27, CDCP1 , CDK4, CEACAM5, c-erbB-2, cMET, Connexin 37, CSF1R, CT-7, cyclophilin b, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, EBV-encoded nuclear antigen (EBNA)-I, E-cadherin, EDNRB, EPHA4, EphB2, etv6, FAP, FcRL5 / FcRH5, Flt3, fodrin, gpA33, GPRC5D, Her2 (ErbB2 / Neu), HER-3 / ERBB3, HER-4 / ERBB4, IL-1 accessory protein, IL-1 accessory Protein, IL-6 receptor, IL-6 receptor, imp-1 , Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), Lewis-Y, MAGE, MAGE-A3, mesothelin (MSLN), MICA, MICB, mmp9, MUC1 , MUC1 -C, MUM-1 , NAG, NTRKR1 (EC 2.7.10.1), NY-ESO-1 , OLR1 , p120ctn, p15, PD1 , PDGF alpha receptor, PDGF beta receptor, PD-L1 , PLAUR, PRAME, protein tyrosine kinase 7(PTK7), RAGE, Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), SCP-1 , SLAMF7, SSTR2, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, sVE-cadherin, T-cell receptor / CD3-zeta chain, TGF-alpha, TMEFF2, TRAILR1 , TRAILR2, TROP2, VEGF, a-catenin, a-fetoprotein, p-catenin, and y-catenin.

[0163] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one antigen-binding region that is obtained / obtainable from a cytotoxic monoclonal antibody against a TAA as is known in the art. In one embodiment, the at least one antigen-binding region at least comprises the six CDR sequences that are obtained / obtainable from a monoclonal antibody against a TAA as is known in the art. In one embodiment, the at least one antigen-binding region at least comprises the variable light (VL) domain and variable heavy (VH) domain sequences or the VHH domain sequences that are obtained / obtainable from a monoclonal antibody against a TAA as is known in the art. Examples of such monoclonal antibodies against TAAs include: Trastuzumab, Cetuximab, Rituximab, Daratumumab, Avelumab, Atezolizumab, Durvalumab, Cosibelimab, Margetuximab, Pertuzumab, Enoblituzumab, Necitumumab, Panitumumab, Amivantamab, Zolbetuximab, Dinutuximab, Naxitamab , Enfortumab, Farletuzumab, Tisotumab, Mirvetuximab, Sacituzumab, Vobramitamab, Onartuzumab, Sibrotuzumab, Olaratumab, Rovalpituzumab, Adebrelimab, Alemtuzumab, Belantamab, Bevacizumab, Brentuximab, Camrelizumab, Cemiplimab, Dostarlimab, Emapalumab, Enlonstobart, Gemtuzumab, Ibritumomab, Inotuzumab, Ipilimumab, Isatuximab, Loncastuximab, Mogamulizumab, Moxetumomab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Pembrolizumab, Penpulimab, Polatuzumab, Prolgolimab, Pucotenlimab, Racotumomab, Ramucirumab, Relatlimab, Retifanlimab, Ripertamab, Serplulimab, Sintilimab, Socazolimab, Sugemalimab, Tafasitamab, Tagitanlimab, Tebentafusp, Tislelizumab, Toripalimab, Zuberitamab, Benmelstobart, Iparomlimab, Tuvonralimab, Anvatabart, Apamistamab, Bemarituzumab, Cetrelimab, Cobolimab, Datopotamab, Domvanalimab, Emactuzumab, Favezelimab, Felzartamab, Fianlimab, Finotonlimab, Geptanolimab, Gotistobart, Ivuxolimab, Lemzoparlimab, Luveltamab, Magrolimab, Mecbotamab, Monalizumab, Nofazinlimab, Nurulimab, Ociperlimab, Oleclumab, Onfekafusp, Patritumab, Pivekimab, Quavonlimab, Retlirafusp, Rosopatamab, Rulonilimab, Sabatolimab, Sasanlimab, Telisotuzumab, Tiragolumab, Tusamitamab, Vibostolimab, Vobramitamab, Zilovertamab, Suvemcitug, Becotatug, Tifcemalimab, Blinatumomab, Cadonilimab, Disitamab, Edrecolomab, Elranatamab, Epcoritamab, Glofitamab, Mosunetuzumab, Talquetamab, Teclistamab, Tositumomab, Tremelimumab, Zimberelimab, Odronextamab, Ivonescimab, Anbenitamab, Izalontamab, Linvoseltamab, Tarlatamab, Zanidatamab, Volrustomig, Zenocutuzumab, Botensilimab, Izalontamab, Rilvegostomig, Envafolimab, Erfonrilimab, Erfonrilimab and Ozekibart.

[0164] In one embodiment therefore, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA, which antigen-binding region comprises a combination of complementarity-determining regions (CDRs) CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 2 (trastuzumab); b) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 3, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 4 (cetuximab); c) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 5, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 6 (rituximab); d) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 7, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 8 (daratumumab); e) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 9, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 10 (avelumab); f) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 11 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 12 (atezolizumab); g) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 13, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 14 (durvalumab); h) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 15, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 16 (cosibelimab); i) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 17, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 18 (margetuximab); j) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 19, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 20 (pertuzumab); k) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 21 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 22 (enoblituzumab); I) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 23, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 24 (necitumumab); m) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 25, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 26 (panitumumab); n) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 27, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 28 (amivantamab EGFR-binding); o) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 29, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 30 (amivantamab cMet-binding); p) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 31 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 32 (zolbetuximab); q) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 33, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 34 (dinutuximab); r) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 35, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 36 (naxitamab); s) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 37, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 38 (enfortumab); t) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 39, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 40 (farletuzumab); u) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 41 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 42 (tisotumab); v) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 43, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 44 (mirvetuximab); w) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 45, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 46 (sacituzumab); x) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 47, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 48 (vobramitamab); y) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 49, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 50 (Onartuzumab); z) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 51 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 52 (sibrotuzumab) aa) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 53, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 54 (olaratumab); ab) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 55, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 56 (rovalpituzumab); ac) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 57, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 58 (adebrelimab); ad) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 59, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 60 (alemtuzumab); ae) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 61 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 62 (belantamab); at) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 63, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 64 (Bevacizumab); ag) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 65, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 66 (brentuximab); ah) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 67, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 68 (camrelizumab); ai) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 69, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 70 (cemiplimab); aj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 71 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 72 (dostarlimab); ak) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 73, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 74 (emapalumab); al) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 75, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 76 (enlonstobart); am) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 77, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 78 (gemtuzumab); an) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 79, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 80 (ibritumomab); ao) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 81 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 82 (inotuzumab); ap) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 83, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 84 (ipilimumab); aq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 85, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 86 (isatuximab); ar) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 87, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 88 (loncastuximab); as) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 89, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 90 (mogamulizumab); at) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 91 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 92 (moxetumomab); au) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 93, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 94 (nimotuzumab); av) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 95, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 96 (nivolumab); aw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 97, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 98 (obinutuzumab); ax) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 99, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 100 (ofatumumab); ay) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 101 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 102 (pembrolizumab); az) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 103, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 104 (Penpulimab); ba) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 105, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 106 (polatuzumab); bb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 107, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 108 (prolgolimab); be) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 109, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 110 (pucotenlimab); bd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 111 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 112 (racotumomab) be) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 113, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 114 (ramucirumab); bf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 115, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 116 (relatlimab); bg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 117, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 118 (retifanlimab); bh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 119, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 120 (ripertamab); bi) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 121 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 122 (serplulimab); bj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 123, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 124 (sintilimab); bk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 125, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 126 (socazolimab); bl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 127, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 128 (sugemalimab); bm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 129, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 130 (tafasitamab); bn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 131 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 132 (tagitanlimab); bo) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 133, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 134 (tebentafusp); bp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 135, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 136 (Tislelizumab); bq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 137, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 138 (Toripalimab); br) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 139, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 140 (zuberitamab); bs) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 141 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 142 (benmelstobart); bt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 143, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 144 (iparomlimab); bu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 145, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 146 (tuvonralimab); bv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 147, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 148 (anvatabart); bw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 149, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 150 (apamistamab); bx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 151 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 152 (bemarituzumab); by) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 153, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 154 (cetrelimab); bz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 155, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 156 (cobolimab); ca) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 157, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 158 (datopotamab); cb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 159, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 160 (domvanalimab); cd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 161 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 162 (emactuzumab); ce) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 163, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 164 (favezelimab); cf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 165, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 166 (felzartamab); eg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 167, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 168 (fianlimab); ch) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 169, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 170 (finotonlimab); ci) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 171 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 172 (geptanolimab); cj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 173, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 174 (gotistobart); ck) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 175, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 176 (ivuxolimab); cl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 177, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 178 (lemzoparlimab); cm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 179, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 180 (luveltamab); cn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 181 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 182 (magrolimab); co) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 183, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 184 (meebotamab); cp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 185, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 186 (monalizumab); cq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 187, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 188 (nofazinlimab); cr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 189, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 190 (nurulimab); cs) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 191 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 192 (ociperlimab); ct) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 193, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 194 (oleclumab); cu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 195, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 196 (onfekafusp); cv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 197, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 198 (patritumab); cw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 199, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 200 (pivekimab); ex) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 201 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 202 (quavonlimab); cy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 203, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 204 (retlirafusp); cz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 205, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 206 (rosopatamab); da) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 207, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 208 (rulonilimab); db) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 209, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 210 (sabatolimab); de) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 211 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 212 (sasanlimab); dd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 213, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 214 (telisotuzumab); de) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 215, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 216 (tiragolumab); df) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 217, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 218 (tusamitamab); dg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 219, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 220 (vibostolimab); dh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 221 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 222 (vobramitamab); di) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 223, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 224 (zilovertamab); dj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 225, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 226 (suvemcitug); dk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 227, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 228 (becotatug); dl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 229, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 230 (tifcemalimab); dm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 231 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 232 (blinatumomab - CD19); dn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 233, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 234 (blinatumomab - CD3); do) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 235, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 236 (cadonilimab - PD-1); dp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 237, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 238 (cadonilimab - CTLA4); dq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 239, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 240 (disitamab); dr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 241 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 242 (edrecolomab); ds) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 243, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 244 (elranatamab - BCMA); dt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 245, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 246 (elranatamab - DC3); du) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 247, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 248 (epcoritamab - CD20); dv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 249, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 250 (epcoritamab - CD3); dw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 251 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 252 (glofitamab - VH1 / VL1); dx); the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 253, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 254 (glofitamab -VH2 / VL2); dy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 255, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 256 (glofitamab -VH3 / VL3); dz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 257, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 258 (mosunetuzumab - CD20); ea) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 259, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 260 (mosunetuzumab - CD3); eb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 261 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 262 (talquetamab - GPCR5D); ec) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 263, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 264 (talquetamab - CD3); ed) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 265, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 266 (teclistamab - BCMA); ee) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 267, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 268 (teclistamab - CD3); ef) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 269, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 270 (tositumomab); eg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 271 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 272 (tremelimumab); eh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 273, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 274 (zimberelimab); ei) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 275, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 276 (odronextamab - CD20); ej) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 277, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 278 (odronextamab - CD3); ek) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 279, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 280 (ivonescimab - PD-1); el) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 281 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 282 (ivonescimab - VEGF); em) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 283, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 284 (anbenitamab - VH1 / VL1); en) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 285, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 286 (anbenitamab - VH2 / VL2); eo) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 287, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 288 (izalontamab - EGFR); ep) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 289, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 290 (izalontamab - HER3); eq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 291 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 292 (linvoseltamab - BCMA); er) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 293, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 294 (linvoseltamab - CD3); es) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 295, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 296 (tarlatamab - DLL3); et) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 297, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 298 (tarlatamab - CD3); eu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 299, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 300 (zanidatamab - VH1 / VL1); ev) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 301 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 302 (zanidatamab - VH2 / VL2); ew) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 303, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 304 (volrustomig - PD-1); ex) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 305, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 306 (volrustomig - CTLA-4); ey) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 307, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 308 (zenocutuzumab - HER2); ez) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 309, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 310 (zenocutuzumab - HER3); fa) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 311 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 312 (botensilimab - VH1 / VL1); fb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 313, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 314 (botensilimab - VH2 / VL2); fc) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 315, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 316 (izalontamab - EGFR); fd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 317, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 318 (izalontamab - HER3); fe) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 319, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 320 (rilvegostomig - TIGIT); and, ff) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 321 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 322 (rilvegostomig - PD-1).

[0165] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA, which antigenbinding region is an ISVD comprising a combination of CDRs CDR-1 , CDR-2 and CDR-3 selected from the group consisting of: a) the CDR-1 , CDR-2 and CDR-3 sequences as comprised in SEQ ID NO: 323 (Envafolimab); b) the CDR-1 , CDR-2 and CDR-3 sequences as comprised in SEQ ID NO: 324 (Erfonrilimab); c) the CDR-1 , CDR-2 and CDR-3 sequences as comprised in SEQ ID NO: 325 (Erfonrilimab); and) the CDR-1 , CDR-2 and CDR-3 sequences as comprised in SEQ ID NO: 326 (Oze ki bai ).

[0166] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA, which antigenbinding region comprises a combination of variable heavy (VH) and variable light (VL) domain sequences that have, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the VH and VL sequence combinations selected from the group consisting of: a) the VH sequence as comprised in SEQ ID NO: 1 , and the VL sequence as comprised in SEQ ID NO: 2 (trastuzumab); b) the VH sequence as comprised in SEQ ID NO: 3, and the VL sequence as comprised in SEQ ID NO: 4 (cetuximab); c) the VH sequence as comprised in SEQ ID NO: 5, and the VL sequence as comprised in SEQ ID NO: 6 (rituximab); d) the VH sequence as comprised in SEQ ID NO: 7, and the VL sequence as comprised in SEQ ID NO: 8 (daratumumab); e) the VH sequence as comprised in SEQ ID NO: 9, and the VL sequence as comprised in SEQ ID NO: 10 (avelumab); f) the VH sequence as comprised in SEQ ID NO: 11 , and the VL sequence as comprised in SEQ ID NO: 12 (atezolizumab); g) the VH sequence as comprised in SEQ ID NO: 13, and the VL sequence as comprised in SEQ ID NO: 14 (durvalumab); h) the VH sequence as comprised in SEQ ID NO: 15, and the VL sequence as comprised in SEQ ID NO: 16 (cosibelimab); i) the VH sequence as comprised in SEQ ID NO: 17, and the VL sequence as comprised in SEQ ID NO: 18 (margetuximab); j) the VH sequence as comprised in SEQ ID NO: 19, and the VL sequence as comprised in SEQ ID NO: 20 (pertuzumab); k) the VH sequence as comprised in SEQ ID NO: 21 , and the VL sequence as comprised in SEQ ID NO: 22 (enoblituzumab); I) the VH sequence as comprised in SEQ ID NO: 23, and the VL sequence as comprised in SEQ ID NO: 24 (necitumumab); m) the VH sequence as comprised in SEQ ID NO: 25, and the VL sequence as comprised in SEQ ID NO: 26 (panitumumab); n) the VH sequence as comprised in SEQ ID NO: 27, and the VL sequence as comprised in SEQ ID NO: 28 (amivantamab EGFR-binding); o) the VH sequence as comprised in SEQ ID NO: 29, and the VL sequence as comprised in SEQ ID NO: 30 (amivantamab cMet-binding); p) the VH sequence as comprised in SEQ ID NO: 31 , and the VL sequence as comprised in SEQ ID NO: 32 (zolbetuximab); q) the VH sequence as comprised in SEQ ID NO: 33, and the VL sequence as comprised in SEQ ID NO: 34 (dinutuximab); r) the VH sequence as comprised in SEQ ID NO: 35, and the VL sequence as comprised in SEQ ID NO: 36 (naxitamab); s) the VH sequence as comprised in SEQ ID NO: 37, and the VL sequence as comprised in SEQ ID NO: 38 (enfortumab); t) the VH sequence as comprised in SEQ ID NO: 39, and the VL sequence as comprised in SEQ ID NO: 40 (farletuzumab); u) the VH sequence as comprised in SEQ ID NO: 41 , and the VL sequence as comprised in SEQ ID NO: 42 (tisotumab); v) the VH sequence as comprised in SEQ ID NO: 43, and the VL sequence as comprised in SEQ ID NO: 44 (mirvetuximab); w) the VH sequence as comprised in SEQ ID NO: 45, and the VL sequence as comprised in SEQ ID NO: 46 (sacituzumab); x) the VH sequence as comprised in SEQ ID NO: 47, and the VL sequence as comprised in SEQ ID NO: 48 (vobramitamab); y) the VH sequence as comprised in SEQ ID NO: 49, and the VL sequence as comprised in SEQ ID NO: 50 (Onartuzumab); z) the VH sequence as comprised in SEQ ID NO: 51 , and the VL sequence as comprised in SEQ ID NO: 52 (sibrotuzumab) aa) the VH sequence as comprised in SEQ ID NO: 53, and the VL sequence as comprised in SEQ ID NO: 54 (olaratumab); ab) the VH sequence as comprised in SEQ ID NO: 55, and the VL sequence as comprised in SEQ ID NO: 56 (rovalpituzumab); ac) the VH sequence as comprised in SEQ ID NO: 57, and the VL sequence as comprised in SEQ ID NO: 58 (adebrelimab); ad) the VH sequence as comprised in SEQ ID NO: 59, and the VL sequence as comprised in SEQ ID NO: 60 (alemtuzumab); ae) the VH sequence as comprised in SEQ ID NO: 61 , and the VL sequence as comprised in SEQ ID NO: 62 (belantamab); at) the VH sequence as comprised in SEQ ID NO: 63, and the VL sequence as comprised in SEQ ID NO: 64 (Bevacizumab); ag) the VH sequence as comprised in SEQ ID NO: 65, and the VL sequence as comprised in SEQ ID NO: 66 (brentuximab); ah) the VH sequence as comprised in SEQ ID NO: 67, and the VL sequence as comprised in SEQ ID NO: 68 (camrelizumab); ai) the VH sequence as comprised in SEQ ID NO: 69, and the VL sequence as comprised in SEQ ID NO: 70 (cemiplimab); aj) the VH sequence as comprised in SEQ ID NO: 71 , and the VL sequence as comprised in SEQ ID NO: 72 (dostarlimab); ak) the VH sequence as comprised in SEQ ID NO: 73, and the VL sequence as comprised in SEQ ID NO: 74 (emapalumab); al) the VH sequence as comprised in SEQ ID NO: 75, and the VL sequence as comprised in SEQ ID NO: 76 (enlonstobart); am) the VH sequence as comprised in SEQ ID NO: 77, and the VL sequence as comprised in SEQ ID NO: 78 (gemtuzumab); an) the VH sequence as comprised in SEQ ID NO: 79, and the VL sequence as comprised in SEQ ID NO: 80 (ibritumomab); ao) the VH sequence as comprised in SEQ ID NO: 81 , and the VL sequence as comprised in SEQ ID NO: 82 (inotuzumab); ap) the VH sequence as comprised in SEQ ID NO: 83, and the VL sequence as comprised in SEQ ID NO: 84 (ipilimumab); aq) the VH sequence as comprised in SEQ ID NO: 85, and the VL sequence as comprised in SEQ ID NO: 86 (isatuximab); ar) the VH sequence as comprised in SEQ ID NO: 87, and the VL sequence as comprised in SEQ ID NO: 88 (loncastuximab); as) the VH sequence as comprised in SEQ ID NO: 89, and the VL sequence as comprised in SEQ ID NO: 90 (mogamulizumab); at) the VH sequence as comprised in SEQ ID NO: 91 , and the VL sequence as comprised in SEQ ID NO: 92 (moxetumomab); au) the VH sequence as comprised in SEQ ID NO: 93, and the VL sequence as comprised in SEQ ID NO: 94 (nimotuzumab); av) the VH sequence as comprised in SEQ ID NO: 95, and the VL sequence as comprised in SEQ ID NO: 96 (nivolumab); aw) the VH sequence as comprised in SEQ ID NO: 97, and the VL sequence as comprised in SEQ ID NO: 98 (obinutuzumab); ax) the VH sequence as comprised in SEQ ID NO: 99, and the VL sequence as comprised in SEQ ID NO: 100 (ofatumumab); ay) the VH sequence as comprised in SEQ ID NO: 101 , and the VL sequence as comprised in SEQ ID NO: 102 (pembrolizumab); az) the VH sequence as comprised in SEQ ID NO: 103, and the VL sequence as comprised in SEQ ID NO: 104 (Penpulimab); ba) the VH sequence as comprised in SEQ ID NO: 105, and the VL sequence as comprised in SEQ ID NO: 106 (polatuzumab); bb) the VH sequence as comprised in SEQ ID NO: 107, and the VL sequence as comprised in SEQ ID NO: 108 (prolgolimab); be) the VH sequence as comprised in SEQ ID NO: 109, and the VL sequence as comprised in SEQ ID NO: 110 (pucotenlimab); bd) the VH sequence as comprised in SEQ ID NO: 111 , and the VL sequence as comprised in SEQ ID NO: 112 (racotumomab) be) the VH sequence as comprised in SEQ ID NO: 113, and the VL sequence as comprised in SEQ ID NO: 114 (ramucirumab); bf) the VH sequence as comprised in SEQ ID NO: 115, and the VL sequence as comprised in SEQ ID NO: 116 (relatlimab); bg) the VH sequence as comprised in SEQ ID NO: 117, and the VL sequence as comprised in SEQ ID NO: 118 (retifanlimab); bh) the VH sequence as comprised in SEQ ID NO: 119, and the VL sequence as comprised in SEQ ID NO: 120 (ripertamab); bi) the VH sequence as comprised in SEQ ID NO: 121 , and the VL sequence as comprised in SEQ ID NO: 122 (serplulimab); bj) the VH sequence as comprised in SEQ ID NO: 123, and the VL sequence as comprised in SEQ ID NO: 124 (sintilimab); bk) the VH sequence as comprised in SEQ ID NO: 125, and the VL sequence as comprised in SEQ ID NO: 126 (socazolimab); bl) the VH sequence as comprised in SEQ ID NO: 127, and the VL sequence as comprised in SEQ ID NO: 128 (sugemalimab); bm) the VH sequence as comprised in SEQ ID NO: 129, and the VL sequence as comprised in SEQ ID NO: 130 (tafasitamab); bn) the VH sequence as comprised in SEQ ID NO: 131 , and the VL sequence as comprised in SEQ ID NO: 132 (tagitanlimab); bo) the VH sequence as comprised in SEQ ID NO: 133, and the VL sequence as comprised in SEQ ID NO: 134 (tebentafusp); bp) the VH sequence as comprised in SEQ ID NO: 135, and the VL sequence as comprised in SEQ ID NO: 136 (Tislelizumab); bq) the VH sequence as comprised in SEQ ID NO: 137, and the VL sequence as comprised in SEQ ID NO: 138 (Toripalimab); br) the VH sequence as comprised in SEQ ID NO: 139, and the VL sequence as comprised in SEQ ID NO: 140 (zuberitamab); bs) the VH sequence as comprised in SEQ ID NO: 141 , and the VL sequence as comprised in SEQ ID NO: 142 (benmelstobart); bt) the VH sequence as comprised in SEQ ID NO: 143, and the VL sequence as comprised in SEQ ID NO: 144 (iparomlimab); bu) the VH sequence as comprised in SEQ ID NO: 145, and the VL sequence as comprised in SEQ ID NO: 146 (tuvonralimab); bv) the VH sequence as comprised in SEQ ID NO: 147, and the VL sequence as comprised in SEQ ID NO: 148 (anvatabart); bw) the VH sequence as comprised in SEQ ID NO: 149, and the VL sequence as comprised in SEQ ID NO: 150 (apamistamab); bx) the VH sequence as comprised in SEQ ID NO: 151 , and the VL sequence as comprised in SEQ ID NO: 152 (bemarituzumab); by) the VH sequence as comprised in SEQ ID NO: 153, and the VL sequence as comprised in SEQ ID NO: 154 (cetrelimab); bz) the VH sequence as comprised in SEQ ID NO: 155, and the VL sequence as comprised in SEQ ID NO: 156 (cobolimab); ca) the VH sequence as comprised in SEQ ID NO: 157, and the VL sequence as comprised in SEQ ID NO: 158 (datopotamab); cb) the VH sequence as comprised in SEQ ID NO: 159, and the VL sequence as comprised in SEQ ID NO: 160 (domvanalimab); cd) the VH sequence as comprised in SEQ ID NO: 161 , and the VL sequence as comprised in SEQ ID NO: 162 (emactuzumab); ce) the VH sequence as comprised in SEQ ID NO: 163, and the VL sequence as comprised in SEQ ID NO: 164 (favezelimab); cf) the VH sequence as comprised in SEQ ID NO: 165, and the VL sequence as comprised in SEQ ID NO: 166 (felzartamab); eg) the VH sequence as comprised in SEQ ID NO: 167, and the VL sequence as comprised in SEQ ID NO: 168 (fianlimab); ch) the VH sequence as comprised in SEQ ID NO: 169, and the VL sequence as comprised in SEQ ID NO: 170 (finotonlimab); ci) the VH sequence as comprised in SEQ ID NO: 171 , and the VL sequence as comprised in SEQ ID NO: 172 (geptanolimab); cj) the VH sequence as comprised in SEQ ID NO: 173, and the VL sequence as comprised in SEQ ID NO: 174 (gotistobart); ck) the VH sequence as comprised in SEQ ID NO: 175, and the VL sequence as comprised in SEQ ID NO: 176 (ivuxolimab); cl) the VH sequence as comprised in SEQ ID NO: 177, and the VL sequence as comprised in SEQ ID NO: 178 (lemzoparlimab); cm) the VH sequence as comprised in SEQ ID NO: 179, and the VL sequence as comprised in SEQ ID NO: 180 (luveltamab); cn) the VH sequence as comprised in SEQ ID NO: 181 , and the VL sequence as comprised in SEQ ID NO: 182 (magrolimab); co) the VH sequence as comprised in SEQ ID NO: 183, and the VL sequence as comprised in SEQ ID NO: 184 (meebotamab); cp) the VH sequence as comprised in SEQ ID NO: 185, and the VL sequence as comprised in SEQ ID NO: 186 (monalizumab); cq) the VH sequence as comprised in SEQ ID NO: 187, and the VL sequence as comprised in SEQ ID NO: 188 (nofazinlimab); cr) the VH sequence as comprised in SEQ ID NO: 189, and the VL sequence as comprised in SEQ ID NO: 190 (nurulimab); cs) the VH sequence as comprised in SEQ ID NO: 191 , and the VL sequence as comprised in SEQ ID NO: 192 (ociperlimab); ct) the VH sequence as comprised in SEQ ID NO: 193, and the VL sequence as comprised in SEQ ID NO: 194 (oleclumab); cu) the VH sequence as comprised in SEQ ID NO: 195, and the VL sequence as comprised in SEQ ID NO: 196 (onfekafusp); cv) the VH sequence as comprised in SEQ ID NO: 197, and the VL sequence as comprised in SEQ ID NO: 198 (patritumab); cw) the VH sequence as comprised in SEQ ID NO: 199, and the VL sequence as comprised in SEQ ID NO: 200 (pivekimab); ex) the VH sequence as comprised in SEQ ID NO: 201 , and the VL sequence as comprised in SEQ ID NO: 202 (quavonlimab); cy) the VH sequence as comprised in SEQ ID NO: 203, and the VL sequence as comprised in SEQ ID NO: 204 (retlirafusp); cz) the VH sequence as comprised in SEQ ID NO: 205, and the VL sequence as comprised in SEQ ID NO: 206 (rosopatamab); da) the VH sequence as comprised in SEQ ID NO: 207, and the VL sequence as comprised in SEQ ID NO: 208 (rulonilimab); db) the VH sequence as comprised in SEQ ID NO: 209, and the VL sequence as comprised in SEQ ID NO: 210 (sabatolimab); de) the VH sequence as comprised in SEQ ID NO: 211 , and the VL sequence as comprised in SEQ ID NO: 212 (sasanlimab); dd) the VH sequence as comprised in SEQ ID NO: 213, and the VL sequence as comprised in SEQ ID NO: 214 (telisotuzumab); de) the VH sequence as comprised in SEQ ID NO: 215, and the VL sequence as comprised in SEQ ID NO: 216 (tiragolumab); df) the VH sequence as comprised in SEQ ID NO: 217, and the VL sequence as comprised in SEQ ID NO: 218 (tusamitamab); dg) the VH sequence as comprised in SEQ ID NO: 219, and the VL sequence as comprised in SEQ ID NO: 220 (vibostolimab); dh) the VH sequence as comprised in SEQ ID NO: 221 , and the VL sequence as comprised in SEQ ID NO: 222 (vobramitamab); di) the VH sequence as comprised in SEQ ID NO: 223, and the VL sequence as comprised in SEQ ID NO: 224 (zilovertamab); dj) the VH sequence as comprised in SEQ ID NO: 225, and the VL sequence as comprised in SEQ ID NO: 226 (suvemcitug); dk) the VH sequence as comprised in SEQ ID NO: 227, and the VL sequence as comprised in SEQ ID NO: 228 (becotatug); dl) the VH sequence as comprised in SEQ ID NO: 229, and the VL sequence as comprised in SEQ ID NO: 230 (tifcemalimab); dm) the VH sequence as comprised in SEQ ID NO: 231 , and the VL sequence as comprised in SEQ ID NO: 232 (blinatumomab - CD19); dn) the VH sequence as comprised in SEQ ID NO: 233, and the VL sequence as comprised in SEQ ID NO: 234 (blinatumomab - CD3); do) the VH sequence as comprised in SEQ ID NO: 235, and the VL sequence as comprised in SEQ ID NO: 236 (cadonilimab - PD-1); dp) the VH sequence as comprised in SEQ ID NO: 237, and the VL sequence as comprised in SEQ ID NO: 238 (cadonilimab - CTLA4); dq) the VH sequence as comprised in SEQ ID NO: 239, and the VL sequence as comprised in SEQ ID NO: 240 (disitamab); dr) the VH sequence as comprised in SEQ ID NO: 241 , and the VL sequence as comprised in SEQ ID NO: 242 (edrecolomab); ds) the VH sequence as comprised in SEQ ID NO: 243, and the VL sequence as comprised in SEQ ID NO: 244 (elranatamab - BCMA); dt) the VH sequence as comprised in SEQ ID NO: 245, and the VL sequence as comprised in SEQ ID NO: 246 (elranatamab - DC3); du) the VH sequence as comprised in SEQ ID NO: 247, and the VL sequence as comprised in SEQ ID NO: 248 (epcoritamab - CD20); dv) the VH sequence as comprised in SEQ ID NO: 249, and the VL sequence as comprised in SEQ ID NO: 250 (epcoritamab - CD3); dw) the VH sequence as comprised in SEQ ID NO: 251 , and the VL sequence as comprised in SEQ ID NO: 252 (glofitamab - VH1 / VL1); dx); the VH sequence as comprised in SEQ ID NO: 253, and the VL sequence as comprised in SEQ ID NO: 254 (glofitamab - VH2 / VL2); dy) the VH sequence as comprised in SEQ ID NO: 255, and the VL sequence as comprised in SEQ ID NO: 256 (glofitamab - VH3 / VL3); dz) the VH sequence as comprised in SEQ ID NO: 257, and the VL sequence as comprised in SEQ ID NO: 258 (mosunetuzumab - CD20); ea) the VH sequence as comprised in SEQ ID NO: 259, and the VL sequence as comprised in SEQ ID NO: 260 (mosunetuzumab - CD3); eb) the VH sequence as comprised in SEQ ID NO: 261, and the VL sequence as comprised in SEQ ID NO: 262 (talquetamab - GPCR5D); ec) the VH sequence as comprised in SEQ ID NO: 263, and the VL sequence as comprised in SEQ ID NO: 264 (talquetamab - CD3); ed) the VH sequence as comprised in SEQ ID NO: 265, and the VL sequence as comprised in SEQ ID NO: 266 (teclistamab - BCMA); ee) the VH sequence as comprised in SEQ ID NO: 267, and the VL sequence as comprised in SEQ ID NO: 268 (teclistamab - CD3); ef) the VH sequence as comprised in SEQ ID NO: 269, and the VL sequence as comprised in SEQ ID NO: 270 (tositumomab); eg) the VH sequence as comprised in SEQ ID NO: 271, and the VL sequence as comprised in SEQ ID NO: 272 (tremelimumab); eh) the VH sequence as comprised in SEQ ID NO: 273, and the VL sequence as comprised in SEQ ID NO: 274 (zimberelimab); ei) the VH sequence as comprised in SEQ ID NO: 275, and the VL sequence as comprised in SEQ ID NO: 276 (odronextamab - CD20); ej) the VH sequence as comprised in SEQ ID NO: 277, and the VL sequence as comprised in SEQ ID NO: 278 (odronextamab - CD3); ek) the VH sequence as comprised in SEQ ID NO: 279, and the VL sequence as comprised in SEQ ID NO: 280 (ivonescimab - PD-1); el) the VH sequence as comprised in SEQ ID NO: 281 , and the VL sequence as comprised in SEQ ID NO: 282 (ivonescimab - VEGF); em) the VH sequence as comprised in SEQ ID NO: 283, and the VL sequence as comprised in SEQ ID NO: 284 (anbenitamab - VH1 / VL1); en) the VH sequence as comprised in SEQ ID NO: 285, and the VL sequence as comprised in SEQ ID NO: 286 (anbenitamab - VH2 / VL2); eo) the VH sequence as comprised in SEQ ID NO: 287, and the VL sequence as comprised in SEQ ID NO: 288 (izalontamab - EGFR); ep) the VH sequence as comprised in SEQ ID NO: 289, and the VL sequence as comprised in SEQ ID NO: 290 (izalontamab - HER3); eq) the VH sequence as comprised in SEQ ID NO: 291 , and the VL sequence as comprised in SEQ ID NO: 292 (linvoseltamab - BCMA); er) the VH sequence as comprised in SEQ ID NO: 293, and the VL sequence as comprised in SEQ ID NO: 294 (linvoseltamab - CD3); es) the VH sequence as comprised in SEQ ID NO: 295, and the VL sequence as comprised in SEQ ID NO: 296 (tarlatamab - DLL3); et) the VH sequence as comprised in SEQ ID NO: 297, and the VL sequence as comprised in SEQ ID NO: 298 (tarlatamab - CD3); eu) the VH sequence as comprised in SEQ ID NO: 299, and the VL sequence as comprised in SEQ ID NO: 300 (zanidatamab - VH1 / VL1); ev) the VH sequence as comprised in SEQ ID NO: 301 , and the VL sequence as comprised in SEQ ID NO: 302 (zanidatamab - VH2 / VL2); ew) the VH sequence as comprised in SEQ ID NO: 303, and the VL sequence as comprised in SEQ ID NO: 304 (volrustomig - PD-1); ex) the VH sequence as comprised in SEQ ID NO: 305, and the VL sequence as comprised in SEQ ID NO: 306 (volrustomig - CTLA-4); ey) the VH sequence as comprised in SEQ ID NO: 307, and the VL sequence as comprised in SEQ ID NO: 308 (zenocutuzumab - HER2); ez) the VH sequence as comprised in SEQ ID NO: 309, and the VL sequence as comprised in SEQ ID NO: 310 (zenocutuzumab - HER3); fa) the VH sequence as comprised in SEQ ID NO: 311 , and the VL sequence as comprised in SEQ ID NO: 312 (botensilimab - VH1 / VL1); fb) the VH sequence as comprised in SEQ ID NO: 313, and the VL sequence as comprised in SEQ ID NO: 314 (botensilimab - VH2 / VL2); fc) the VH sequence as comprised in SEQ ID NO: 315, and the VL sequence as comprised in SEQ ID NO: 316 (izalontamab - EGFR); fd) the VH sequence as comprised in SEQ ID NO: 317, and the VL sequence as comprised in SEQ ID NO: 318 (izalontamab - HER3); fe) the VH sequence as comprised in SEQ ID NO: 319, and the VL sequence as comprised in SEQ ID NO: 320 (rilvegostomig - TIGIT); and, ff) the VH sequence as comprised in SEQ ID NO: 321 , and the VL sequence as comprised in SEQ ID NO: 322 (rilvegostomig - PD-1).

[0167] In one embodiment, a multispecific antigen binding protein as described herein comprises at least one (first and / or second) antigen-binding region that specifically binds to a TAA, which antigenbinding region is an ISVD comprising a VHH sequence that has, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with a VHH sequence selected from the group consisting of: a) SEQ ID NO: 323 (Envafolimab); b) (Erfonrilimab); c) SEQ ID NO: 325 (Erfonrilimab); and) SEQ ID NO: 326 (Ozekibart).

[0168] Antigen binding regions binding to yd T cell receptors

[0169] In one embodiment, a multispecific antigen binding protein described herein comprises a third antigen-binding region that specifically binds an epitope of a y6 T cell receptor (TOR). The third antigen-binding region as used in a multispecific antigen binding protein described herein can be derived from any of a variety of immunoglobulin or non-immunoglobulin scaffolds, as described above for the first and second antigen binding regions. In a preferred embodiment, a third antigen-binding region as used in a multispecific antigen binding protein described herein comprises or consists of an immunoglobulin variable region. Such immunoglobulin variable regions can comprise or consist of variable domains derived from antibodies (immunoglobulin chains), e.g. in the form of associated VL and VH domains found on two polypeptide chains, such as present in a Fab. Alternatively, immunoglobulin variable domains can comprise or consist of a single chain antigen-binding domain such as a scFv, a VH domain, a VL domain, or an immunoglobulin single variable domain (ISVD) such as a dAb, a V-NAR domain or a VHH domain. An immunoglobulin variable region to be used as third antigen-binding region in a multispecific antigen binding protein described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as herein defined above.

[0170] In one embodiment, the third antigen-binding region that specifically binds an epitope of a y6 TCR is an antigen-binding region derived from immunoglobulin or non-immunoglobulin scaffolds as defined above. Preferably, the third antigen-binding region comprises or consists of at least one immunoglobulin variable domain. More preferably, the third antigen-binding region comprises or consists of a Fab that specifically binds an epitope of a y6 TCR or an immunoglobulin single variable domain (ISVD) that specifically binds an epitope of a y6 TCR. In one embodiment, the third antigenbinding region binds the epitope of a y6 TCR with a KD value of no more than 1CM M, as may be determined as herein described above.

[0171] In one embodiment, a multispecific antigen binding protein described herein comprises a third antigen-binding region that specifically binds an epitope of at least one of: a) a variable region (V) of a delta (6) chain selected from the group consisting of: V61 , V62 and V63 chains; b) a V region of a gamma (y) chain selected from the group consisting of: Vy2, Vy3, Vy4, Vy5, Vy8, and Vy9 chains; c) a constant (C) region of a y chain; and, d) a C region of a 6 chain.

[0172] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a y6 TCR, preferably to any yb TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / derived from a monoclonal antibody selected from the group consisting of: clone 5A6.E9 (TCR1061 , Thermo Fisher Sci.), clone B1 .1 (Thermo Fisher Sci .), clone gamma 3.20 (TCR 1153, Thermo Fisher Sci.), clone IMMU510 (Product No: IM1571U, Beckman Coulter Life Sci.), and clone 11F2 (MUB1809P, Thermo Fisher Sci.). All of these monoclonal antibodies are mouse monoclonal antibodies. Hence, the skilled person will understand that their CDR amino acid sequences preferably are grafted into human framework regions, so as to humanize these monoclonal antibodies for incorporation of into a third antigen-binding region of a multispecific antigen binding protein as described herein.

[0173] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a V52 TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / derived from a monoclonal antibodies selected from the group consisting of: clone 15D (TCR1732, Thermo Fisher Sci.) and clone B6 (MA5-44049, Thermo Fisher Sci.). Both of these monoclonal antibodies are mouse monoclonal antibodies. Hence, the skilled person will understand that their CDR amino acid sequences preferably are grafted into human framework regions, so as to humanize these monoclonal antibodies for incorporation of into a third antigen-binding region of a multispecific antigen binding protein as described herein.

[0174] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a Vy9 TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / derived from a monoclonal antibodies selected from the group consisting of: clone B3 (MA5-44047, Thermo Fisher Sci.) and clone 7A5 (TCR1720, Thermo Fisher Sci.). Both of these monoclonal antibodies are mouse monoclonal antibodies. Hence, the skilled person will understand that their CDR amino acid sequences preferably are into human framework regions, so as to humanize these monoclonal antibodies for incorporation of into a third antigen-binding region of a multispecific antigen binding protein as described herein.

[0175] In a preferred embodiment, a multispecific antigen binding protein described herein comprises a third antigen-binding region that specifically binds an epitope in the V region of a V61 chain of a y6 TCR. In one embodiment, a third antigen-binding region that specifically binds an epitope in the V region of a V61 chain of a y6 TCR, does not interact with other delta chains such as V62 or V63. In one embodiment, a third antigen-binding region that specifically binds an epitope in the V region of a V61 chain of a y6 TCR, does not interact with gamma chains such as Vy2, Vy3, Vy4, Vy5, Vy8, and Vy9. In one embodiments, a third antigen-binding region that specifically binds an epitope in the V region of a V61 chain of a y6 TCR, also does not bind or interact with other domains found within a y6 TCR, such as TRDJ, TRDC, TRGJ or TRGC.

[0176] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a V61 TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / derived from a monoclonal antibodies selected from the group consisting of: clone TS8.2 (TCR1730, Thermo Fisher Sci.), clone TS-1 (TCR 1055, Thermo Fisher Sci.), and clone R9.12 (Product No: IM1761 , other name: ZAP-70, Beckman Coulter Life Sci.). All three of these monoclonal antibodies are mouse monoclonal antibodies. Hence, the skilled person will understand that their CDR amino acid sequences preferably are grafted into human framework regions, so as to humanize these monoclonal antibodies for incorporation of into a third antigen-binding region of a multispecific antigen binding protein as described herein.

[0177] In one embodiment, a multispecific antigen binding protein described herein comprises a third antigen-binding region that specifically binds an activating epitope of a y5 T cell. In a preferred embodiment, the third antigen-binding region specifically binds an activating epitope in the V region of a V51 chain of a y5 TCR.

[0178] An “activating” epitope can include, for example, stimulating a TCR function, such as cell degranulation, TCR downregulation, cytotoxicity, proliferation, mobilization, increased survival or resistance to exhaustion, intracellular signaling, cytokine or growth factor secretion, phenotypic change, or a change in gene expression. For example, the binding of the activating epitope may stimulate expansion (i.e. proliferation) of the yb T cell population, preferably the Vb1 + T cell population. Accordingly, a multispecific antigen binding protein described herein comprising a third antigen-binding region that specifically binds an activating epitope of a yb T cell, can be used to modulate yb T cell activation, and, thereby, to modulate the immune response. Therefore, in one embodiment, binding of the activating epitope by the third antigen-binding region downregulates the yb TCR. In an additional or alternative embodiment, binding of the activating epitope by the third antigen-binding region activates degranulation of the yb T cell. In a further additional or alternative embodiment, binding of the activating epitope by the third antigen-binding region promotes yb T cell mediated killing of cells expressing the antigen (e.g. TAA) targeted by the multispecific antigen binding protein.

[0179] In one embodiment, an activating epitope of TRDV1 is one that, upon being bound by a third antigen-binding region in a multispecific antigen binding protein described herein, results in downregulation of the receptor and optionally activates the Vb1 cell. In some embodiments said downregulation of the receptor also results in the down-regulation of associated CD3 molecules. In some embodiments, the activating epitope is one that, upon binding by the third antigen-binding region, upregulates expression of activatory markers on the Vb1 cell, for example CD107a, CD25, CD69 and / or Ki67. In some embodiments, an activating epitope is one that, upon binding by the third antigen-binding region, upregulates expression of activatory markers on the Vb1 cell, for example CD107 and CD25, and optionally CD69 and / or Ki67. In some embodiments, upregulation of the one or more activatory markers (such as CD107a) may be upregulation in the presence of cancer cells.

[0180] As T-cell receptors are often complexed with other proteins, downregulation of the T-cell receptor via binding of a third antigen-binding region to a Vb1 domain may cause downregulation of other proteins associated with the T-cell receptor (i.e. the binding of the third antigen-binding region to a Vb1 domain causes down regulation of the T-cell receptor complex). For example, in some embodiments, an activating epitope of TRDV1 is one that upon binding by a third antigenbinding region, down-regulates the TCR / CD3 receptor complex. In this way, a multispecific antigen binding protein as described herein, may cause indirect downregulation of cell surface proteins that are not bound by the protein, but are complexed to the T-cell receptor. Given T- cells expressing gamma delta 1 chains (i.e. Vb1 cells) represent only a small number of the total T-cell population, the multispecific antigen binding proteins described herein can be used to selectively (and indirectly) downregulate proteins in the TCR complex, such as CD3, by only downregulating them in Vb1 cells.

[0181] In one embodiment, a T-cell receptor complex activating epitope is one that upon activation, by being bound by a third antigen-binding region, downregulates the T-cell receptor complex, whilst not downregulating CD3 molecules not associated with said TRDV1 TCR complex.

[0182] In one embodiment, a third antigen-binding region preferably binds an epitope that is comprised of at least one extracellular, soluble, hydrophilic or external portion of the Vb1 chain of a yb TCR.

[0183] In a particular embodiment, a third antigen-binding region binds an epitope that does not comprise an epitope found in a hypervariable region of the Vb1 chain of the yb TCR, in particular not in the CDR3 of the Vb1 chain. In a preferred embodiment, a third antigen-binding region binds an epitope that is located within the non-variable region of the Vb1 chain of the yb TCR. It will be appreciated that such binding allows for the unique recognition of the Vb1 chain without the restriction to the sequences of the TCR which are highly variable (in particular CDR3). Various yb TCR complexes which recognize antigen may be recognized in this way, solely by presence of the Vb1 chain. As such, it will be appreciated that any Vb1 chain-comprising yb TCR may be recognized using the multispecific antigen binding proteins described herein, irrespective of the specificity of the yb TCR. In one embodiment, the third antigen-binding region binds an epitope that comprises one or more amino acid residues within amino acid regions 1-24 and / or 35-90 of SEQ ID NO: 375, e.g. the portions of the Vb1 chain which are not part of the CDR1 and / or CDR3 sequences. In one embodiment, the third antigen-binding region binds an epitope that does not comprise amino acid residues within amino acid region 91-105 (CDR3) of SEQ ID NO: 375. In some embodiments, the third antigen-binding region binds an the epitope that comprises amino acids in the TRDV-1 CDR2 sequence.

[0184] In a similar manner to the well characterized ap T cells, yb T cells utilize a distinct set of somatically rearranged variable (V), diversity (D), joining (J), and constant (C) genes, although yb T cells contain fewer V, D, and J segments than ap T cells. In one embodiment, the epitope bound by the third antigen-binding region, does not comprise an epitope found in the J region of the Vb1 chain or in the C-region of the Vb1 chain. In one embodiment, the epitope bound by the third antigen-binding region binds an epitope found in the N-terminal leader sequence of the Vb1 chain. The third antigen-binding region may therefore only bind in the V region of the Vb1 chain. Thus, in one embodiment, the epitope consists of an epitope in the V region of the yb TCR (e.g. amino acid residues 1 -90 of SEQ ID NO: 375).

[0185] Reference to the epitope are made in relation to the Vb1 sequence derived from the sequence described in Luoma et al. (2013) Immunity 39: 1032-1042, and RCSB Protein Data Bank entries: 4MNH and 3OMZ, shown as SEQ ID NO: 375. SEQ ID NO: 375 represents a soluble TCR comprising a V region (also referred to as the variable domain), a D region, a J region and a TCR constant region. The V region comprises amino acid residues 1-90, the D region comprises amino acid residues 91 -104, the J region comprises amino acid residues 105-115 and the constant region (derived from T-cell receptor alpha) comprises amino acid residues 116-209. Within the V region, CDR1 is defined as amino acid residues 25-34 of SEQ ID NO: 375, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO: 375, and CDR3 is defined as amino acid residues 93-104 of SEQ ID NO: 375 (Xu et al., PNAS USA 108(6):2414-2419 (2011)).

[0186] Therefore, in one embodiment, there is provided a multispecific antigen binding protein described herein comprising a third antigen-binding region that binds the epitope of a yb TCR with a binding affinity (KD) as measured by surface plasmon resonance of less than 1.5 x 107M (i.e.150 nM). In one embodiment, the KD of the third antigen-binding region for the epitope of a yb TCR is (with increasing preference) 1.5 x 10-7M (i.e.150 nM) or less, 1.3 x 10-7M (i.e.130 nM) or less, 1.0 x 10-7M (i.e.100 nM) or less, 5.0 x 10-8M (i.e.50 nM) or less, 2.0 x 10-8M (i.e. 20 nM) or less, 1.0 x 10-8M (i.e. 10 nM) or less, 5 x 10-9M (i.e. 5 nM) or less, 2 x 10-9M (i.e. 2 nM) or less, or 1 x 10-9M (i.e. 1 nM) or less.

[0187] In one embodiment, there is provided a multispecific antigen binding protein described herein comprising a third antigen-binding region that binds the epitope of a y5 TCR with a binding affinity (KD) as measured by surface plasmon resonance of higher than 1.5 x 107M (i.e.150 nM). In one embodiment, the KD of the third antigen-binding region for the epitope of a y5 TCR is (with increasing preference) 1.5 x 10-7M (i.e.150 nM) or more, 3 x 10-7M (i.e. 300 nM) or more, 1.0 x 10-6M (i.e.1 pM) or more, 2.0 x 10-6M (i.e. 2 pM) or more, 5.0 x 10-6M (i.e. 5 pM) or more, 1.0 x 10-5M (i.e. 10 pM) or more, 2 x 10-5M (i.e. 20 pM) or more, 5 x 10-5M (i.e. 50 pM) or more, or 1 x 10-4M (i.e. 100 pM) or more.

[0188] In one embodiment, the binding affinity of the third antigen-binding region in a multispecific antigen binding protein described herein, is established by coating the third antigen-binding region or the multispecific antigen binding protein comprising the third antigen-binding region directly or indirectly (e.g. by capture with an antibody) onto the surface of a sensor (e.g. an amine high capacity chip or equivalent), wherein the target bound by the third antigen-binding region (e.g. the V51 chain of a y5 TCR) is flowed over the chip to detect binding. Suitably, a MASS-2 instrument (which may also be referred to as Sierra SPR-32) is used at 25 °C in PBS + 0.02 % Tween 20 running buffer at 30 pl / min.

[0189] In one embodiment, a multispecific antigen binding protein described herein is used to modulate or useful for modulating delta variable 1 chain (V51) T cells in a patient in situ (i.e. in vivo), via the TRDV1 -binding third antigen-binding region. The multispecific antigen binding proteins can be comprised in medicaments for such purposes.

[0190] Modulation of V51 T cells may include:

[0191] - expansion of the V51 T cells, e.g. by selectively increasing the number of V51 T cells or promotion of survival ofV51 T cells;

[0192] - directing and binding of the V51 T cells to TAA expressing target cells, therefore inducing cytotoxicity against the target cells simultaneously to activation of the V51 T cells;

[0193] - stimulation of the V51 T cells, e.g. by increasing V51 T cell potency, i.e. increasing target cell killing, and which can include increasing killing of TAA negative target cells through innate mechanisms;

[0194] - prevention of V51 T cell exhaustion, e.g. by increasing persistence of the V51 T cells; - degranulation of V51 T cells; - increase in NCR expression;

[0195] - immunomodulation of the V51 T cells, e.g. by downregulation of V51 TCR cell surface expression, i.e. by causing V51 TCR internalization or reduced expression of V51 TCR protein, or blocking the V51 TCR from binding; and / or

[0196] - downregulation of a TCR / CD3 complex.

[0197] Unlike anti-V51 antibodies of the prior art which focus on depletion of V51 T-cells or attempt activation of V51 T-cells via the TCR without any appropriate co stimulation or synapse formation, the multispecific antigen binding proteins provided herein are useful for the activation ofV51 T-cells via the TRDV1 -binding third antigen-binding region. Although they may cause downregulation of the TCRs on T-cells to which they bind, they do not cause V61 T-cell depletion, but rather they stimulate the T-cells and hence may be useful in therapeutic settings that would benefit from the activation of this compartment of T-cells. Activation of V51 T-cells is evident through TCR downregulation, CD3 downregulation, changes in activation markers such as CD25 and Ki67 and degranulation marker CD107a. Activation of V61 T-cell in turn triggers release of inflammatory cytokines such as INFy and TNFa to promote immune licensing.

[0198] In one embodiment, there is provided a multispecific antigen binding protein comprising a TRDV1 -binding third antigen-binding region, characterized in that it: a. does not exhibit CDC or ADCC; and b. does not deplete V61 T-cells.

[0199] In one embodiment, a multispecific antigen binding protein as described herein also stimulates V61 T-cell proliferation. T-cell depletion is the process of T cell death, removal or reduction. References to the multispecific antigen binding protein not depleting the V61 T cells refers to a depletion of less than about 30% or less than about 20% (preferably less than about 10%) of the viable V51 T+ cell population when incubated by one or more of the multispecific antigen binding proteins as described herein, and as measured by any via suitable means in a controlled study (for example via controlled flow cytometry methodology or via other established controlled assays). ADCC and CDC are mechanisms by which T-cell depletion may occur. Reference to the multispecific antigen binding proteins as described herein not causing ADCC or CDC refers to a depletion of less than about 30% or less than about 20% (preferably less than about 10%) of the viable V51 T+ cell population via ADCC and / or CDC when incubated by one or more of the antibodies as described herein, as measured by any via suitable means (for example via controlled flow cytometry methodology or via other established controlled assays).

[0200] In one embodiment, there is provided a multispecific antigen binding protein as described herein, characterized in that it does not induce secretion of IL-17A. IL-17A (lnterleukin-17A) is a pro-tumorigenic cytokine which is produced by activated T-cells. IL-17A can enhance tumor growth and dampen the anti- cancer immune response. Reference to the multispecific antigen binding protein as described herein not inducing secretion of IL-17A refers to inducing less than about 30%, or less than about 20%, or less than about 10% of the IL-17A secretion induced by equivalent anti-CD3 multispecific antigen binding proteins.

[0201] Some of the following sections relate to anti-V61 third antigen binding region when provided in a monospecific format, whose use in multispecific formats is specifically contemplated herein. Suitably, the functional properties of the third antigen binding region in a monospecific format are shared or enabled by the multispecific antigen binding protein as described herein that additionally specifically bind to a further antigens as described herein (e.g. TAAs), and / or that comprise agonists as described herein and / or co-stimulatory moieties as described herein.

[0202] In one embodiment, there is provided a multispecific antigen binding protein as described herein that modulates immune cell markers of V61+ T cells upon administration to a patient. A multispecific antigen binding protein as described herein can also be assessed for its suitability for therapeutic use by measuring yb T modulation, and such an assessment may be carried out when the anti-V51 third antigen binding region is provided in a monospecific, FC competent format. For example, by measuring a change in the levels of CD25 or CD69 or CD107a present on a V51 + T-cell or cells in a model system. Such markers are often used as markers of lymphocyte modulation (e.g. proliferation or degranulation) and can be measured following application of a multispecific antigen binding protein or anti-V61 third antigen binding region as described herein, e.g. by flow cytometry. Optionally, the change in phenotype of a V61 + T cell or population thereof tested in the model system can then be compared to the change in phenotype when an alternative comparator antibody is applied (e.g. OKT-3, TS8.2, etc.) to said equivalent y6 T cells.

[0203] Hence, in one embodiment, there is provided a method of assessing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) which binds to the V61 chain of a y6 TCR for therapeutic use comprising administering the antigen binding region to a cell population comprising V61+ cells or administering the multispecific antigen binding protein to a cell population comprising V61 + cells and TAA expressing target cells and determining the effect on the level of CD25 and / or CD69 and / or CD107a on the surface of the V61+ cells. The effect on the level of CD25, CD69 and / or CD107a may be determined / measured over a period of time. It will be understood that the effect can be measured in comparison to the level of CD25 and / or CD69 and / or CD107a on the surface of the V61 + cell when said antibody is not applied to said cell over the same period of time. In a further embodiment, there is provided a method of selecting or characterizing or comparing the (third) antigen binding region (or a multispecific antigen binding protein comprising said region) which binds to the V61 chain of a y6 TCR as described herein by adding said antigen binding region (or the multispecific antigen binding protein) to a cell population comprising V61 + cells and target cells and then measuring the level (or expression) of CD25 or CD69 or CD107a on the surface of said V61+ cells.

[0204] In one embodiment, a monospecific, FC competent anti-V61 third antigen binding monomer modulates the growth properties of V61+ cells in a model system when provided in an immobilized way, bound to beads or bound to cells expressing FC gamma receptors in the presence of V61 + cells.

[0205] In one embodiment, a multispecific antigen binding protein described herein modulates the growth properties of V61+ cells upon administration to a patient. For example, the multispecific antigen binding protein can expand V61+ cells. An alternate approach to measuring y6 T proliferation may include measuring the change in relative number of V61 + cells over time when applying a multispecific antigen binding protein as described herein to model systems containing said cells. Preferably, the model system also contains tumor cells, more preferably tumor cell expressing the TAA bound by the first and / or second antigen-binding region of the multispecific antigen binding protein. Optionally this change in number can then be compared to the change in number observed when an alternative comparator antibody is applied (e.g. anti- OKT3) to said model systems.

[0206] In one embodiment, a multispecific antigen binding protein described herein modulates the growth properties ofV61+ cells upon administration to a patient when used in an FC mutated format. An alternate approach to measuring y6 T proliferation may include measuring the change in relative number of V61 + cells overtime when applying a multispecific antigen binding protein as described herein to model systems containing said cells with TAA expressing target cells.

[0207] Hence, in another aspect, there is provided a method of assessing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) comprising administering the antigen binding region (or said multispecific antigen binding protein) to a cell population comprising V61+ cells together with TAA expressing target cells and determining the effect on the number of V51+ cells in the population. The effect on cell number can be determined / measured over a period of time. It will be understood that the effect can be measured in comparison to the effect on cell numbers observed when said antigen binding region or protein is not applied to the cell population for the same period of time. In a further embodiment, there is provided a method of selecting or characterizing or comparing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) by applying said antigen binding region or protein to a cell population comprising V61+ cells and then measuring the number of said cells over time. Medicaments that modulate the proliferative capacity and numbers of V51+ cells.

[0208] A preferred therapeutic (third) antigen binding region (or a multispecific antigen binding protein comprising said region) is one that is capable of enhancing the proliferation ofV51+ cells in vivo. Multispecific antigen binding proteins comprising such antigen binding regions can then be employed as medicaments designed to specifically increase the V61+ cell number in a subject or patient.

[0209] For example, in cancer relative increases in the numbers of V51+ cells have been reported as a positive prognostic indicator associated with improved outcomes for many cancer (for example see Gentles et al (2015) Nature Immunology 21 : 938-945; Wu et al. (2019) Sci. Trans. Med.

[0210] 11(513): eaax9364; Catellani et al. (2007) Blood 109(5): 2078-2085). In one embodiment, presented herein is a medicament capable of increasing the relative or absolute numbers of V51 + cells in situ within in a cancer patient.

[0211] For example, in the case of pathogenic parasitic, viral or microbial infections, V51+ cell enrichment is observed during host defense against numerous acquired pathogenic parasitic, viral or microbial infections. For recent general review see Zhao et al. (2018) Immunol. Res.

[0212] 2018:5081634. Furthermore, increased numbers V51+ are also considered protective against a variety of DNA and RNA viral infections. For example, increased numbers are also considered protective during CMV infections associated with allogeneic transplants (see van Dorp et al. (2011) Biology of Blood and Marrow Transplantation 17(2): S217). Additionally, V5+ cell numbers increase in patients with coronavirus infection (Poccia et al. (2006) J. Infect. Dis.193(9): 1244-1249). In another embodiment, presented herein is a medicament capable of increasing the relative or absolute numbers of V61+ cells in a subject or patient harboring a pathogenic infection.

[0213] For example, in the case of a stem cell transplant, increased numbers of V61+ cells have also been associated with less disease relapse, fewer viral infections, higher overall and disease-free survival and favorable clinical outcomes in general during hematopoietic stem cell transplant (for example see Aruda et al. (2019) Blood 3(21): 3436-3448 and see Godder et al. (2007) Bone Marrow Transplantation 39: 751-757). Hence another embodiment, presented herein is a medicament capable of increasing the relative or absolute numbers of V51 + cells in a subject as part of a treatment regimen supporting a stem cell transplant. Consequently, a medicament capable of preferentially or specifically increasing the numbers of V51+ cells in-situ is highly desirable.

[0214] In one embodiment, a multispecific antigen binding protein described herein maintains or induces or increases V51+ T cell cytokine secretion. Cytokines are a large group of proteins, peptides or glycoproteins that are secreted by specific cells of immune system. They are a category of signaling molecules that mediate and regulate immunity, inflammation, and hematopoiesis. A number of cytokines have been implicated in ameliorating signs and symptoms of disease through either direct or indirect modulation of the tumor and cellular microenvironment, autoimmune tissue and associated microenvironment, or virally infected tissue or cellular environment. Exemplar pro-inflammatory cytokines include tumor necrosis factor-alpha (TNFa) and Interferon-gamma (IFNy). However, many such cytokines exhibit unfavorable toxicity when dosed systemically. Hence, there is a need for more in situ controlled, more localized, more tissue or cell specific production of such cytokines. For example, more controlled expression or induction of pro-inflammatory cytokines is proposed as one approach whereby “cold” tumors can be turned “hot”. Hot tumors are also sometimes termed “T-cell-inflamed” because of an increase in the number or density of CD45+ T-cells also observed. See Bonaventura et al. (2019) Front. Immunol.10: 168 for a recent review. For such reasons, in one embodiment, a multispecific antigen binding protein described herein which binds to the V51 chain of a y5 TCR is one that can maintain or enhance or induce the secretion of cytokines in V51+ cells in vivo. Such multispecific antigen binding proteins can then be employed as medicaments designed to specifically increase or induce cytokines in a subject or patient and in a more localized, less systemic manner and one which better correlates with the distribution of V51+cells in said subject or patient.

[0215] Hence, In one embodiment, there is provided a method of assessing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) comprising administering the antigen binding region (or said multispecific antigen binding protein) to a cell population comprising V51+ cells and determining the amount of at least one cytokine produced by the cell population. The amount of cytokine produced can be determined / measured over a period of time and optionally compared to the amount observed when said antigen binding region is not applied to the cell population for the same period oftime. In one embodiment, the observed level of cytokine produced when the antigen binding region is administered to the cell population is more than about 10%, more than about 20%, more than about 30%, more than about 50%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 350%, more than about 400%, more than about 450%, more than about 500%, more than about 1000%, relative to the level of cytokine produced when the antigen binding region is not applied. In a further embodiment, the cytokine is a pro-inflammatory cytokine. In a further embodiment, the cytokine is a TNF-a cytokine. In a further embodiment, the cytokine is a IFN-y cytokine.

[0216] In one embodiment there is provided a method of selecting or characterizing or comparing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) by applying said antigen binding region (or said multispecific antigen binding protein) to a cell population comprising V61 + cells and then measuring the level of at least one cytokine generated.

[0217] In one embodiment the cytokine measured is TNF-a cytokine and / or IFN-y cytokine. In a further embodiment, there is provided a method of assessing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) by applying said antigen binding region (or said multispecific antigen binding protein) to a cell population comprising V61 + cells and measuring the effect of the antigen binding region or protein on modulating a colder or cold tumor to become a hotter or hot tumor by determining the quantity of proinflammatory cytokines produced and / or the number or density of CD45+ T-cells present in the tumor or tumor microenvironment.

[0218] In one embodiment there is provided a method of selecting or characterizing or comparing assessing a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) by applying said antigen binding region (or said antigen binding protein) to a co-culture comprising V61 + cells and diseased cells and then measuring the quantity or activity of Granzyme B in the diseased cell.

[0219] In one embodiment, a multispecific antigen binding protein described herein expands polyclonal V61 + T cell populations. Thus, a (third) antigen binding region (or a multispecific antigen binding protein comprising said region) can also be one designed to ensure the expanding V61 + cells do not become too clonally focused at the hypervariable CDR3 sequence level. Hence, in one embodiment, a (third) antigen binding region can be designed such to avoid inducing proliferation V61 + cells by binding to specific or ‘private’ 61+ CDR3 sequence paratopes. Rather, the third antigen binding region antibody can bind via conserved germline sequences present on all V61+ T cell receptors and in a gamma-chain independent manner, rather than bind to sequences presented only a sub-set of V61+ cells. Hence a preferred third antigen binding region antibody (or a multispecific antigen binding protein described herein comprising said antigen binding region) stimulates the expansion V61+ cells to generate a plurality of V61+ cells containing a mixture of CDR3 sequences. This in turn would result in an in vivo expanded heterogenous polyclonal population of V61+ cells displaying different CDR3 sequences on delta variable 1 chains.

[0220] The extend of polyclonality of expanded V61+ cell populations generated by a method of adding a third antigen binding region (or a multispecific antigen binding protein comprising said region) as described herein to a starting population of immune cells containing V61+ cells can be analyzed by RNAseq based methodologies designed to sequence through the CDR3 hypervariable regions of RNA extracted. Accordingly in one aspect, there is provided a method of assessing an (third) antigen binding region which binds to the V61 chain of a y6 TCR (or a multispecific antigen binding protein comprising said region) comprising administering said antigen binding region (or said antigen binding protein) to a cell population comprising V61+ T cells and determining the polyclonality of the expanded V61+ cells. It is desirable for a multispecific antigen binding protein as described herein to generate an expanded polyclonal population containing a plurality of V61 + CDR3 sequences. Polyclonality can be determined using methods known in the art, such as by nucleic acid sequencing approaches capable of analyzing the V61 chain hypervariable CDR3 content of said V61+ cells. In one embodiment, a multispecific antigen binding protein as described herein expands polyclonal Vb1 + cells for extended periods of time. Thus, in one embodiment a multispecific antigen binding protein as described herein is able to enhance or promote or stimulate the proliferation of primary Vb1 + cells without exhausting such cells in vivo. For example, and by way of comparison, anti-CD3 medicaments such as OKT3 (e.g. Muronomab), whilst capable of expanding CD3 positive T-cells may also exhaust or induce anergy.

[0221] To assess the capacity of monospecific versions of the multispecific antigen binding proteins as described herein, i.e. the third antigen binding region which bind to the Vb1 chain of a yb TCR to drive continued cell division of viable Vb1 + cells, longer term proliferation studies were undertaken using monospecific antibodies / antigen binding regions. Thus, in one embodiment, there is provided a method of assessing an antibody or antigen binding region which binds to the Vb1 chain of a yb TCR comprising applying the antibody or region to a cell population and monitoring the length of time Vb1 + cell division occurs. Ideally, the antibody or antigen binding region is capable of stimulating Vb1 + cell division for a period of 5 to 60 days, such as at least 7 to 45 days, 7 to 21 days, 7 to 18 days, or for as long as the multispecific antigen binding protein and TAA expressing target cells are available.

[0222] In a further embodiment, there provided a third antigen binding region which binds to the Vb1 chain of a yb TCR, or a multispecific antigen binding proteins as described herein comprising said antigen binding region, which when administered to a patient is capable of stimulating Vb1 + cell division to increase the number by at least 2-fold in number, at least 5-fold in number, at least 10-fold in number, at least 25-fold in number, at least 50-fold in number, at least 60-fold in number, at least 70-fold in number, at least 80-fold in number, at least 90-fold in number, at least 100-fold in number, at least 200-fold in number, at least 300-fold in number, at least 400-fold in number, at least 500-fold in number, at 600-fold in number, at least 1 ,000-fold in number.

[0223] In one embodiment, there is provided a method of selecting or characterizing or comparing antigen binding regions as described herein which bind to the Vb1 chain of a yb TCR by applying said antigen binding regions to Vb1 + cells or mixed cell population containing Vb1 + cells, and optionally contain TAA expressing target cells, and then measuring Vb1 + cell numbers over time.

[0224] In one embodiment, a multispecific antigen binding protein as described herein modulates non-V61+ immune cells through targeting Vb1 + immune cells. Thus, in one embodiment, a third antigen binding region which binds to the Vb1 chain of a yb TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region can also be assessed by measuring V61+ cell mediated modulation of other immune cells. For example, a change observed in a non-yb T cell ‘fraction’ can be measured following application of a third antigen binding region or a multispecific antigen binding protein comprising said antigen binding region to a model system comprising mixed population of immune cells such as one comprising human ap T cells and yb T cells. Further, the effect on non-yb cell types in said models can be measured by flow cytometry. For example, by measuring the relative change in numbers of CD8+ ap T cells upon addition of a third antigen binding region as described herein (or an antigen binding protein comprising said region) to mixed cultures comprising yb T cells and non-yb T cells and TAA expressing target cells. Optionally, the observed change in number or phenotype of a non-yb T-cell CD8+ lymphocyte population can then be compared to the change in number when an alternate anti-V51 antibody is applied (e.g. TS8.2) to said mixed population.

[0225] Hence, in one embodiment, there is provided a method of assessing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region comprising administering the antigen binding region (or said antigen binding protein) to a mixed population of immune cells or tissues comprising Vb1+ cells and Vb1 -negative immune cells and measuring the effect on the Vb1-negative immune cells. The effect can be determined / measured over a period of time and optionally compared to the effect observed in Vb1 -negative cells when said antigen binding region (or said antigen binding protein) is not applied for the same period of time. The effect may be measured as a change in the number of Vb1- negative immune cells. For example, the antigen binding region (or antigen binding protein) can increase the number Vb1 -negative immune cells by more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 500%, relative to the levels observed when said antigen binding region (or said antigen binding protein) is not applied.

[0226] In one embodiment, the modulated V51 -negative cell is a CD45+ cell. In a further embodiemnt the modulated cell is a ap T-cell. In a further aspect, the modulated ap+ cell is CD8+ lymphocyte. In a further embodiment the modulated ap T-cell, or population thereof, exhibits evidence of enhanced cell division. In one embodiment, there is provided a method of selecting or characterizing or comparing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region by administering said antigen binding region (or said antigen binding protein) to a population of mixed immune cells comprising V51+ cells and V51 -negative immune cells and then measuring an effect conferred on the V51- negative cell population by V51+ cells modulated by said antigen binding region (or said antigen binding protein).

[0227] Optionally, and during “V51+ cell mediated immune system modulation” as conferred by an antigen binding region (or antigen binding protein) as described herein, a concomitant increase in V51+ cell number is also observed. And whilst not being bound by this theory, it is possible that said increase in V51+ cell number may be causal in driving the concomitant expansion of co-present V51 -negative immune cells, such as ap T-cells. An alternate hypothesis may be that antibody-induced cytokine secretions from the V51+ T cells stimulate the expansion of V51 -negative immune cells.

[0228] In one embodiment, the observed increase in ap+ CD8+ lymphocyte population is compared to a comparator antibody such as OKT3 antibody or alternate anti-V51 antibody (e.g. TS8.2). In one embodiment, there is provided a method of selecting or characterizing or comparing assessing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein by applying said antigen binding region (or said antigen binding protein) to a population of mixed immune cells comprising V51+ T-cells and ap T-cells and then measuring the numbers of CD8+ ap+ T-cells lymphocytes over time.

[0229] In one embodiment, a multispecific antigen binding protein as described herein modulates Tumor Infiltrating Lymphocytes (TILs). Thus, in one embodiment, a third antigen binding region which binds to the V61 chain of a y6 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region can also be assessed by measuring the effect conferred on tumor-infiltrating populations (TILs) in model systems. In one embodiment, monospecific versions of the multispecific antigen binding protein as described herein, i.e. comprising a third antigen binding region as described herein, can be assessed for their ability to measurably modulated TIL populations in human tumors. For example, a change in either the number or phenotype of y5+ lymphocyte TIL population or the non-yb lymphocyte TIL population is measured following application of a third antigen binding region or a multispecific antigen binding protein as described herein comprising said antigen binding region to a human tumor such as a human renal cell carcinoma. Optionally, the observed change in number or phenotype of either the y5+ lymphocyte TIL population or non-yb lymphocyte TIL population can then be compared to the change observed when an alternative comparator antibody is applied (e.g. OKT-3) to said model system.

[0230] Hence, in one embodiment, there is provided a method of assessing a third antigen binding region which binds to the Vb1 chain of a yb TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region comprising administering the antigen binding region (or an antigen binding protein comprising said region) to TILs located in, or derived from, a human tumor and determining the effect on the number of TILs. The effect can be determined / measured over a period of time and optionally compared to the TIL number observed when said antigen binding region or protein is not applied over the same period of time. The effect may be an increase in the number of TILs. For example, the antigen binding region or protein may increase the number of TILs more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100% relative to the number of TILs observed when said antigen binding region or protein is not applied. In a further aspect, the TILs in which the number observed are y5+ lymphocyte TIL cells and / or non-y5 lymphocyte TIL cells. In a further aspect, there is provided a method of selecting or characterizing or comparing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region by applying said antigen binding region or protein to TIL or TILs located in or derived from a human tumor and then measuring the change in number of TIL or TILs cells over a period of time.

[0231] In one embodiment, a multispecific antigen binding protein as described herein modulates human V51+ cytotoxicity. Thus, in one embodiment, a third antigen binding region which binds to the V51 chain of a y5 TCR, ora multispecific antigen binding protein as described herein comprising said antigen binding region can also be assessed by measuring the conferred effect on V51 + mediated cell cytotoxicity. For example, a reduction in the number of cancer cells or an increase in the number of killed cancer cells is observed following application of a third antigen binding region or a multispecific antigen binding protein comprising said antigen binding region to a model system comprising a mixed culture comprising V61+ cells and said cancer cells. Optionally, the reduction in the number of cancer cells or the increase in the number of killed cancer cells can then be compared to the outcome when an alternative comparator antibody is applied (e.g. TS8.2) to said model systems.

[0232] Hence, in one embodiment, there is provided a method of assessing a third antigen binding region which binds to the V61 chain of a y6 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region comprising applying the antigen binding region or protein to a mixed population of cells comprising V61+ cells and cancer cells and measuring the cytotoxicity of the V61+ cells towards the cancer cells. The cytotoxicity may be measured by an increase in the number of dead cancer cells over a period of time, optionally compared to the number of dead cancer cells observed when said antigen binding region or protein is not applied to the mixed population of cells over the same period of time. For example, the observed increase in dead cells when said antigen binding region or protein is applied may be more than about 10%, by more than about 20%, by more than about 30%, by more than about 40%, by more than about 50%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 200%, more than about 500%, relative to the number of dead cells observed when said antigen binding region or protein is not applied.

[0233] In one embodiment there is provided a method of selecting or characterizing or comparing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region by adding said antigen binding region or protein to said population of mixed immune cells comprising human V51+ cells and cancer cells and then measuring an increase in dead cancer cells overtime.

[0234] In one embodiment, a multispecific antigen binding protein as described herein modulates V51+ cell target-to-effector cell ratios (T:E ratios). Thus, in one embodiment, a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region can be assessed by measuring how said antigen binding region or protein enhances V51+ mediated cancer cell cytotoxicity by determining the target cell to effector cell ratio wherein the 50% of the target cells (EC50) are killed in a model system to assess said antigen binding regions or said proteins as potential medicaments. In one embodiment, multispecific antigen binding proteins as described herein (as well as monospecific versions thereof binding to the V51 chain of a y5 TCR) favorably modify the EC50 T:E ratio in model systems, for example mixed cultures comprising target cancer cells with human V51+ effector cells. Such modifications of T:E ratios can be measured as numbers of V51+ cells required to observe 50% killing of cancer cells over a set time. This can also be reported as change or as foldimprovements or as percent-improvements in cytotoxicity towards said cancer cells. Optionally, the T:E ratio conferred by a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region can then be compared to the T:E ratios when an alternative comparator antibody is applied (e.g. OKT-3 or TS8.2) to said model systems. In some scenarios, the multispecific antigen binding proteins as described herein present opportunities for improved cancer cell cytotoxicity even at lower E:T ratios, compared to monospecific antigen binding proteins that binds to the V61 chain of a y6 TCR.

[0235] Hence, in one embodiment, there is provided a method of assessing a third antigen binding region which binds to the V61 chain of a y6 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region, comprising applying the antigen binding region or protein to a mixed population of cells comprising human V61+ cells and cancer cells and measuring the number of V51 + cells required to kill 50% of the cancer cells. This may be measured relative to the number V51 + cells required to kill 50% of cancer cells without application of said antigen binding region or protein, optionally over the same period of time. For example, the reduction in the number of V51+ cells required to kill 50% of the cancer cells when said antigen binding region or protein is applied may be greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater about 200%, greater than about 500%, relative to the number of V51+ cells required to kill 50% of the cancer cells when said antigen binding region or protein is not applied.

[0236] In one embodiment, there is provided a method of selecting or characterizing or comparing a third antigen binding region which binds to the V51 chain of a y5 TCR, or a multispecific antigen binding protein as described herein comprising said antigen binding region by adding said antigen binding region or protein to said population of cells comprising V51+ cells plus cancer cells and then measuring the numbers of V51+ cells required to kill 50% of the cancer cells.

[0237] In one embodiment, a multispecific antigen binding protein as described herein enhances V51+ cell EC50 cytotoxicity. An alternate way to measure the observed enhanced cytotoxicity of human V51+ cells or population thereof is to measure the number of cells required to kill 50% of the cancer cells over a set period of time in condition A (such as starting control) and compare this to the number of cells required to kill 50% of the cancer cells over a set period of time in condition B (such as upon application of a multispecific antigen binding protein as described herein, or a monospecific version thereof binding to theV51 chain of a y5 TCR). In one embodiment, the number of V51+ cells required to kill 50% of target cancer cells over a set period of time in the absence of a multispecific antigen binding protein as described herein, or a monospecific version thereof binding to the V51 chain of a y5 TCR, is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 100%, greater about 200%, greater than about 500%, relative to the number of V51+ cells required to kill 50% of the cancer cells when said antigen binding region or protein is present.

[0238] In one embodiment, there is provided a method of selecting or characterizing or comparing a multispecific antigen binding protein as described herein, or a monospecific version thereof binding to the V51 chain of a y5 TCR by adding said antigen binding protein or monospecific version thereof to said population of mixed immune cells comprising V51+ cells and cancer cells and determining the relative or percent-change in cytotoxicity versus an equivalent or control experiment wherein there is no application of said antibody to said mixture of cells.

[0239] In one embodiment, a multispecific antigen binding protein as described herein enhances V61+ cells diseased-cell specificity whilst sparing healthy cells. Another approach to assess a multispecific antigen binding protein as described herein, or a monospecific version thereof binding to the V61 chain of a y6 TCR is to measure how said antigen binding protein or monospecific version thereof modulate diseased-cell specific cytotoxicity. A multispecific antigen binding protein as described herein, when administered to a patient to ameliorate a symptom of cancer, will confer enhanced cytotoxicity specifically towards diseased cells whilst sparing healthy cells.

[0240] Medicaments which enhance effector cell cytotoxicity specifically towards diseased cells, such as cancer cells, can be said to exhibit an enhanced therapeutic index (Tl) over medicaments which do not selectively enhance effector cell cytotoxicity specifically towards said diseased cells. The therapeutic index is also referred to as therapeutic ratio and is a quantitative measurement of the relative safety of a drug. It is a comparison of the amount of a therapeutic agent that causes the therapeutic effect to the amount that causes toxicity e.g. by conferring undesirable death in related or relevant healthy cell populations. A multispecific antigen binding protein as described herein can be assessed by measuring its ability to change or to enhance or to fold-improve V61+ cell capacity to selectivity kill diseased cells over and above healthy cells in model systems. For example, said model systems may comprise V61+ effector cells, cancer cells, and control cells (such as healthy cells). Optionally, the fold-improvement in selective diseased-cell killing conferred by a multispecific antigen binding protein as described herein can then be compared to the fold-improvement observed when an alternative comparator antibody is applied (e.g. OKT-3 or TS8.2) to said model systems.

[0241] The diseased-cell specificity and diseased-cell specificity-enhancement of V61+ cells can be measured in cultures comprising V61+ cells, diseased cells, and healthy cells. For example, V61 + specificity towards diseased cells can be measured by observing the number of cancer cells killed by the V61 + cells and then comparing the number of healthy cells killed by the V61 + cells. Such comparisons can be controlled by including equivalent numbers of diseased cells and healthy cells in a model system also containing V61 + cells e.g. “tricultures”. Alternative comparison methodology can also be considered - for example when analytical or equipment limitations reduce the ability to distinguish and track all three cell types or more in parallel in a single assay (inclusive of V61+ cells, diseased cells, and non-diseased cells). In said instances, comparing V61+ cell cytotoxicity towards diseased cells in one experiment and then comparing V61+ cell cytotoxicity towards healthy cells in a separate equivalent experiment offers an alternate approach to such studies.

[0242] In one embodiment, there is provided a method of assessing a multispecific antigen binding protein as described herein or a monospecific version thereof which binds to the V61 chain of a y6 TCR, comprising administering the multispecific antigen binding protein or a monospecific version thereof to a cell population comprising V61+ cells and target cells and measuring the cell cytotoxic specificity towards the target cells. In one embodiment, the cell cytotoxicity specificity to a first target cell type can be compared to the cytotoxicity observed towards a second target cell type, therefore the method may be repeated using different target cell types. In a further embodiment the first target cell type is a diseased cell and the second target cell type is a control cell such as a healthy cell or a cell with a different disease to the first target cell type.

[0243] In one embodiment there is provided a method for selecting or characterizing or comparing a multispecific antigen binding protein as described herein or a monospecific version thereof which binds to the V61 chain of a y6 TCR, wherein the effect conferred by said antigen binding protein or monospecific version thereof, on V61+ cell cytotoxicity towards (i) a first cell type and (ii) a second cell type is measured and compared. In a further aspect, an antibody is thereby selected which enhances the specific cytotoxicity towards the first cell type more so than towards the second cell type. In a further embodiment the first cell type is a diseased-cell and the second cell type is a healthy cell.

[0244] As described herein, the multispecific antigen binding proteins or monospecific versions thereof used in the assays may be presented on a surface, for example the surface of a cell, such as a cell comprising an a TAA. For example, the multispecific antigen binding proteins or monospecific versions thereof can be presented on the surface of SKOV3 cells, such as HTB-77™ cells (available from American Type Culture Collection (ATCC)). Alternatively, the multispecific antigen binding proteins or monospecific versions thereof may be used directly in the assays.

[0245] In such functional assays, output may be measured by calculating the half maximal concentration, also referred to as “EC50” or “effective concentration at 50 percent”. The term "IC50" refers to the inhibitory concentration. Both EC50 and IC50 may be measured using methods known in the art, such as flow cytometry methods. In some instances, EC50 and IC50 are the same value or can be considered equivalent. For example, the effective concentration (EC) of effector cells required to inhibit (e.g. kill) 50% of a certain cell type may also be considered the 50% inhibitory concentration (IC). For the avoidance of doubt, the values of EC50 in the present application are provided using IgG 1 formatted antibody when referring to an antibody. Such values can be easily converted based on the molecular weight of the antibody format for equivalent values as follows:

[0246] (pg / ml) / (MW in kDa) = pM.

[0247] The EC50 for downregulation of the y5 TCR upon binding by a multispecific antigen binding protein or a monospecific version thereof which binds to the V51 chain of a y5 TCR, can be less than 0.50 pg / ml, such as less than 0.40 pg / ml, 0.30 pg / ml, 0.20 pg / ml, 0.15 pg / ml, 0.10 pg / ml, 0.06 pg / ml or 0.05 pg / ml. In a preferred embodiment, the EC50 for downregulation of the y6 TCR upon binding by a multispecific antigen binding protein or a monospecific version thereof is less than 0.10 pg / ml. In particular, the EC50 for downregulation of the y6 TCR upon binding by a multispecific antigen binding protein or a monospecific version thereof may be less than 0.06 pg / ml, such as less than 0.05 pg / ml, 0.04 pg / ml or 0.03 pg / ml. In particular, said EC50 values are when the multispecific antigen binding protein or the monospecific version thereof is measured in an lgG1 format. For example, the EC50 y6 TCR downregulation value can be measured using flow cytometry.

[0248] The EC50 for y6 T cell degranulation upon binding by a multispecific antigen binding protein described herein or a monospecific version thereof which binds to the V61 chain of a y6 TCR can be less than 0.050 pg / ml, such as less than 0.040 pg / ml, 0.030 pg / ml, 0.020 pg / ml, 0.015 pg / ml, 0.010 pg / ml or 0.008 pg / ml. In particular, the EC50 for y6 T cell degranulation upon binding by a multispecific antigen binding protein ora monospecific version thereof can be less than 0.050 pg / ml, less than 0.020 pg / ml, less than 0.010 pg / ml or less than 0.005 pg / ml, such as less than 0.002 pg / ml. In a preferred embodiment, the EC50 for yb T cell degranulation upon binding by a multispecific antigen binding protein or a monospecific version thereof is less than 0.007 pg / ml. In particular, said EC50 values are when the multispecific antigen binding protein or the monospecific version thereof is measured in an lgG1 format. For example, the yb T cell degranulation EC50 value can be measured by detecting CD107a expression (i.e. a marker of cell degranulation) using flow cytometry (e.g. as described in the assay of Example 7). In one embodiment, CD107a expression is measured using an anti-CD107a antibody, such as anti-human CD107a BV421 (clone H4A3) (BD Biosciences).

[0249] The EC50 for yb T cell killing of (TAA expressing) target cells upon binding by a multispecific antigen binding protein described herein or a monospecific version thereof which binds to the Vb1 chain of a yb TCR, can be less than 0.50 pg / ml, such as less than 0.40 pg / ml, 0.30 pg / ml, 0.20 pg / ml, 0.15 pg / ml, 0.10 pg / ml or 0.07 pg / ml. In a preferred embodiment, the EC50 for yb T cell killing upon binding by the multispecific antigen binding protein or monospecific version thereof is less than 0.10 pg / ml. In particular, the EC50 for yb T cell killing upon binding by the multispecific antigen binding protein or monospecific version thereof can be less than 0.060 pg / ml, such as less than 0.055 pg / ml, in particular less than 0.020 pg / ml. In particular, said EC50 values are when the multispecific antigen binding protein or the monospecific version thereof is measured in an IgG 1 format. For example, the EC50 yb T cell killing value can be measured by detecting proportion of dead cells (i.e. using a cell viability dye) using flow cytometry following incubation of the multispecific antigen binding protein or a reference protein, yb T cell and target cells (e.g. as described in the assay of the Examples herein). In one embodiment, death of the target cell is measured using a cell viability dye is Viability Dye eFluor™ 520 (ThermoFisher).

[0250] In the assays described in these aspects, the antigen bound by the multispecific antigen binding protein or the monospecific version thereof may be presented on the surface of a cell, such as a SKOV3 cell, for example HTB-77™ (ATCC). The SKOV3 cells are optionally labelled with a dye, such as CellTracker™ Orange CMTMR (ThermoFisher).

[0251] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a Vb1 TCR, which antigen-binding region comprises a combination of complementarity-determining regions (CDRs) CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 sequence of SEQ ID NO: 380, the CDR-H2 sequence of SEQ ID NO: 381 , CDR-H3 sequence of SEQ ID NO: 382, the CDR-L1 sequence of SEQ ID NO: 383, the CDR-L2 sequence of SEQ ID NO: 384 (amino acids YDS) and the CDR-L3 sequence of SEQ ID NO: 385; b) the CDR-H1 sequence of SEQ ID NO: 386, the CDR-H2 sequence of SEQ ID NO: 387, CDR-H3 sequence of SEQ ID NO: 388, the CDR-L1 sequence of SEQ ID NO: 389, the CDR-L2 sequence of SEQ ID NO: 390 (amino acids DAS) and the CDR-L3 sequence of SEQ ID NO: 391 ; c) the CDR-H1 sequence of SEQ ID NO: 392, the CDR-H2 sequence of SEQ ID NO: 393, CDR-H3 sequence of SEQ ID NO: 394, the CDR-L1 sequence of SEQ ID NO: 395, the CDR-L2 sequence of SEQ ID NO: 396 (amino acids VAS) and the CDR-L3 sequence of SEQ ID NO: 397.

[0252] In one embodiment, a multispecific antigen binding protein as described herein comprises a third antigen-binding region that specifically binds to a V61 TOR, which antigen-binding region comprises a combination of variable heavy (VH) and variable light (VL) domain sequences that have, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the VH and VL sequence combinations selected from the group consisting of: a) the VH sequence as comprised in SEQ ID NO: 398, and the VL sequence as comprised in SEQ ID NO: 399; b) the VH sequence as comprised in SEQ ID NO: 400, and the VL sequence as comprised in SEQ ID NO: 401 ; c) the VH sequence as comprised in SEQ ID NO: 402, and the VL sequence as comprised in SEQ ID NO: 403.

[0253] y<5 T cell-activating agonists

[0254] A multispecific antigen binding protein as described herein, thus further comprises at least one y6 T cell-activating agonist. In one embodiment, the y6 T cell-activating agonist is an agonist that induces at least one of STAT5 and STAT3 activation. Thus, in one embodiment, the y6 T cellactivating agonist is selected from the group consisting of: a) an interleukin 2 receptor (IL-2R) agonist; b) an interleukin 6 receptor (IL-6R) agonist; c) an interleukin 7 receptor (IL-7R) agonist; d) an interleukin 9 receptor (IL-9R) agonist; e) an interleukin 12 receptor (IL-12R) agonist; f) an interleukin 15 receptor (IL-15R) agonist; g) an interleukin 18 receptor (IL-18R) agonist; h) an interleukin 21 receptor (IL-21 R) agonist; i) an interleukin 23 receptor (IL-23R) agonist; and, j) an interleukin 27 receptor (IL-27R) agonist.

[0255] In one embodiment, a multispecific antigen binding protein as described herein comprises y6 T cell-activating agonist that is selected from the group consisting of: a) an IL-2R agonist that comprises or consists of an IL-2 polypeptide or an agonistic antigen-binding region that specifically binds IL-2R; b) an IL-6R agonist that comprises or consists of an IL-6 polypeptide or an agonistic antigen-binding region that specifically binds IL-6R; c) an IL-7R agonist that comprises or consists of an IL-7 polypeptide or an agonistic antigen-binding region that specifically binds IL-7R; d) an IL-9R agonist that comprises or consists of an IL-9 polypeptide or an agonistic antigen-binding region that specifically binds IL-9R; e) an IL-12R agonist that comprises or consists of an IL-12 polypeptide or an agonistic antigen-binding region that specifically binds IL-12R; f) an IL-15R agonist that comprises or consists of an IL-15 polypeptide or an agonistic antigen-binding region that specifically binds IL-15R; g) an IL-18R agonist that comprises or consists of an IL-18 polypeptide or an agonistic antigen-binding region that specifically binds IL-18R; h) an IL-21 R agonist that comprises or consists of an IL-21 polypeptide or an agonistic antigen-binding region that specifically binds IL-21 R; i) an IL-23R agonist that comprises or consists of an IL-23 polypeptide or an agonistic antigenbinding region that specifically binds IL-23R; and, j) an IL-27R agonist that comprises or consists of an IL-27 polypeptide or an agonistic antigen-binding region that specifically binds IL-27R.

[0256] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-2 polypeptide. In one embodiment, the IL-2 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 351 (i.e. the functional domain of IL-2, corresponding to amino acids 21-153 of Uniprot acc. No. P60568). The IL-2 polypeptide preferably has IL-2 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT5 phosphorylation in a bio-assay, using reporter cells (e.g. https: / / nld.promeqa.com / products / reporter-bioassavs / cytokine-and-qrowth-factor-bioassavs / il2-bioassay / ?catNum=JA2201 ). The IL-2 polypeptide preferably has the ability to specifically bind to an IL-2 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0257] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-6 polypeptide. In one embodiment, the IL-6 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 367. The IL-6 polypeptide preferably has IL-6 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT-3 activation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il6). The IL-6 polypeptide preferably has the ability to specifically bind to an IL-6 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0258] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-7 polypeptide. In one embodiment, the IL-7 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 368 (i.e. the functional domain of IL-7, corresponding to amino acids 26-177 of Uniprot acc. No. P13232). The IL-7 polypeptide preferably has IL-7 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT-5 activation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il7). The IL-7 polypeptide preferably has the ability to specifically bind to an IL-7 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0259] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-9 polypeptide. In one embodiment, the IL-9 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 369 (i.e. the functional domain of IL-9, corresponding to amino acids 19-144 of Uniprot acc. No. P15248). The IL-9 polypeptide preferably has IL-9 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT-5 activation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il9). The IL-9 polypeptide preferably has the ability to specifically bind to an IL-9 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0260] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-12 polypeptide. IL-12 is a heterodimeric cytokine composed of an IL-12a subunit and an IL-120 subunit. In one embodiment, the IL-12 polypeptide comprises i) an IL-12a polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 352 (i.e. the functional domain of IL-12 a, corresponding to amino acids 23-219 of Uniprotacc. No. P29459), and, ii) an IL-120 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 353 (i.e. the functional domain of IL-120, corresponding to amino acids 23-328 of Uniprot acc. No. P29460). An IL-12 polypeptide can comprise, in an N-to C-terminal direction, an aforementioned IL-12a polypeptide linked to an aforementioned IL-120 polypeptide, whereby the IL-12a polypeptide is linked to the IL-120 polypeptide through a peptidyl linker as herein described. Alternatively, an IL-12 polypeptide can comprise, in an N- to C-terminal direction, an aforementioned IL-120 polypeptide linked to an aforementioned IL-12a polypeptide, whereby the IL-120 polypeptide is linked to the IL-12a polypeptide through a peptidyl linker as herein described. Preferred, peptidyl linkers include (GGGGS)n, (GGGSS)n, (GGSSS)nor (GSSSS)n, whereby n = 3, 4, 5, 6 or 7, of which 4 is preferred and of which (GGGGS)4 is more preferred. The IL-12 polypeptide preferably has IL-12 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT4 phosphorylation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il12). The IL-12 polypeptide preferably has the ability to specifically bind to an IL-12 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0261] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-15 polypeptide. In one embodiment, the IL-15 polypeptide comprises an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 350 (i.e. the functional domain of IL-15, corresponding to amino acids 49-162 of Uniprot acc. No. P40933). The IL-15 polypeptide preferably has IL-15 receptor agonist activity as can be assayed as described in the art (see e.g. US 20230158164), e.g. by assaying STAT5 phosphorylation in a bio-assay, using reporter cells (e.g. https: / / nld.promeqa.com / products / reporter-bioassavs / cvtokine-and-qrowth-factor-bioassays / il-15-bioassay / ?catNum=JA2011). The IL-15 polypeptide preferably has the ability to specifically bind to an IL-15 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0262] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-18 polypeptide. In one embodiment, the IL-18 polypeptide comprises an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 354 (i.e. the functional domain of IL-18, corresponding to amino acids 37-193 of Uniprot acc. No. Q14116). The IL-18 polypeptide preferably has IL-18 receptor agonist activity as can be assayed as described in the art, e.g. by assaying NF-kB activation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il18). The IL-18 polypeptide preferably has the ability to specifically bind to an IL-18 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0263] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-23 polypeptide. IL-23 is a heterodimeric cytokine composed of an IL-12B (IL-12p40) subunit (which is shared with IL-12) and an IL-23A (IL-23p19) subunit. In one embodiment, the IL-23 polypeptide comprises i) an IL-23a polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 370 (i.e. the functional domain of IL-23a, corresponding to amino acids 20-189 of Uniprot acc. No. Q9NPF7); and, ii) an IL-120 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 353 (i.e. the functional domain of IL-120, corresponding to amino acids 23-328 of Uniprot acc. No. P29460). An IL-23 polypeptide can comprise, in an N- to C-terminal direction, an aforementioned IL-23a polypeptide linked to an aforementioned IL-120 polypeptide, whereby the IL-23a polypeptide is linked to the IL-120 polypeptide through a peptidyl linker as herein described. Alternatively, an IL-23 polypeptide can comprise, in an N- to C-terminal direction, an aforementioned IL-120 polypeptide linked to an aforementioned IL-23a polypeptide, whereby the IL-120 polypeptide is linked to the IL-23a polypeptide through a peptidyl linker as herein described. Preferred, peptidyl linkers include (GGGGS)n, (GGGSS)n, (GGSSS)nor (GSSSS)n, whereby n = 3, 4, 5, 6 or 7, of which 4 is preferred and of which (GGGGS)4 is more preferred. The IL-23 polypeptide preferably has IL-23 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT-5 activation in a bio-assay, using reporter cells (e.g. https: / / www.invivogen.com / hek-blue-il23). The IL-23 polypeptide preferably has the ability to specifically bind to an IL-23 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0264] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-27 polypeptide. IL-27 is a heterodimeric cytokine consisting of a four-helix bundle, IL-27a (also referred to as p28), with similarity to IL-6, complexed with a secreted binding protein, IL-270 (also referred to as Epstein-Barr Virus-Induced 3, Ebi3), with homology to type I cytokine receptors (Pflanz et al., 2002, Immunity. 2002;16:779-790). In one embodiment, the IL-27a polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 371 ((i.e. the functional domain of IL-27a, corresponding to amino acids 29-243 of Uniprot acc. No. Q8NEV9). In one embodiment, the comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 372 (i.e. the functional domain of IL-270, corresponding to amino acids 21-229 of Uniprot acc. No. Q14213). An IL-27 polypeptide can comprise, in an N- to C-terminal direction, an aforementioned IL-27a polypeptide linked to an aforementioned IL-270 polypeptide, whereby the IL-27a polypeptide is linked to the IL-270 polypeptide through a peptidyl linker as herein described. Alternatively, an IL-27 polypeptide can comprise, in an N- to C-terminal direction, an aforementioned IL-270 polypeptide linked to an aforementioned IL-27a polypeptide, whereby the IL-270 polypeptide is linked to the IL-27a polypeptide through a peptidyl linker as herein described. Preferred, peptidyl linkers include (GGGGS)n, (GGGSS)n, (GGSSS)nor (GSSSS)n, whereby n = 3, 4, 5, 6 or 7, of which 4 is preferred and of which (GGGGS)4 is more preferred. The IL-27 polypeptide preferably has IL-27 receptor agonist activity as can be assayed as described in the art, e.g. by assaying induced receptor dimerisation of IL-27 receptor alpha and gp130 in a bio-assay, using reporter cells (e.g. https: / / www.discoverx.com / product / pathhunter-u2os-il27ra-il6st-dimerization-cell-line / ); or assaying STAT-3 activation in a bio assay, using reporter cells for IL-19 and IL-20, reporting cross reactivity of the IL-20 type 1 receptor to IL-27 (e.g. https: / / www.invivogen.com / hek-blue-il20). The IL-27 polypeptide preferably has the ability to specifically bind to an IL-27 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.

[0265] In one embodiment, the at least one y6 T cell-activating agonist comprised in a multispecific antigen binding protein as described herein is an agonist that has a reduced or enhanced affinity for its receptor, wherein preferably the agonist that has affinity for its receptor that is reduced or enhanced as compared to the affinity of the corresponding wild type agonist for its receptor. In one embodiment, the at least one y6 T cell-activating agonist has an amino acid sequence that is modified as compared to the amino acid sequence of corresponding wild type agonist, which modification reduces or enhances the affinity of the modified agonist for its receptor, as compared to the affinity of the corresponding wild type agonist for that receptor. In one embodiment, the at least one y6 T cell-activating agonist has an amino acid sequence that is modified as compared to the amino acid sequence of corresponding wild type agonist, which modification reduces the affinity of the modified agonist for its receptor, as compared to the affinity of the corresponding wild type agonist for that receptor.

[0266] Thus, in one embodiment, a multispecific antigen binding protein as described herein comprises a y6 T cell-activating agonist that has a reduced affinity for its cognate receptor, preferably as compared to the corresponding wild type y6 T cell-activating agonist. Multispecific antigen binding proteins comprising such y6 T cell-activating agonists with reduced affinity fortheir cognate receptor, will have reduced pleiotropic- and undesired side-effects, when present in the bloodstream, while, when present at targeted sites, their avidity will ensure their local efficacy. Without being bound by theory, the multispecific antigen binding protein as described herein are designed to utilize the immune potentiating activity of the y6 T cell-activating agonist (which may be prerequisite to address toxicity and off-target immune suppression), to maximize efficacy at targeted loci, and at the same time improve the feasibility of dosing in the clinic.

[0267] Therefore, in one embodiment, a multispecific antigen binding protein as described herein comprises: a) an IL-2 polypeptide that is an IL-2 mutein that is modified to reduce or enhance affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide; b) an IL-6 polypeptide that is an IL-6 mutein that is modified to reduce or enhance affinity for IL-6R, relative to a corresponding wild type IL-6 polypeptide; c) an IL-7 polypeptide that is an IL-7 mutein that is modified to reduce or enhance affinity for IL-7R, relative to a corresponding wild type IL-7 polypeptide; d) an IL-9 polypeptide that is an IL-9 mutein that is modified to reduce or enhance affinity for IL-9R, relative to a corresponding wild type IL-9 polypeptide; e) an IL-12 polypeptide that is an IL-12 mutein that is modified to reduce or enhance affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide; f) an IL-15 polypeptide that is an IL-15 mutein that is modified to reduce or enhance affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide; g) an IL-18 polypeptide that is an IL-18 mutein that is modified to reduce or enhance affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide; h) an IL-21 polypeptide that is an IL-21 mutein that is modified to reduce or enhance affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide; i) an IL-23 polypeptide that is an IL-23 mutein that is modified to reduce or enhance affinity for IL-23R, relative to a corresponding wild type IL-23 polypeptide; and, j) an IL-27 polypeptide that is an IL-27 mutein that is modified to reduce or enhance affinity for IL-27R, relative to a corresponding wild type IL-27 polypeptide.

[0268] In a preferred embodiment, a multispecific antigen binding protein as described herein comprises: a) an IL-2 polypeptide that is an IL-2 mutein that is modified to reduce affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide; b) an IL-6 polypeptide that is an IL-6 mutein that is modified to reduce affinity for IL-6R, relative to a corresponding wild type IL-6 polypeptide; c) an IL-7 polypeptide that is an IL-7 mutein that is modified to reduce affinity for IL-7R, relative to a corresponding wild type IL-7 polypeptide; d) an IL-9 polypeptide that is an IL-9 mutein that is modified to reduce affinity for IL-9R, relative to a corresponding wild type IL-9 polypeptide; e) an IL-12 polypeptide that is an IL-12 mutein that is modified to reduce affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide; f) an IL-15 polypeptide that is an IL-15 mutein that is modified to reduce affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide; g) an IL-18 polypeptide that is an IL-18 mutein that is modified to reduce affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide; h) an IL-21 polypeptide that is an IL-21 mutein that is modified to reduce affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide; i) an IL-23 polypeptide that is an IL-23 mutein that is modified to reduce affinity for IL-23R, relative to a corresponding wild type IL-23 polypeptide; and, j) an IL-27 polypeptide that is an IL-27 mutein that is modified to reduce affinity for IL-27R, relative to a corresponding wild type IL-27 polypeptide.

[0269] In one embodiment, the IL-15 mutein has reduced affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide, and wherein the IL-15 mutein has a mutation (i.e. amino acid substitution, deletion or insertion) of one or more amino acids selected from the group consisting of: N1 , D8, D30, H32 L45, E46, I50, V49, S51 , L52, V63, E64, N65, I68, L69, Q108, M109, N112 (amino acid positions referring to position in SEQ ID NO: 350 or a corresponding position in an IL-15 allelic variant). Preferably, the IL-15 mutein comprises one or more amino acid substitutions selected from the group consisting of: N1G, D8S, D30N, D30T, H32D, H32E, H32N, H32Q, E46G, E46K, V49R, V49D, I50D, V63F, V63A, V63K, V63R, E64Q, E64K, E64R, N65D, N65E, N65K, N65R, I68F, I68H, I68D, I68K, I68R, I68E, I68Q, I68G, L69D, L69E, L69K, L69R, Q108A, Q108D, Q108E, Q108F, Q108H, Q108K, Q108L, Q108M, Q108N, Q108S, Q108T, Q108Y, M109A, M109H, M109R, N112D, N112G, N112P and N112R.

[0270] In one embodiment, the IL-15 mutein has reduced affinity for at least one of IL-15R0 and IL-15Ry, relative to a corresponding wild type IL-15 polypeptide. IL-15 muteins with an amino acid substitution in D8 or Q108 (e.g. D8S or Q108S) have a reduced affinity for IL-15R0 and IL-15Ry, respectively.

[0271] In a preferred embodiment, the IL-15 mutein has reduced affinity for IL-15R0, relative to a corresponding wild type IL-15 polypeptide. Thus, a preferred IL-15 mutein has an amino acid substitution in at least one of D8 (e.g. D8S), E64, N65, I68, and L69.

[0272] Thus, in one embodiment, a multispecific antigen binding protein as described herein comprises an IL-15 polypeptide that is an IL-15 mutein that is modified to reduce affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide. IL-15 muteins with reduced affinity for IL-15R as compared to human wild type IL-15 are described in Pettit et al. (J. Biol. Chem. 1997, 272: 2312-2318), W02005 / 085282, Xu et al. (Cancer Immunol. Res. 2021 , 9:1141-57) and WO2024 / 054425.

[0273] In one embodiment, a multispecific antigen binding protein as described herein has a valency of at least one agonist that induces at least one of STAT5 and STAT3 as described above, that is higher than one, which understood to mean that the multispecific antigen binding protein comprises more than one, e.g. two, three, four or five of agonist-moieties. Preferably, the more than one agonist-moieties are identical agonist-moieties.

[0274] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-21 polypeptide. Interleukin 21 (IL-21) is a protein that in humans is encoded by the IL-21 gene (Entrez Gene ID: 59067). IL-21 is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and which induces cell division / proliferation in its target cells. Amino acid sequences for human IL-21 precursor (including its signal sequence) are described in NCBI accession numbers NP_001193935 and NP_068575, the disclosures of which are incorporated herein by reference. IL-21 (mature / processed) comprises amino acids 30 - 153 of NP_001193935 or amino acids 30 - 162 of NP_068575 (i.e. SEQ ID NO: 355). IL-21 exerts its effects on target cells through the IL-21 receptor (IL-21 R) is expressed on the surface of T, B and NK cells. IL-21 R is similar in structure to the receptors for other type I cytokines like IL-2R or IL-15 and requires dimerization with the common gamma chain (yc) in order to bind IL-21. IL-21 R is encoded in humans by the IL-21R gene (Entrez Gene ID: 50615). Amino acid sequences for human IL-21 R are described in NCBI accession numbers NP_068570, NP_851564 and NP_851565, the disclosures of which are incorporated herein by reference. An example of an IL-21 R alpha chain is herein provided as SEQ ID NO: 356 and an example of an IL-21 R gamma chain is herein provided as SEQ ID NO: 357.

[0275] As used herein, an “IL-21 R agonist” is an agent that has “agonist” activity at the IL-21 receptor, which means that the agent that can cause or increase "IL-21 R signaling". “IL-21 R signaling” refers to an ability of IL-21 R, e.g. when expressed on the surface of T, B and NK cells and triggered by its natural ligand IL-21 , to activate or transduce an intracellular signaling pathway. The “natural ligand IL-21” is herein understood as a human wild type IL-21 comprising or consisting of an amino acid sequence as indicated above. When bound to IL-21 , the IL-21 receptor acts through the Jak / STAT pathway, utilizing Jak1 and Jak3 and a STAT3 homodimer to activate its target genes. IL-21 R agonist activity, i.e. changes in IL-21 R signaling activity, can be measured, for example, by assays designed to measure changes in the IL-21 R signaling pathways, e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or assays designed to measure expression of reporter genes under control of IL-21 R-sensitive promoters and enhancers, or indirectly by a downstream effect mediated by IL-21 R (e.g. activation of specific cytolytic machinery in NK cells). A suitable cell-based assay for biological activity of an IL-21 R agonist, is e.g. described in Maurer et al. (mAbs. 2012; 4(1): 69-83.), wherein a murine pre-B-cell line is transfected with both the human IL-21 R and a STAT-responsive luciferase reporter gene. IL-21 R agonist activity can be determined using this cell line by measuring the level of STAT3 phosphorylation using anti-pSTAT3 antibody-conjugated beads and / or by detecting luciferase luminescence, upon contacting the cell line with an IL-21 R agonist. The natural ligand IL-21 can serve as a positive control in an assay for IL-21 R agonist activity and can also be used as a reference for the amount of IL-21 R agonist activity of a given non-natural IL-21 R agonist, such as a multispecific antigen binding protein as described herein comprising an IL-21 R agonist.

[0276] In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-21 R agonist that is an IL-21 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 355, and preferably having an IL-21 R agonist activity as defined above, and / or preferably having an affinity for the IL-21 R as can be determined using methods generally known in the art, such as surface plasmon resonance. In one embodiment, a multispecific antigen binding protein as described herein comprises an IL-21 mutein, as described herein.

[0277] When IL-21 binds to IL-21 R, the Jak / STAT signaling pathway is activated to activate target genes. While IL-21-induced signaling may be therapeutically desirable, careful consideration of the timing and the location of the signaling is needed, given IL-21 R's broad expression profile and due to the fact that IL-21 has the ability to potentiate CD8 T cell responses as well as to suppress antigen presentation and T cell priming. The data presented herein supports the use of carefully designed IL-21 muteins to achieve IL-21 R signaling at the appropriate time and place and to improve pharmacokinetics of therapeutics comprising such IL-21 muteins.

[0278] Thus, in one embodiment, a multispecific antigen binding protein as described herein comprises an IL-21 polypeptide that is an IL-21 mutein that is modified to reduce affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide. IL-21 muteins with reduced affinity for IL-21 R as compared to human wild type IL-21 are described in Shen et al. (Front Immunol. 2020; 11 : 832), WO2019 / 028316, W02006 / 111524, W02008 / 049920 and the co-pending application EP24163865.9. In one embodiment, the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has a mutation (i.e. amino acid substitution, deletion or insertion) of one or more amino acids selected from the group consisting of: D4, R5, I8, R9, R11, Q12, L13, 114, D15, 116, D18 Q19, L20, K21 , Y23, R65, I66, I67, N68, V69, S70, K72, K73, L74, K75, R76, K77, P78, P79, S80, E100, E109, R110, K112, S113, Q116, K117, 1119, H120, and L123 (amino acid positions referring to position in SEQ ID NO: 355 or a corresponding position in an IL-21 allelic variant). Preferably, the IL-21 mutein comprises one or more amino acid substitutions selected from the group consisting of: I8H, I8Q, I8V, I8A, D4H, R11E, Q12K, L13E, Q19S, L20S, L20N, L20W, K21 H, I67T, I67N, K75E, K112H, I14D, D18A, E100R, I119D, K72D, K73A, K73Y, K75D, R76H, K77D, L13D, P78D, Q12A, Q19D, R5A, R65D, R76A, R9A, S70E, S80G, V69D, Y23D, I16E, I66G, I8D, K72G, K73D, K75G, K77G, P79D, Q12D, R5D, R65G, R76D, R9D, S70G, S80P, V69G, I66P, I8E, K72P, K73E, K75P, K77P, Q12E, R5E, R65P, R76E, R9E, S70P, V69P, I8G, K73G, Q12N, R5G, R76G, R9G, S70Y, I8N, K73H, Q12S, R5H, R76H, R9H, I8S, K73I, Q12T, R5I, R76I, R9I, K73N, Q12V, R5K, R76K, R9K, K73P, R5L, R76L, R9L, K73Q, R5M, R76M, R9M, K73S, R5N, R76N, R9N, K73V, R5Q, R76P, R9Q, R5S, R76Q, R9S, R5T, R76S, R9T, R5V, R76T, R9V, R5Y, R76V, R9Y and R76Y.

[0279] In one embodiment, the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has at least one of: a) a deletion of at least one, two, three, four, five, six, seven or eight amino acids in the region of N59 to I66; b) a deletion of at least one, two, three, four, five, six, seven or eight amino acids in the region of N82 to R90; and c) a deletion of amino acids K75 and R76 or a deletion of amino acids K75, R76 and R77. In one embodiment, the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has at least one of the deletions (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (N63-, E64-, R65- and I66-), (N82-, A83-, G84-, R85-, R86-, Q87- and K88-) and (N82-, A83-, G84-, R85-, R86-, Q87-, K88-, H89- and R90-).

[0280] In one embodiment, the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has an insertion of at least two, three or four amino acids immediately C-terminal to G84 and / or an insertion K75 insX, wherein X is one, two or three amino acids selected from the group consisting of: G, S and D.

[0281] In a further aspect, the present disclosure provides IL-21 muteins comprising at least one amino acid substitution, deletion and / or insertion. Amino acid substitutions, deletions and insertions in an IL-21 mutein provided herein are indicated relative to the wild-type human IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 355. Hence, to allow for allelic variation, a wildtype human IL-21 preferably comprises an amino acid sequence having, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 355.

[0282] In one embodiment, there is provided an IL-21 mutein comprising at least one amino acid substitution, deletion or insertion selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11E, R11Q, R11N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21E, K21D, I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L74N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, R76-, K112H, K112N, K112Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D). In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution, deletion or insertion selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11E, R11Q, R11N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21 E, K21D, I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L74N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, R76-, K112H, K112N, K112Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65-and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D).

[0283] In one embodiment, there is provided an IL-21 mutein comprising a combination of at least two, three, four, five, six, seven or eight amino acid substitutions, deletions and / or insertions selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R11 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21 E, K21D, I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L74N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, R76-, K112H, K112N, K112Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D). In one embodiment, the IL-21 mutein comprises no other modifications than the at least two, three, four, five, six, seven or eight amino acid substitutions, deletions and / or insertions selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11E, R11Q, R11N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21H, K21N, K21Q, K21 E, K21D, I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L74N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, R76-, K112H, K112N, K112Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65-and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D). In one embodiment, there is provided an IL-21 mutein comprising a deletion of at least one amino acid in the region of N59 to I66. In one embodiment, there is provided an IL-21 mutein comprising a deletion of at least two, three, four, five, six, seven or eight amino acids in the region of N59 to I66. In one embodiment, there is provided an IL-21 mutein comprising the deletions (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-). In one embodiment, there is provided an IL-21 mutein comprising the deletions (N63-, E64-, R65- and I66-).

[0284] In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to an IL-21 receptor (IL-21 R), preferably an IL-21 R having an amino acid sequence of SEQ ID NO: 356. In another embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to an IL-21 R gamma chain, preferably an IL-21 R gamma chain having an amino acid sequence of SEQ ID NO: 357.

[0285] In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the human IL-21 R with a reduced affinity, relative to the affinity of wildtype IL-21 for the human IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the alpha chain of the human IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the alpha chain of the human IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the gamma chain of the human IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the gamma chain of the human IL-21 R.

[0286] In one embodiment, the IL-21 mutein having a with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution or deletion selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; K75E; L13E; D4H; I8H; (N63- E64- R65- 166-); I67N; L74D; L74F; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; K75E; L13E; D4H; I8H; (N63- E64- R65- 166-); I67N; L74D; L74F; L20S; and L20N.

[0287] The IL-21 muteins provided herein bind to IL-21 R in a non-covalent and reversible manner. In one embodiment, the binding strength of an IL-21 mutein to IL-21 R may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the mutein and the IL-21 R. In one embodiment, an IL-21 mutein provided herein has a low-affinity for IL-21 R and thus will bind a lesser amount of IL-21 R than a wild type IL-21. In one embodiment, an IL-21 mutein provided herein has an equilibrium association constant, KA, which is, with decreasing preference, at least 105M1, at least 106M1, at least 107M1, at least 108M1, at least 109M1, or at least 1010M L As understood by the artisan of ordinary skill, KA can be influenced by factors including pH, temperature and buffer composition.

[0288] In one embodiment, the binding strength of an IL-21 mutein provided herein to IL-21 R may be described in terms of its sensitivity or affinity. KD is the equilibrium dissociation constant, a ratio of koff / kon, between the IL-21 mutein and IL-21 R. KD and KA are inversely related. The KD value relates to the concentration of the mutein (the amount of mutein needed for a particular experiment or application) and so the lower the KD value (lower concentration needed) the higher the affinity of the mutein. In one embodiment, the binding strength of an IL-21 mutein provided herein to IL-21 R may be described in terms of KD. In one embodiment, the KD of an IL-21 mutein provided herein is about IO4M, about 105M, about 106M, or less. In one embodiment, the KD of an IL-21 mutein provided herein is micromolar, nanomolar, or picomolar. In one embodiment, the KD of an IL-21 mutein provided herein is within a range of about 10 to 10-6M, or 10-7to 10-9M. In one embodiment, an IL-21 mutein provided herein binds to the human IL-21 R with a Kothat is greater than oris about 0.04 nM. In one embodiment, an IL-21 mutein provided herein binds to the human IL-21 R with a KD of about 0.01 nM to about 20 nM, 0.02 nM to 20 nM, 0.05 nM to 20 nM, 0.05 nM to 15 nM, 0.1 nM to 15 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM.

[0289] In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity forthe human IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000.000-fold reduction in binding affinity for IL-21 R, relative to, relative to the affinity of wild-type IL-21 for IL-21 R. In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity forthe alpha chain of IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in binding affinity for the alpha chain of IL-21 R, relative to, relative to the affinity of wild-type IL-21 for the alpha chain of IL-21 R. In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity forthe gamma chain of IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in binding affinity for the gamma chain of IL-21 R, relative to, relative to the affinity of wild-type IL-21 for the gamma chain of IL-21 R.

[0290] In one embodiment, the binding affinity of an IL-21 mutein provided herein is determined by SPR, e.g. as described in the Examples herein. In one embodiment, the binding affinity of an IL-21 mutein provided herein is thus determined when the IL-21 mutein is present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate can further comprise a trimer of a 4-1 BB ligand extracellular domain (4-1 BBL ECD).

[0291] In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity forthe human IL-21 R, expressed in pKo, that is at least 0.5 lower than the pKo of wild-type IL-21 for the IL-21 R. pKo is understood herein to be -logio(Ko). In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 0.5 lower than the pKo of wild-type IL-21 forthe IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N.

[0292] In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R, In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N.

[0293] In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.6 lower than the pKo of wild-type IL-21 for the IL-21 R, In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 1.6 lower than the pKo of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; Q12K; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; Q12K; I67N; L74D; L20S; and L20N.

[0294] In one embodiment, an IL-21 mutein provided herein exhibits a reduction in activity as measured by an in vitro STAT3 phosphorylation assay, relative to the activity of wild-type IL-21 under corresponding conditions. In one embodiment, an IL-21 mutein provided herein exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in activity as measured by a STAT3 phosphorylation assay, relative to the activity of wild-type IL-21 under corresponding conditions.

[0295] In one embodiment, an IL-21 mutein provided herein exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold reduction in activity as measured by a NK cell or y5 T cell proliferation assay, relative to the activity of wild-type IL-21 under corresponding conditions. In one embodiment, the NK cell or y5 T cell proliferation assay is a short-term proliferation assay, measuring proliferation over the course of less than one week, e.g. 3, 4, 5 or 6 days, e.g. as described in the Examples herein. In one embodiment, the NK cell or y5 T cell proliferation assay is a long-term proliferation assay, measuring proliferation over the course of more than one week, e.g. at least 10, 12 or 14 days, e.g. as described in the Examples herein.

[0296] In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with a monoclonal antibody against a TAA (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of tumor cells expressing the TAA (e.g. SKOV3 cells), that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L13E; I8H; (N63- E64- R65- I66-); I67T; I67N; L74G; L74D; L74F; L20S; L20N; and G84insGGGGG, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L13E; I8H; (N63- E64- R65- I66-); I67T; I67N; L74G; L74D; L74F; L20S; L20N; and G84insGGGGG.

[0297] In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2 higher than the EC50 of a corresponding control conjugate comprising wildtype IL-21 in the same assay. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; I8H; (N63- E64-R65- I66-); I67T; I67N; L74G; L74D; L74F; and L20N, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; I8H; (N63- E64- R65- 166-); I67T; I67N; L74G; L74D; L74F; and L20N.

[0298] In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) has a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- 166-); and L74F, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- I66-); and L74F.

[0299] In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.0 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) has a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; I8H; (N63- E64- R65- 166-); and L74F, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; I8H; (N63- E64- R65- 166-); and L74F. In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity, expressed in pKo, for the human IL-21 R that is at least 0.5 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; and I8H, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; and I8H.

[0300] In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; and L13E, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; and L13E.

[0301] In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.0 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; and I67N, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; and I67N.

[0302] In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with trastuzumab, which conjugate further comprises a trimer of an 4-1 BBL ECD mutein comprising a substitution A154D, induces a maximal proliferation of NK cells at a saturating concentration of 25 nM of the conjugate in a normalized 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, which proliferation is at least 65%, 70%, 75%, 80%, 85%, or 89% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and wild type 4-1 BBL in the same assay. Hence, in one embodiment, the IL-21 mutein comprises the amino acid substitution L20W, or the IL-21 mutein comprises no other modifications than the amino acid substitution L20W.

[0303] In one embodiment, there is provided an IL-21 mutein as described herein, which, when present in a conjugate with an antigen-binding protein that specifically binds a (target) antigen, as a result of the reduced affinity of the IL-21 mutein for the IL-21 R, produces reduced (little or no) agonist activity at an IL-21 R expressed at the surface of a cell, in the absence of the antigen or cells carrying the antigen. However, when the conjugate is bound to the antigen or to cells carrying the antigen, the IL-21 mutein in the conjugate manifests significant agonist activity. This activity is the result of the IL-21 mutein being present in high local density on the surface of the target cells, which leads to an enhanced apparent affinity for the IL-21 R on local NK cells (see Figure 1) or y6 T cells, primarily through the mechanism of avidity. As such, an IL-21 mutein as described herein in a conjugate with an antigen-binding protein broadens the therapeutic window as compared to a corresponding conjugate comprising a wild type IL-21. The term “therapeutic window” is herein understood as the ratio (or fold-difference) of the EC50 values obtained from a functional assay (e.g., a proliferation assay) comparing conditions in which cancer cells are absent to conditions in which they are present. A therapeutic window of 10 (or 1 log) would mean that the EC50 without cancer cells was 10 times higher ( a less potent effect) as compared to when the cancer cells were present. Upon systemic administration the conjugate comprising the IL-21 mutein will have little or no effect on cells in the periphery, including T-, B- or NK cells, while remaining effective in stimulating these immune cells, in particular NK cells or y6 T cells, at a tumor site or a site of infection or inflammation. This is because the target antigen bound by the antigen-binding protein is present at high local concentrations only in these specific areas, enabling the effect of avidity.

[0304] Thus, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a conjugate with an antigen binding protein that specifically binds an antigen, wherein the conjugate optionally further comprises a 4-1 BBL extracellular domain (ECD), has an EC50 in an NK cell or y6 T cell proliferation assay in the presence of the antigen or cells expressing the antigen that is, with increasing preference, at least a factor 2, 5, 10, 20, 50, 100, 200, 500, 1 ,000, 2,000, 5,000, 10,000, 20,000, 50,000 or 100,000 lower than the EC50 in a corresponding NK cell proliferation assay in the absence of the antigen or cells expressing the antigen.

[0305] In one embodiment, the difference in induction of NK cell or y5 T cell proliferation between the presence and absence of the antigen is determined using a reference multispecific antigen binding protein, such as AVC1 and reference tumor cells such as SK-OV-3 cells expressing HER2. The AVC1 multispecific antigen binding protein consists of a first trastuzumab heavy chain fused to the 4-1 BB ligand extracellular domain (SEQ ID NO: 468), a second trastuzumab heavy chain fused to wild type IL-21 (SEQ ID NO: 469) and trastuzumab light chains (SEQ ID NO: 470), wherein the constant regions of the first and second heavy chains are distinguished using knob-in-hole technology (see WO2024 / 056862). The wild type IL-21 amino acid sequence in the second heavy chain amino acid sequence of SEQ ID NO: 469 can be replaced by an amino acid sequence of an IL-21 mutein to be assayed, e.g. for its ability to induce NK cell or y6 T cell proliferation in the presence and absence of tumor cells expressing HER2, such as the SK-OV-3 cells.

[0306] In one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470, has an EC50 in an NK cell or y6 T cell proliferation assay in the presence of SK-OV-3 cells that is, with increasing preference, at least a factor 25, 50, 100, 200, 500, 1 ,000, 2,000, 5,000, 10,000, 20,000, 50,000 or 100,000 lower than the EC50 in a corresponding NK cell or y6 T cell proliferation assay in the absence of the SK-OV-3 cells.

[0307] In one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding proteins consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470, produces a difference in EC50 in an NK cell or y6 T cell proliferation assay in the presence vs absence of SK-OV-3 cells that is, with increasing preference, at least a factor 2, 5, 10, 20, 50, 100, 200, 500, 1 ,000, 2,000 or 5,000 higher than the difference in EC50 in an NK cell or y6 T cell proliferation assay in the presence vs absence of SK-OV-3 cells as produced by a reference multispecific antigen binding proteins consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470.

[0308] In the above embodiments, the NK cell or y6 T cell proliferation assays are preferably performed using NK cells or y6 T cells isolated from healthy donors. In the above embodiments, the EC50 values in the NK cell or y6 T cell proliferation assays are preferably determined on the basis of the average values using NK cells or y6 T cell isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or y6 T cell proliferation assays are preferably performed essentially as described in the examples herein.

[0309] While the reduced affinity of the IL-21 muteins described herein when present in a conjugate with an antigen binding protein increases the therapeutic window as compared to a corresponding conjugate comprising a wild type IL-21 , at the same time the ability of a conjugate with an IL-21 muteinto induce NK cell or y6 T cell cytotoxicity against cells carrying the antigen that is bound by the antigen binding protein, preferably remains essentially unaffected.

[0310] Hence, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470, has an EC50 in an NK cell or y6 T cell cytotoxicity assay in the presence of SK-OV-3 cells that is, with increasing preference, at least equal to, or at least 2-fold, at least 5-fold or at least 10-fold higher than the EC50 of a reference multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470, in the same assay. The NK cell or or yb T cell cytotoxicity assay in the presence of SK-OV-3 cells preferably performed using NK cells or yb T cells isolated from healthy donors. Preferably, the EC50 values in the NK cell or yb T cell cytotoxicity assays are determined on the basis of the average values using NK cells or yb T cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or yb T cell cytotoxicity assays are preferably performed essentially as described in the examples herein.

[0311] Also the ability of a conjugate between an IL-21 mutein as described herein and an antigen binding protein to support long-term expansion of NK cells or yb T cell in the presence of cells carrying the antigen that is bound by the antigen binding protein, preferably remains essentially unaffected.

[0312] Hence, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470, induces a fold expansion of NK cells or yb T cells in the presence of SK-OV-3 cells in an NK cell expansion assay, that is, with increasing preference, at least equal to, or at least 2-fold, at least 5-fold or at least 10-fold higher than the fold expansion induced by a reference multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 468; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 469; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 470 in the same assay. The NK cell or yb T cell expansion assay in the presence of SK-OV-3 cells preferably performed using NK cells or yb T cells isolated from healthy donors. Preferably, the fold expansion of NK cells or yb T cells in the assays is determined on the basis of the average values using NK cells or yb T cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or yb T cell expansion assays are preferably performed essentially as described in the examples herein.

[0313] A further advantage of the IL-21 muteins as described herein is that their reduced affinity for IL-21 R the improves pharmaco-kinetics of therapeutics comprising the IL-21 muteins. In the body many cells, including T-, B- or NK cells, are present that express IL-21 R molecules at their surfaces. These IL-21 R molecules act as a sink for therapeutics comprising a moiety with affinity for IL-21 R such as an IL-21 mutein. Upon binding to a surface expressed IL-21 R, the therapeutic comprising the IL-21-moiety will be internalized and will therefore no longer be available for exerting its therapeutic effect, e.g. in the tumor microenvironment. Hence, the reduced affinity for IL-21 R of the IL-21 muteins as described herein reduces or prevents their disappearance in this sink and thereby improves pharmaco-kinetics of therapeutics comprising the IL-21 muteins.

[0314] In addition, the reduced affinity IL-21 muteins as described herein for IL-21 R will diminish its potential apoptosis-inducing effect on dendritic cells, which can therefore remain active in antigen presentation and subsequent induction of T cell responses.

[0315] In specific embodiments, the multispecific antigen binding protein comprises a combination of an IL-21 mutein as described herein above, and a 4-1 BBL ECD mutein as described herein below. In specific embodiments, the multispecific antigen binding protein comprises a combination of a 4-1 BBL ECD mutein and an IL-21 mutein selected from the group consisting of: 4-1 BBL mutein A154D and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein A154D and IL-21 mutein L20W; 4-1 BBL mutein A154D and IL-21 mutein L74D; 4-1 BBL mutein A154D and IL-21 mutein L20N; 4-1 BBL mutein A154D and IL-21 mutein I67N; 4-1 BBL mutein A154D and IL-21 mutein L20S; 4-1 BBL mutein A154D and IL-21 mutein L13E; 4-1 BBL mutein A154D and IL-21 mutein I8H; 4-1 BBL mutein A154D and IL-21 mutein (N63- E64- R65- 166-); 4-1 BBL mutein A154D and IL-21 mutein L74F; 4-1 BBL mutein A154E and IL-21 mutein (N82- A83- G84- R85- R86- Q87-K88-); 4-1 BBL mutein A154E and IL-21 mutein L20W; 4-1 BBL mutein A154E and IL-21 mutein L74D; 4-1 BBL mutein A154E and IL-21 mutein L20N; 4-1 BBL mutein A154E and IL-21 mutein I67N; 4-1 BBL mutein A154E and IL-21 mutein L20S; 4-1 BBL mutein A154E and IL-21 mutein L13E; 4-1BBL mutein A154E and IL-21 mutein I8H; 4-1 BBL mutein A154E and IL-21 mutein (N63- E64-R65- 166-); 4-1 BBL mutein A154E and IL-21 mutein L74F; 4-1 BBL mutein A154D + G155Q and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein A154D + G155Q and IL-21 mutein L20W; 4-1 BBL mutein A154D + G155Q and IL-21 mutein L74D; 4-1 BBL mutein A154D + G155Q and IL-21 mutein L20N; 4-1 BBL mutein A154D + G155Q and IL-21 mutein I67N; 4-1 BBL mutein A154D + G155Q and IL-21 mutein L20S; 4-1 BBL mutein A154D + G155Q and IL-21 mutein L13E; 4-1 BBL mutein A154D + G155Q and IL-21 mutein I8H; 4-1 BBL mutein A154D + G155Q and IL-21 mutein (N63- E64- R65- I66-); 4-1 BBL mutein A154D + G155Q and IL-21 mutein L74F; 4-1BBL mutein V153Q and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein V153Q and IL-21 mutein L20W; 4-1 BBL mutein V153Q and IL-21 mutein L74D; 4-1 BBL mutein V153Q and IL-21 mutein L20N; 4-1 BBL mutein V153Q and IL-21 mutein I67N; 4-1 BBL mutein V153Q and IL-21 mutein L20S; 4-1 BBL mutein V153Q and IL-21 mutein L13E; 4-1 BBL mutein V153Q and IL-21 mutein I8H; 4-1 BBL mutein V153Q and IL-21 mutein (N63- E64- R65- I66-); 4-1BBL mutein V153Q and IL-21 mutein L74F; 4-1 BBL mutein Q227E and IL-21 mutein (N82- A83-G84- R85- R86- Q87- K88-); 4-1 BBL mutein Q227E and IL-21 mutein L20W; 4-1 BBL mutein Q227E and IL-21 mutein L74D; 4-1 BBL mutein Q227E and IL-21 mutein L20N; 4-1 BBL mutein Q227E and IL-21 mutein I67N; 4-1 BBL mutein Q227E and IL-21 mutein L20S; 4-1 BBL mutein Q227E and IL-21 mutein L13E; 4-1 BBL mutein Q227E and IL-21 mutein I8H; 4-1 BBL mutein Q227E and IL-21 mutein (N63- E64- R65- I66-); 4-1 BBL mutein Q227E and IL-21 mutein L74F; 4-1 BBL mutein Q227R and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein Q227R and IL-21 mutein L20W; 4-1 BBL mutein Q227R and IL-21 mutein L74D; 4-1 BBL mutein Q227R and IL-21 mutein L20N; 4-1 BBL mutein Q227R and IL-21 mutein I67N; 4-1 BBL mutein Q227R and IL-21 mutein L20S; 4-1 BBL mutein Q227R and IL-21 mutein L13E; 4-1 BBL mutein Q227R and IL-21 mutein I8H; 4-1 BBL mutein Q227R and IL-21 mutein (N63- E64- R65- 166-); 4-1 BBL mutein Q227R and IL-21 mutein L74F; 4-1 BBL mutein L101 N and IL-21 mutein (N82- A83- G84- R85- R86- Q87-K88-); 4-1 BBL mutein L101N and IL-21 mutein L20W; 4-1 BBL mutein L101N and IL-21 mutein L74D; 4-1 BBL mutein L101 N and IL-21 mutein L20N; 4-1 BBL mutein L101 N and IL-21 mutein I67N; 4-1 BBL mutein L101N and IL-21 mutein L20S; 4-1 BBL mutein L101N and IL-21 mutein L13E; 4-1BBL mutein L101N and IL-21 mutein I8H; 4-1 BBL mutein L101 N and IL-21 mutein (N63- E64-R65- 166-); 4-1 BBL mutein L101 N and IL-21 mutein L74F; 4-1 BBL mutein Y110Q and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein Y110Q and IL-21 mutein L20W; 4-1 BBL mutein Y110Q and IL-21 mutein L74D; 4-1 BBL mutein Y110Q and IL-21 mutein L20N; 4-1 BBL mutein Y110Q and IL-21 mutein I67N; 4-1 BBL mutein Y110Q and IL-21 mutein L20S; 4-1 BBL mutein Y110Q and IL-21 mutein L13E; 4-1 BBL mutein Y110Q and IL-21 mutein I8H; 4-1 BBL mutein Y110Q and IL-21 mutein (N63- E64- R65- 166-); 4-1 BBL mutein Y110Q and IL-21 mutein L74F; 4-1BBL mutein Q230K and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein Q230K and IL-21 mutein L20W; 4-1 BBL mutein Q230K and IL-21 mutein L74D; 4-1 BBL mutein Q230K and IL-21 mutein L20N; 4-1 BBL mutein Q230K and IL-21 mutein I67N; 4-1 BBL mutein Q230K and IL-21 mutein L20S; 4-1 BBL mutein Q230K and IL-21 mutein L13E; 4-1 BBL mutein Q230K and IL-21 mutein I8H; 4-1 BBL mutein Q230K and IL-21 mutein (N63- E64- R65- I66-); 4-1BBL mutein Q230K and IL-21 mutein L74F; 4-1 BBL mutein V100Q and IL-21 mutein (N82- A83-G84- R85- R86- Q87- K88-); 4-1 BBL mutein V1 OOQ and IL-21 mutein L20W; 4-1 BBL mutein V1 OOQ and IL-21 mutein L74D; 4-1 BBL mutein V100Q and IL-21 mutein L20N; 4-1 BBL mutein V100Q and IL-21 mutein I67N; 4-1 BBL mutein V100Q and IL-21 mutein L20S; 4-1 BBL mutein V100Q and IL-21 mutein L13E; 4-1 BBL mutein V100Q and IL-21 mutein I8H; 4-1 BBL mutein V100Q and IL-21 mutein (N63- E64- R65- 166-); and, 4-1 BBL mutein V100Q and IL-21 mutein L74F.

[0316] Co-stimulatory agonist

[0317] A multispecific antigen binding protein as described herein, can further comprise at least one co-stimulatory agonist of a y6 T cell. A co-stimulatory agonist of a y6 T cell can advantageously be included in the multispecific antigen binding protein amongst others to promote longevity and / or to avoid terminal differentiation of y6 T cells. In one embodiment, a multispecific antigen binding protein as described herein comprises a co-stimulatory agonist that is at least one of: i) a 4-1 BB agonist; ii) a CD27 agonist; and iii) a GITR agonist.

[0318] In one embodiment, a multispecific antigen bin...

Claims

Claims1. A multispecific antigen binding protein comprising:a) a first antigen-binding region and an optional second antigen-binding region that specifically bind a tumor or pathogen associated antigen (TAA);b) a third antigen-binding region that specifically binds an epitope of a y6 T cell receptor (TCR);c) a y6 T cell-activating agonist that induces at least one of STAT5 and STAT3 activation; and,d) a y6 T cell co-stimulatory agonist that is at least one of:i) a 4-1 BB agonist;ii) a CD27 agonist; and,iii) a GITR agonist.

2. A multispecific antigen binding protein according to claim 1 , wherein at least one of the first, optional second and third antigen-binding region comprise an immunoglobulin-derived antigen-binding region, wherein preferably the immunoglobulin-derived antigen-binding region comprises or consists of a Fab, a single-chain Fv (scFv) or an immunoglobulin single variable domain (ISVD).

3. A multispecific antigen binding protein according to any one of the preceding claims, wherein the TAA is selected from the group consisting of: Her2 (ErbB2 / Neu), Receptor Tyrosine Kinase-like Orphan Receptor 1 (ROR1), Crypto, CD2, CD4, CD20, CD30, CD19, CD38, CD40, CD47, Glycoprotein NMB, CanAg, CD22 (Siglec2), CD33 (Siglec3), CD79, CD123, CD138, CD171 , CTLA-4 (CD152), PD1 , PSCA, L1-CAM, EpCAM, PSMA (prostate specific membrane antigen), BCMA, TROP2, STEAP1 , CD52, CD56, CD80, CD70, E-selectin, EphB2, EPHA4, Melanotransferrin, Mud 6, TMEFF2, Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), B7.1 , B7.2, B7-H3, B7-H4, B7-H6, PD-L1 , IL-6 receptor, IL1 accessory Protein, MAGE, MART-1 / Melan-A, gp100, MICA, MICB, adenosine deaminase- binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-C017- 1A / GA733, protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), NaPi2b, TYRP1 , nectin-4, a UL16-binding protein (ULBP), a RAET1 protein, carcinoembryonic antigen (CEA), CEACAM5, etv6, aml1 , prostate specific antigen (PSA), T- cell receptor / CD3-zeta chain, MAGE-A3, a GAGE-tumor antigen, anti-Mullerian hormone Type II receptor, delta-like ligand 3 (DLL3), delta-like ligand 4 (DLL4), DR5, NTRKR1 (EC 2.7.10.1), SLAMF7, TRAILR1 , TRAILR2, BAGE, RAGE, LAGE-1 , NAG, GnT-V, MUM-1 , CDK4, MUC1 , MUC1-C, VEGF, VEGFR2, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor (EGFR / ERBB1), HER-3 / ERBB3, HER-4 / ERBB4, a heterodimeric receptor comprised of at least one HER subunit, gastrin releasing peptide receptor antigen, cMET, integrin receptors, a5p3 integrins, a5p1 integrins, allbp3-integrins, PDGF alpha receptor,PDGF beta receptor, sVE-cadherin, IL-8 receptor, hCG, CSF1R, a-fetoprotein, mesothelin (MSLN), Isoform 2 of Claudin-18 (Claudin 18.2, CLDN18)), folate receptor alpha (FRa, FOLR1), tissue factor (TF, CD142), P-cadherin, E-cadherin, a-catenin, p-catenin and y- catenin, Plexin-A1 , TNFRSF10B, AXL, EDNRB, OLR1 , ADAM12, PLAUR, CCR4, CCR6, p120ctn, PRAME, NY-ESO-1 , cdc27, CDCP1 , adenomatous polyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, a GM2 ganglioside, a GD2 ganglioside, a human papillomavirus protein, imp-1 , P1A, EBV-encoded nuclear antigen (EBNA)-I, brain glycogen phosphorylase, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, FcRL5 / FcRH5, Flt3, mud 6, mu 7, mmp9, FAP, Lewis-Y, EGFRvlll, GPC3, gpA33, 5T4, SSTR2, CD73, CD25, CD45, CD133, FGFR2b, CD79B, BTLA, Fibronectin extra-domain B, GM3, LAG-3, 0X40, PDGFRa, TIGIT, VEGF-a, GPCR5D and scatter factor receptor kinase.

4. A multispecific antigen binding protein according to any one of claims 1 - 3, wherein the third antigen-binding region specifically binds an epitope in the V region of a V61 chain of a y6 TCR.

5. A multispecific antigen binding protein according to any one of the preceding claims, wherein the y6 T cell-activating agonist is selected from the group consisting of:a) an IL-2 polypeptide that is an IL-2 mutein that is modified to reduce (or enhance) affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide;b) an IL-6 polypeptide that is an IL-6 mutein that is modified to reduce (or enhance) affinity for IL-6R, relative to a corresponding wild type IL-6 polypeptide;c) an IL-7 polypeptide that is an IL-7 mutein that is modified to reduce (or enhance) affinity for IL-7R, relative to a corresponding wild type IL-7 polypeptide;d) an IL-9 polypeptide that is an IL-9 mutein that is modified to reduce (or enhance) affinity for IL-9R, relative to a corresponding wild type IL-9 polypeptide;e) an IL-12 polypeptide that is an IL-12 mutein that is modified to reduce (or enhance) affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide;f) an IL-15 polypeptide that is an IL-15 mutein that is modified to reduce (or enhance) affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide;g) an IL-18 polypeptide that is an IL-18 mutein that is modified to reduce (or enhance) affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide;h) an IL-21 polypeptide that is an IL-21 mutein that is modified to reduce (or enhance) affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide;i) an IL-23 polypeptide that is an IL-23 mutein that is modified to reduce (or enhance) affinity for IL-23R, relative to a corresponding wild type IL-23 polypeptide; and, j) an IL-27 polypeptide that is an IL-27 mutein that is modified to reduce (or enhance) affinity for IL-27R, relative to a corresponding wild type IL-27 polypeptide.

6. A multispecific antigen binding protein according to claim 5, wherein:i) the IL-15 mutein has reduced affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide, and wherein the IL-15 mutein has a mutation in at least one amino acid position selected from the group consisting of: N1 , D8, D30, H32 L45, E46, 150, V49, S51 , L52, V63, E64, N65, I68, L69, Q108, M109 and N112, of which D8, E64, N65, I68, and L69 are preferred; and,ii) the IL-21 mutein has reduced affinity for IL-21 R, relative to a corresponding wild type IL-21 polypeptide, and wherein the IL-21 mutein has at least one of:a) a mutation in one or more amino acid positions selected from the group consisting of: D4, R5, I8, R9, R11 , Q12, L13, 114, D15, 116, D18 Q19, L20, K21 , Y23, R65, I66, I67, N68, V69, S70, K72, K73, L74, K75, R76, K77, P78, P79, S80, E100, E109, R110, K112, S113, Q116, K117, 1119, H120, and L123;b) a deletion of at least one, two, three, four, five, six, seven or eight amino acids in the region of N59 to I66 or a deletion of amino acids N63 to I66; and / or a deletion of amino acids K75 and R76 or a deletion of amino acids K75, R76 and R77; and, c) an insertion of at least two, three or four amino acids immediately C-terminal to G84 and / or an insertion K75 insX, wherein X is one, two or three amino acids selected from the group consisting of: G, S and D.

7. A multispecific antigen binding protein according to any one of the preceding claims, wherein at least one of:i) the 4-1 BB agonist comprises or consists of at least one 4-1 BB ligand (4-1 BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds 4-1 BB;ii) the CD27 agonist comprises or consists of at least one CD70 extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds CD27; and, iii) the GITR agonist comprises or consists of at least one GIRTL extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds GITR.

8. A multispecific antigen binding protein according to claim 7, wherein at least one of:i) the 4-1 BBL ECD is a mutein with reduced affinity for 4-1 BB, relative to a corresponding wild type 4-1 BBL ECD, and wherein the 4-1 BBL ECD mutein comprises the amino acid sequence:REGPELSPDD PAGLLDLRQG MFAQLVAQNX XLIDGPLSWX SDPXXXGVSL TGGLSYKEDT KELVVAKAGV YYVFFQLELR RVXXGEGSGS VSLALHLQPL XSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWXLTX GATVLGLFRV TPEIPAGLPS PRSE (SEQ ID NO: 362), wherein X represents any amino acid, and wherein the 4-1 BBL ECD mutein amino acid sequence differs from the amino acid sequence of the wild type human 4-1 BBL ECD (SEQ ID NO: 359) by at least 1 amino acid, and wherein preferably, the 4-1 BBL ECD comprises one or more amino acid substitutions selected from the group consisting of: V100T,V100Q, L101 N, Y110Q, G114K, L115R, A116D, V153Q, A154D, R171 D, Q227E, Q227R, Q230S, Q230K, V100T, Q227R, Q230S and Q230K; and,ii) the CD70 ECD is a mutein with reduced affinity for CD27 relative to a corresponding wild type CD70 ECD, and wherein CD70 ECD mutein comprises at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 365 selected from the group consisting of: Q61 , A80, S137, S146 and H148, wherein preferably, the CD70 ECD mutein comprises at least one amino acid substitution selected from the group consisting of: Q61A, A80F, A80R, S137A, S137E, S137K, S146A, H148A, H148E and H148D; and, iii) the 4-1 BBL ECD is a mutein with reduced affinity for 4-1 BB, relative to a corresponding wild type 4-1 BBL ECD.

9. A multispecific antigen binding protein according to claim 7 or 8, wherein at least one of: i) the 4-1 BB agonist comprises or consists of a fusion protein comprising three 4-1 BBL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three 4-1 BBL ECD monomers are connected by polypeptide linkers; and,ii) the CD27 agonist comprises or consists of a fusion protein comprising three CD70 ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three CD70 ECD monomers are connected by polypeptide linkers; and,iii) the GITR agonist comprises or consists of a fusion protein comprising three GITRL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three 4-1 BBL ECD monomers are connected by polypeptide linkers.

10. A multispecific antigen binding protein according to any one of the preceding claims, wherein at least one of the first, second and third antigen-binding region is a Fab, wherein the Fab is operably linked to a polypeptide chain of a dimeric Fc region, wherein preferably, the Fc region has reduced affinity for at least one of the human Fc y receptor (FcyR) and human C1q, relative to a corresponding wild-type Fc region, wherein more preferably, the Fc region is a variant human lgG1 Fc region comprising at least one modification selected from the group consisting of: L234A, L235A and G237A.

11. A multispecific antigen binding protein according to any one of the preceding claims, wherein the multispecific antigen binding protein has at least one biological activity selected from: a) the multispecific antigen binding protein causes an increase in at least one y6 T cell activity selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are not brought into contact with the multispecific antigen binding protein; and,b) the multispecific antigen binding protein causes an increase in at least one y6 T cell activity selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are brought into contact with a conventional human lgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the multispecific antigen binding protein.

12. A pharmaceutical composition comprising a multispecific antigen binding protein according to any one of the preceding claims, and a pharmaceutically acceptable carrier.

13. A multispecific antigen binding protein according to any one of claims 1 - 11 , or a composition according to claim 12, for use as a medicament.

14. A multispecific antigen binding protein according to any one of claims 1 - 11 , or a composition according to claim 12, for use in the treatment of a cancer, an infectious disease or an inflammatory disease, wherein preferably, the cancer is a cancer comprising tumor cells expressing the TAA.

15. A multispecific antigen binding protein according to any one of claims 1 - 11 , or a composition according to claim 12, for a use according to claim 14, wherein at least one of:a) the multispecific antigen binding protein is administered as a neoadjuvant therapy before a primary therapy comprising at least one of surgery and radiation therapy of the cancer; and,b) the multispecific antigen binding protein is administered as an adjuvant therapy after a primary therapy comprising at least one of surgery and radiation therapy of the cancer.

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