Muteins of 4-1BB ligand extracellular domain, fusion proteins comprising the same and uses thereof

Abstract

The present invention relates to 4-1BB ligand (4-BBL) extracellular domain (ECD) muteins having reduce affinity for its cognate receptor 4-1BB. The 4-1BBL ECD muteins can be present in homo- or heterotrimeric fusion protein comprising three 4-1BBL ECD monomers. The invention further relates conjugates of such the 4-BBL ECD muteins with a heterologous moiety, such as an antigen binding protein. The antigen-binding regions comprised in the antigen binding protein in the conjugates preferably are specific for a tumor-associated antigen (TAA). In addition to a 4-BBL ECD mutein, the conjugates can comprise further NK cell-activating cytokines, such an IL-21 receptor agonist. The conjugates can further comprise an antigen-binding region that has affinity for a surface antigen expressed on NK cells, e.g. CD16A. Alternatively, the conjugates can further comprise an antigen-binding region that specifically binds an epitope of a γδ TCR. The conjugates of the invention specifically redirect and activate NK cells or γδ T cells to lyse targeted tumor cells. The invention further relates to the use of the 4-BBL ECD muteins and conjugates thereof in the treatment of cancer, preferably a cancer expressing the TAA.

Description

[0001] Muteins of 4-1 BB ligand extracellular domain, fusion proteins comprising the same and uses thereof

[0002] Field of the invention

[0003] The present invention relates to the field of medicine, in particular to the fields of oncology, immunology, inflammation and immunotherapy of tumors. Specifically, the invention relates to muteins of the 4-1 BB ligand (4-1 BBL) extracellular domain (ECD), preferably 4-1 BBL ECD muteins having an altered affinity, relative to the affinity of wild-type 4-1 BBL for 4-1 BB. The invention further relates to fusion proteins comprising such 4-1 BBL ECD muteins and the use of such 4-1 BBL ECD muteins and fusion proteins in medical treatments of e.g. cancer.

[0004] Background of the invention

[0005] 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.

[0006] 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.

[0007] 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. Strategies based on the recruitment of cytotoxic NK cells are currently being developed. One such strategy employs multifunctional antibodies called natural killer cell engagers (NKCEs) have been developed, which simultaneously target tumor-associated antigens (TAAs), and activate receptors on endogenous NK cells. A number of NKCEs that are currently in development for clinical application is reviewed by Demaria et al. (Eur. J. Immunol. 2021 . 51 : 1934-1942). NKCEs are designed to strengthen the interaction between the NK cell and targeted tumor cell and to increase NK cell effector functions towards the tumor cell. However, NK cells in tumors are low in number and of poor functionality (have an exhausted phenotype). NKCE’s developed thus far only address interaction of NK cells with tumor cells and in most cases do not improve their functionality, and in none of the cases improves the NK cell numbers in the tumor.

[0008] Also y6 T cells, predominantly V62 T-cells have been tested as cellular immunotherapies. Despite a great safety profile, induction of growth and / or targeting / redirection in-situ via the V62 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.

[0009] More recently, WO2024 / 056862 and WO2024 / 056861 disclose such multifunctional antigenbinding proteins comprising as NK cell-activating cytokine a 4-1 BB agonist, such as 4-1 BB ligand (4-1 BBL). Co-pending applications EP 24208721.1 and EP24208726.0 disclose multispecific antigen binding proteins, which bind to a tumor-associated antigen of interest and / or to a y6 T cell receptor, and which comprise a y6 T cell-activating agonist, such IL-21 , and optionally a y6 T cell co-stimulatory agonist such as 4-1 BBL. The multifunctional antigen-binding proteins in these applications do address the number, functionality and location of the NK cells and / or y6 T cells. However, the specificity of the 4-BB agonist in these proteins is not yet specific enough for function in the tumor only, potentially causing side effects outside the tumor, e.g. in the periphery.

[0010] 4-1 BB is a member of the TNFR superfamily expressed on dendritic cells, monocytes, macrophages, B cells, T cells and NK cells. Its activation by 4-1 BBL on CD8 T cells and NK cells results in increased proliferation, cytokine production, survival, and anti-tumor cytotoxicity. 4-1 BB activation can also have a profound impact on myeloid cell activation, the humoral immune system and CD4 T cells. 4-1 BB stimulation therefore has been an attractive target fortherapy and achieved objective clinical responses in cancer patients. However, the development of 4-1 BB- or 4-1 BBL- based therapies has been complex due to its broad expression on immune cells in the periphery. In particular, the development of 4-1 BB agonists has been hampered by severe and at times fatal hepatotoxicity in patients due to 4-1 BB-mediated activation of liver monocyte / macrophages and subsequent T cell infiltration and activation (Segal et al. Clin Cancer Res. 2017;23(8):1929-1936; Bartkowiak et al. Clin Cancer Res. 2018;24(5):1138-1151). Accordingly, care must be taken to avoid broad 4-1 BB-activation in leukocytes and the potential toxicity thereof. The therapeutic application of 4-1 BBL signaling must therefore be balanced and targeted, such that the effects triggered by 4-1 BBL are designed to occur at the right time and place in the body.

[0011] There is therefore a need in the art for 4-1 BBL treatment modalities that address these issues. It is thus an object of the present invention to provide for such 4-1 BBL treatment modalities. Description of the invention

[0012] Definitions

[0013] 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 for testing of the present invention, the preferred materials and methods are described herein.

[0014] “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.

[0015] “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.

[0016] “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.

[0017] “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.

[0018] “Exemplary”: this term means “serving as an example, instance, or illustration,” and should not be construed as excluding other configurations disclosed herein.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] “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) I 8 (proteins) and gap extension penalty = 3 (nucleotides) I 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 of the invention 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.

[0025] 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.

[0026] Alternative conservative amino acid residue substitution classes.

[0027] Alternative physical and functional classifications of amino acid residues.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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), s (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.

[0032] 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. 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).

[0033] The term "monospecific" antibody as used herein denotes that the antibody-part of a conjugate 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 conjugate 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 antigen-binding 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.

[0034] 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 conjugate 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 conjugate.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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).

[0039] 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).

[0040] 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.

[0041] 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, conjugate 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.

[0042] 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).

[0043] "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 aspect of the invention, 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 Citype 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).

[0044] 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).

[0045] 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.

[0046] 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 .

[0047] 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).

[0048] 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).

[0049] 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 .

[0050] 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 VHH fragments). Furthermore, the term single variable domain antibody includes an autonomous human heavy chain variable domain (aVH) or VNAR fragments derived from sharks.

[0051] 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. 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).

[0052] Micro bodies 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.

[0053] 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.

[0054] 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 according to the invention include variants containing at least one modification that alters (enhances or diminishes) an Fc associated effector function. Also, Fc regions according to the invention 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 .

[0055] 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.

[0056] 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)).

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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. Table A. CDR definitions1

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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, 6, s, y, and m respectively.

[0065] 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.

[0066] 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.

[0067] Typically, an antigen-binding region of a conjugate of the invention thereof will specifically bind its target molecule (antigen) with a dissociation constant (KD) of about 10-6to 10-12M or less, and preferably 10-8to 10-12M or less, and / or with a binding affinity of at least 10-6M or 10-7M, preferably at least 10-8M, more preferably at least 10-9M, such as at least 1 O-10, 10’11, 10-12M or less. Any KD value greater than 10-4M (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 10-4M, as may be determined as herein described below. Preferably, an antigen-binding region of a conjugate of the invention 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 invention (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.

[0068] 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 5 pl / minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M 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 (kotr) 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.

[0069] 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.

[0070] One class of antigen-binding regions for use in the invention 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 positions) 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 :105-115 1998); 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 of the invention 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.

[0071] "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.

[0072] 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.

[0073] 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.

[0074] 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, V153Q denotes that the original amino acid valine (V) in position 153 is changed to a glutamine (Q).

[0075] 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.

[0076] Amino acid deletions 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.

[0077] As used herein, the phrase “NK cells” refers to a sub-population of lymphocytes that is involved in innate immunity. NK cells can be identified by virtue of certain characteristics and biological properties, such as the expression of specific surface antigens including CD56 and / or NKp46 for human NK cells, the absence of the alpha / beta or gamma / delta TCR complex on the cell surface, the ability to recognize and kill cells that fail to express “self MHC / HLA antigens by the activation of specific cytolytic machinery, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response. Any of these characteristics and activities can be used to identify NK cells, using methods well known in the art. Any subpopulation of NK cells will also be encompassed by the term NK cells. Within the context herein “active” NK cells designate biologically active NK cells, including NK cells having the capacity of lysing target cells or enhancing the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from blood samples, cytapheresis, tissue or cell collections, etc. Useful protocols for assays involving NK cells can be found in Natural Killer Cells Protocols (2000, edited by Campbell KS and Colonna M). Humana Press, pp. 219-238).

[0078] 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.

[0079] 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 of the invention 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. 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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).

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Detailed description of the invention

[0088] We have previously described multispecific antigen binding proteins, which bind to a tumor- associated antigen of interest and / or to an NK cell activating receptor, and which comprise an NK cell-activating cytokine that triggers at least one of the NK cell’s interleukin 21 receptor and 4-1 BB, and that are capable of inducing a hyper-functional phenotype in NK cells (see WO2024 / 056862 and WO2024 / 056861). We have also previously described multispecific antigen binding proteins, which bind to a tumor-associated antigen of interest and / or to a y6 T cell receptor, and which comprise a y6 T cell-activating agonist (e.g. IL-21), and optionally a y6 T cell co-stimulatory agonist (e.g. 4-1 BBL), and that are capable of inducing a hyper-functional phenotype (expansion, activation and / or innate potency) in y6 T cells (see co-pending applications EP 24208721.1 and EP24208726.0). NK cells and y6 T cells having a hyper-functional phenotype proliferate, are resistant to the tumor microenvironment, have an enhanced capability to mediate lysis of target cells, even in the absence of the original tumor associated antigen targeted, hyper-secrete cytokines (e.g. IFN-y) when in contact with target cells and have the ability to prolong these capabilities over time. Agents capable of inducing a hyper-functional phenotype in NK cells and / or in y6 T cells are therefore useful in the treatment of cancers and infectious diseases. However, the systemic administration of agents comprising cytokines with pleiotropic effects, such as 4-1 BB ligand (4-1 BBL), remains challenging because of the risks of toxicity and other undesired sideeffects. There remains therefore a need in the art for 4-1 BBL treatment modalities that reduce the systemic pleiotropic effect of 4-1 BBL, while maintaining their potential when targeted to tumors or sites infected by pathogens. The present invention therefore provides novel muteins of the extracellular domain (ECD) of 4-1 BBL having an altered affinity for 4-1 BB. For example, 4-1 BBL ECD muteins described herein and having a reduced affinity for 4-1 BB, and conjugates described herein comprising such 4-1 BBL ECD muteins, will have reduced pleiotropic- and undesired sideeffects, when present in the bloodstream, while, when present at targeted sites, their avidity will ensure their local efficacy. These novel 4-1 BBL ECD muteins can be combined with further cytokines, such as IL-21 muteins having a reduced affinity for IL-21 R, in conjugates with antigenbinding proteins. Without being bound by a theory, the 4-1 BBL ECD-containing conjugates described herein are designed to utilize the immune potentiating activity of 4-1 BBL ECD (which may be prerequisite to address toxicity and off-target immune suppression), to maximize efficacy at targeted loci, and improve the feasibility of dosing in the clinic.

[0089] 4-1 BB ligand and muteins of the extracellular domain of 4-1 BB ligand

[0090] 4-1 BB is a member of the tumor necrosis factor receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 9 (TNFRSF9), CD137 and induced by lymphocyte activation (ILA). 4-1 BB is encoded by the TNFRSF9 gene (Entrez Gene ID: 3604). An amino acid sequence for human 4-1 BB is described in NCBI accession numbers NP_001552, which is incorporated as SEQ ID NO: 175, wherein the mature 4-1 BB corresponds to positions 24 - 255. 4-1 BB is known as a co-stimulatory immune checkpoint molecule. 4-1 BB is expressed by activated T cells of both the CD4+ and CD8+ lineages, as well as on activated NK cells and on activated y6 T cells. The proliferation and activation of NK cells and y6 T cells at least requires engagement of a costimulatory receptor such as 4-1 BB by its ligand 4-1 BBL. NK cells and y6 T cells with increased 4-1 BB expression are known to be highly active against target cells (e.g. tumor cells) expressing 4- 1 BB ligand. 4-1 BB ligand (4-1 BBL), also known as TNFSF9 or CD137L, is a protein that in humans is encoded by the TNFSF9 gene (Entrez Gene ID: 8744). An amino acid sequence for human 4- 1 BBL is described in NCBI accession numbers NP_003802, the disclosure of which is incorporated herein by reference. The 4-1 BB / 4-1 BBL complex consists of three monomeric 4-1 BBs bound to a trimeric 4-1 BBL. Each 4-1 BB monomer binds to two 4-1 BBLs via cysteine-rich domains (CRDs). The interaction between 4-1 BB and the second 4-1 BBL is required to stabilize their interactions. As used herein, an “4-1 BB agonist” is an agent that has “agonist” activity at the 4-1 BB, which means that the agent that can cause or increase "4-1 BB signaling". “4-1 BB signaling” refers to an ability of 4-1 BB, e.g. when expressed on the surface of T, B and NK cells and triggered by its natural ligand 4-1 BBL, to activate or transduce an intracellular signaling pathway. The “natural 4-1 BB ligand” is herein understood as the extracellular domain (ECD) of a human wild type 4-1 BBL comprising or consisting of an amino acid sequence from position 71 to 254 of the amino acid sequence of human 4-1 BBL (i.e. SEQ ID NO: 37). A 4-1 BBL extracellular domain (ECD) is herein thus understood as a polypeptide comprising or consisting of an amino acid sequence from positions 71 to 254 of human 4-1 BBL, or a fragment thereof having 4-1 BB agonist activity.

[0091] 4-1 BB agonist activity, i.e. changes in 4-1 BB signaling activity, can be measured, for example, by assays designed to measure changes in the 4-1 BB 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 indirectly by a downstream effect mediated by 4-1 BB (e.g. production of specific cytokines). A suitable cell-based assay for in vitro biological activity of a 4-1 BB agonist, is e.g. described in Zhang et al. (Clin Cancer Res ,2007;13(9): 2758- 2767), using measurement of IL-2 production from splenocytes aseptically removed from BALB / c mice in microtiter plates precoated with an anti-CD3 monoclonal antibody (145-11 C clone). Other suitable cell-based assays for in vitro biological activity of a 4-1 BB agonist, are described in WO2016 / 075278, Example 6 (see e.g. Example 6.1). The natural 4-1 BB ligand, a 4-1 BBL ECD trimer as described by Fellermeier et al. (Oncoimmunol. 2016, 5(11): e1238540), e.g. a 4-1 BBL ECD trimer comprising the amino acid sequence of SEQ ID NO: 36, or an anti-CD137 agonist antibody (such the antibody 2A, Epstein et al., Tumor necrosis imaging and treatment of solid tumors. In: V. P. Torchilin, editor. Handbook of targeted delivery of imaging agents, Vol. 16. Boca Raton: CRCPress; 1995. p. 259.) can serve as a positive control in an assay for 4-1 BB agonist activity and can also be used as a reference for the amount of 4-1 BB agonist activity of a given nonnatural 4-1 BB agonist, such as a multispecific antigen binding protein as described herein comprising a 4-1 BB agonist. The data presented herein supports the use of carefully designed 4- 1 BBL ECD muteins to achieve 4-1 BB signalling at the appropriate time and place and to improve pharmacokinetics of therapeutics comprising such 4-1 BBL ECD muteins. In addition, the 4-1 BBL ECD muteins provided herein are designed to reduce potential unwanted effects in the absence of the target cells, e.g. hepatotoxicity and cytokine release.

[0092] In a first aspect, the present disclosure provides muteins of the 4-1 BBL ECD comprising at least one substitution, deletion and / or insertion. Amino acid substitutions, deletions and insertions in a 4-1 BBL ECD mutein provided herein are indicated relative to the wild-type human 4-1 BBL ECD amino acid sequence, which is provided herein as SEQ ID NO: 37. Hence, to allow for allelic variation, a wild-type human 4-1 BBL ECD preferably comprises an amino acid sequence having, with increasing preference, 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 with SEQ ID NO: 37. In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein SEQ ID NO: 49 is

[0093] REGPELSPDD PAGLLDLRQG MFAQLVAQNX XLIDGPLSWX SDPXXXGVSL TGGLSYKEDT KELWAKAGV YYVFFQLELR RVXXGEGSGS VSLALHLQPL XSAAGAAALA LTVDLPPASS EARNSAFGFQ GRLLHLSAGQ RLGVHLHTEA RARHAWXLTX GATVLGLFRV TPEI PAGLPS PRSE (SEQ ID NO: 49), 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: 37) by at least one amino acid. The amino acid positions in the amino acid sequence of the 4-1 BBL ECD muteins of SEQ ID NO: 49, as referred to herein correspond to the amino acid positions of the full- length 4-1 BBL amino acid sequence. Hence, the first amino acid position in SEQ ID NO: 49 is referred to as position 71 , from which the subsequent amino acid positions are counted, up to the last amino acid in position 254.

[0094] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 37 selected from the group consisting of the positions: 154, 153, 110, 227, 101 , 230, 100, 114, 115, 116, and 171 , in decreasing preference. In one embodiment, the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 37 selected from the group consisting of the positions: 154, 153, 110, 227, 101 , , 230, 100, 114, 115, 116, and 171.

[0095] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, wherein SEQ ID NO: 49 differs from SEQ ID NO: 37 by at least one amino acid at a position designated by X in SEQ ID NO: 49. In one embodiment, the 4-1 BBL ECD mutein comprising SEQ ID NO: 49 has, with increasing preference, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.4% sequence identity to SEQ ID NO: 37.

[0096] In one embodiment, there is provided a 4-1 BBL ECD mutein comprises an amino acid sequence having, with increasing preference, 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 with SEQ ID NO: 37, and wherein the amino acid sequence comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101 N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101 E; L101 Q; L101 D; L101 R; L101 K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G1 14K; G1 14R; G1 14Q; G1 14D; G1 14E; G1 14N; G1 14S; G1 14A; G114G; G1 14T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; G155Q; R171 D; R171 E; R171 Q; R171 N; R171 S; R171T; R171 G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T and Q230A. In one embodiment, there is provided a 4- 1BBL ECD mutein comprising at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T and Q230A. In one embodiment, the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A.

[0097] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least two, three, four or five amino acid substitutions selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A. In one embodiment, the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than theat least two, three, four or five amino acid substitutions selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101N; Y110Q; Q230K; V100Q; V100T; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101E; L101Q; L101D; L101R; L101K; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G114K; G114R; G114Q; G114D; G114E; G114N; G114S; G114A; G114G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153N; V153R; V153K; V153D; V153E; V153S; V153G; A154R; A154K; A154Q; A154N; A154Y; A154H; R171D; R171E; R171Q; R171N; R171S; R171T; R171G; R171A; R171Y; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A.

[0098] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution at a position in SEQ ID NO: 37 selected from the substitutions listed in Table B.

[0099] Table B. Additional substitutions, deletions and insertions for 4-1 BBL ECD muteins

[0100] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution at a position in SEQ ID NO: 37 selected from the group consisting of: A154D, A154E, V153Q, Q227E, L101 N, Y110Q, Q230K, and V100Q. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution at a position in SEQ ID NO: 37 selected from the group consisting of: A154D; A154E; V153Q; Q227E; L101 N; Y110Q and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution at a position in SEQ ID NO: 37 selected from the group consisting of: A154D; A154E; V153Q; and Q227E. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution at a position in SEQ ID NO: 37 selected from the group consisting of: A154D and A154E.

[0101] In one embodiment, there is provided a 4-1 BBL ECD mutein, 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: 37) at least at a position corresponding to position 154 of the full-length 4-1 BBL amino acid sequence. In one embodiment, the 4-1 BBL ECD mutein comprises no other differences from the wild type amino acid sequence than the difference at position 154. In one embodiment, the different amino acid at position 154 is aspartate (D) or glutamate (E), of which aspartate (D) is preferred.

[0102] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 154, and wherein preferably the 4-1 BBL ECD mutein comprises a A154D or A154E substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a A154D or A154E substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: V100T; V100Q; V100S; V100A; V100G; V100N; V100D; V100E; V100K; V100R; L101 N; L101 E; L101Q; L101 D; L101 R; L101 K; Y110Q; Y110E; Y110N; Y110D; Y110R; Y110K; Y110S; Y110A; Y110G; Y110T; G1 14K; G1 14R; G1 14Q; G1 14D; G1 14E; G1 14N; G1 14S; G1 14A; G1 14G; G114T; L115R; L115K; L115Q; L115N; L115D; L115E; L115S; L115A; L115G; L115T; A116D; A116E; A116R; A116K; A116Q; A116N; A116Y; A116H; V153Q; V153N; V153R; V153K; V153D; V153E; V153S; V153G; G155Q; R171 D; R171 E; R171 Q; R171 N; R171 S; R171T; R171 G; R171A; R171Y; Q227E; Q227R; Q227K; Q227D; Q227Y; Q227H; Q227G; Q227S; Q227T; Q230S; Q230K; Q230R; Q230N; Q230D; Q230E; Q230G; Q230S; Q230T; and Q230A, of which V153Q; Q227E; L101 N; Y110Q; Q230K; and V100Q, are preferred.

[0103] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: A154D and V153Q; A154D and G155Q; A154D and Q227E; A154D and L101 N; A154D and Y110Q; A154D and Q230K; A154D and V100Q; A154D and V100T; A154D and V100S; A154D and V100A; A154D and V100G; A154D and V100N; A154D and V100D; A154D and V100E; A154D and V100K; A154D and V100R; A154D and L101 E; A154D and L101 Q; A154D and L101 D L101 R; A154D and L101 K; A154D and Y110E; A154D and Y110N; A154D and Y110D; A154D and Y110R; A154D and Y110K; A154D and Y110S; A154D and Y110A; A154D and Y110G; A154D and Y110T; A154D and G1 14K; A154D and G114R; A154D and G114Q; A154D and G114D; A154D and G1 14E; A154D and G114N; A154D and G1 14S; A154D and G114A; A154D and G114G; A154D and G114T; A154D and L115R; A154D and L115K; A154D and L115Q; A154D and L115N; A154D and L115D; A154D and L115E; A154D and L115S; A154D and L115A; A154D and L115G; A154D and L115T; A154D and A116D; A154D and A116E; A154D and A116R; A154D and A116K; A154D and A116Q; A154D and A116N; A154D and A116Y; A154D and A116H; A154D and V153N; A154D and V153R; A154D and V153K; A154D and V153D; A154D and V153E; A154D and V153S; A154D and V153G; A154D and R171 D; A154D and R171 E; A154D and R171 Q; A154D and R171 N; A154D and R171 S; A154D and R171T; A154D and R171 G; A154D and R171A; A154D and R171Y; A154D and Q227R; A154D and Q227K; A154D and Q227D; A154D and Q227Y; A154D and Q227H; A154D and Q227G; A154D and Q227S; A154D and Q227T; A154D and Q230S; A154D and Q230R; A154D and Q230N; A154D and Q230D; A154D and Q230E; A154D and Q230G; A154D and Q230S; A154D and Q230T; A154D and Q230A; A154E and V153Q; A154E and Q227E; A154E and L101 N; A154E and Y110Q; A154E and Q230K; A154E and V100Q; A154E and V100T; A154E and V100S; A154E and V100A; A154E and V100G; A154E and V100N; A154E and V100D; A154E and V100E; A154E and V100K; A154E and V100R; A154E and L101 E; A154E and L101 Q; A154E and L101 D L101 R; A154E and L101 K; A154E and Y110E; A154E and Y110N; A154E and Y110D; A154E and Y110R; A154E and Y110K; A154E and Y110S; A154E and Y110A; A154E and Y110G; A154E and Y110T; A154E and G114K; A154E and G114R; A154E and G114Q; A154E and G114D; A154E and G114E; A154E and G114N; A154E and G114S; A154E and G114A; A154E and G114G; A154E and G114T; A154E and L115R; A154E and L115K; A154E and L1 15Q; A154E and L1 15N; A154E and L115D; A154E and L115E; A154E and L115S; A154E and L1 15A; A154E and L115G; A154E and L115T; A154E and A116D; A154E and A116E; A154E and A116R; A154E and A116K; A154E and A116Q; A154E and A116N; A154E and A116Y; A154E and A116H; A154E and V153N; A154E and V153R; A154E and V153K; A154E and V153D; A154E and V153E; A154E and V153S; A154E and V153G; A154E and R171 D; A154E and R171 E; A154E and R171 Q; A154E and R171 N; A154E and R171 S; A154E and R171T; A154E and R171 G; A154E and R171A; A154E and R171Y; A154E and Q227R; A154E and Q227K; A154E and Q227D; A154E and Q227Y; A154E and Q227H; A154E and Q227G; A154E and Q227S; A154E and Q227T; A154E and Q230S; A154E and Q230R; A154E and Q230N; A154E and Q230D; A154E and Q230E; A154E and Q230G; A154E and Q230S; A154E and Q230T; and A154E and Q230A.

[0104] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises a V100T or a V100Q substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a V153Q substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: V100T; L101 N; Y110Q; G1 14K; L115R; A116D; R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: V153Q and V100T; V153Q and L101 N; V153Q and Y110Q; V153Q and G114K; V153Q and L115R; V153Q and A116D; V153QV153Q and R171 D; V153Q and Q227E; V153Q and Q227R; V153Q and Q230S; and, V153Q and Q230K.

[0105] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises an L101 N substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising an L101 N substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: Y110Q; G1 14K; L115R; A116D; R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of L101 N and Y110Q; L101 N and G114K; L101 N and L115R; L101 N and A116D; L101 N and R171 D; L101 N and Q227E; L101 N and Q227R; L101 N and Q230S; and, L101 N and Q230K.

[0106] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises a Y110Q substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a Y110Q substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: G114K; L115R; A116D; R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of Y110Q and G1 14K; Y1 10Q and L1 15R; Y110Q and A1 16D; Y110Q and R171 D; Y110Q and Q227E; Y110Q and Q227R; Y110Q and Q230S; and, Y110Q and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising the combination of substitutions Y110Q, A154D and Q227E.

[0107] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises a G114K substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a G114K substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: L115R; A116D; R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of G114K and G1 14K; G114K and L1 15R; G1 14K and A1 16D; G114K and R171 D; G114K and Q227E; G114K and Q227R; G114K and Q230S; and, G114K and Q230K.

[0108] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises an L115R substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising an L115R substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: A116D; R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of L115R and A116D; L115R and R171 D; L115R and Q227E; L115R and Q227R; L115R and Q230S; and, L115R and Q230K.

[0109] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises an A116D substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising an A116D substitution in combination with at least one, two, three or four amino acid substitutions selected from the group consisting of: R171 D; Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: A116D and R171 D; A116D and Q227E; A116D and Q227R; A116D and Q230S; and, A116D and Q230K.

[0110] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises an R171 D substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising an R171 D substitution in combination with at least one or two amino acid substitutions selected from the group consisting of: Q227E; Q227R; Q230S; and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: R171 D and Q227E; R171 D and Q227R; R171 D and Q230S; and, R171 D and Q230K.

[0111] In one embodiment, a 4-1 BBL ECD mutein provided herein comprises the amino acid sequence of SEQ ID NO: 49, 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: 37) at least at position 100, and wherein preferably the 4-1 BBL ECD mutein comprises a Q227E or Q227R substitution. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a Q227E substitution in combination with at least one amino acid substitution selected from Q230S and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: Q227E and Q230S; and, Q227E and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a Q227R substitution in combination with at least one amino acid substitution selected from Q230S and Q230K. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions selected from the group consisting of: Q227R and Q230S; and, Q227R and Q230K.

[0112] In one embodiment, there is provided a 4-1 BBL ECD mutein comprising a combination of substitutions at positions in SEQ ID NO: 37, selected from the group consisting of: Y110Q, V153Q and Q227E; L101 N, Y110Q and V153Q; V100Q, Y110Q and V153Q; L101 N, V153Q and Q227E; Y110Q, A154D and Q227E; Y110Q and V153Q; V153Q and Q227E; Y110Q and Q227E; and, L101 N and Q227E.

[0113] Substitutions to aspartate, such A154D, when followed (in an N- to C-terminal direction) by a G-, A- or S-residue, such as G155, can potentially introduce an aspartate isomerization site, which can lead to the instability and / or increased degradation rate of a mutein comprising such isomerization site. In one embodiment therefore, a substitution to aspartate, when followed by a G- , A- or S-residue, the substitution to aspartate is combined with a substitution of the subsequent G- , A- or S-residue to any amino acid residue other than G, A, S or T. Preferably the subsequent G-, A- or S-residue substituted to Q, N, Y, L, V, or F. Hence, in one embodiment, there is provided a 4- 1 BBL ECD mutein comprising a combination of substitutions at positions in SEQ ID NO: 37, selected from the group consisting of: Y110D and S111X; L115D and A116X; A116D and G1 17X; V153D and A154X; A154 D and G155X; R171 D and S172X; and, Q230D andG231X, wherein X is any to any amino acid residue other than G, A, S or T, whereby preferably X is Q, E, N, D, H, K, R or Y. In a preferred embodiment, any 4-1 BBL ECD mutein comprising the A154D substitution is combined with a G155X substitution, wherein X is any to any amino acid residue other than G, A, S or T, whereby preferably X is Q, E, N, D, H, K, R or Y, of which Q is most preferred.

[0114] In one embodiment, there is provided a 4-1 BBL ECD mutein as structurally defined above, which 4-1 BBL ECD mutein binds to its cognate receptor 4-1 BB with a reduced affinity, relative to the affinity of wild-type 4-1 BBL ECD for the 4-1 BB. In one embodiment, there is provided a 4-1 BBL ECD mutein as structurally defined above, which 4-1 BBL ECD mutein binds to its cognate receptor human 4-1 BB with a reduced affinity, relative to the affinity of wild-type 4-1 BBL ECD for the human 4-1 BB, e.g. a human 4-1 BB having an amino acid sequence comprised in SEQ ID NO: 175.

[0115] It is understood herein that a 4-1 BBL ECD mutein with reduced affinity for its cognate receptor 4-1 BB, as compared to a wild type 4-1 BBL ECD mutein, will also have a reduced affinity for 4-1 BB when present in a homotrimeric fusion protein comprising three 4-1 BBL ECD mutein monomers connected through polypeptide linkers, e.g. (GGGGS)4. Generally, the affinity of a 4- 1 BBL ECD mutein for its cognate receptor 4-1 BB is herein defined as the affinity for 4-1 BB as determined when the 4-1 BBL ECD mutein is present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, whereby the homotrimer can be present in a conjugate with an antibody (e.g. trastuzumab), which conjugate can further comprise IL-21 .

[0116] In one embodiment, there is provided a 4-1 BBL ECD mutein having a with a reduced affinity for 4-1 BB, relative to the affinity of wild-type 4-1 BBL ECD, wherein the 4-1 BBL ECD mutein comprises at least one substitution selected from the group consisting of: V100T; V100Q; L101 N; Y110Q; G1 14K; V153Q; R171 D; Q227E; Q227R; Q230S; Q230K; A116D; A154D; and A154E; or wherein the 4-1 BBL ECD mutein comprises a combination of substitutions selected from the group consisting of: Y110Q, V153Q and Q227E; L101 N, Y110Q and V153Q; V100Q, Y110Q and V153Q; L101 N, V153Q and Q227E; Y110Q, A154D and Q227E; Y110Q and V153Q; V153Q and Q227E; A154D and G155Q; Y110Q and Q227E; and, L101 N and Q227E.

[0117] The 4-1 BBL ECD muteins provided herein bind to 4-1 BB in a non-covalent and reversible manner. In one embodiment, the binding strength of a 4-1 BBL ECD mutein to 4-1 BB may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the mutein and 4-1 BB. In one embodiment, a 4-1 BBL ECD mutein provided herein has a low- affinity for 4-1 BB and thus will bind a lesser amount of 4-1 BB than a wild type 4-1 BBL. In one embodiment, a 4-1 BBL ECD mutein provided herein has an equilibrium association constant, KA, which is, with decreasing preference, at least 103M-1,at least 104M-1, at least 105M-1, at least 106M’1, at least 107M-1, at least 108M-1, at least 109M-1, or at least 101° M-1. As understood by the artisan of ordinary skill, KA can be influenced by factors including pH, temperature and buffer composition.

[0118] In one embodiment, the binding strength of a 4-1 BBL ECD mutein provided herein to 4-1 BB may be described in terms of its affinity, i.e. KD. KD is the equilibrium dissociation constant, a ratio of koff / kon, between the 4-1 BBL ECD mutein and 4-1 BB. 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 a 4-1 BBL ECD mutein provided herein to 4-1 BB may be described in terms of KD. In one embodiment, the KD of a 4-1 BBL ECD mutein provided herein is about 10-3M, about 10-4M, about 10-5M, about 10-6M, or less. In one embodiment, the KD of a 4-1 BBL ECD mutein provided herein is micromolar, nanomolar, or picomolar. In one embodiment, the KD of a 4-1 BBL ECD mutein provided herein is within a range of about 103to 10-4M, about 104to 105M, or 105to 10-6M, or 107to 10-8M, 108to 10-9M. In one embodiment, a 4-1 BBL ECD mutein provided herein binds to the human 4-1 BB with a KD that is greater than or is about 140 nM. In one embodiment, a 4-1 BBL ECD mutein provided herein binds to the human 4-1 BB with a KD of about 100 nM to about 4,000 nM, 200 nM to 4,000 nM, 500 nM to 4,000 nM, 1 ,000 nM to 4,000 nM, 120 nM to 3,000 nM, 200 nM to 2,000 nM, 500 nM to 2,000 nM, or 1 ,000 nM to 2,000 nM.

[0119] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits a reduction in binding affinity for human 4-1 BB. In one embodiment, the 4-1 BBL ECD 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, 1000-fold, 2000-fold, 5000-fold or 10.000-fold reduction in binding affinity for 4-1 BB, relative to, relative to the affinity of wild-type 4-1 BBL ECD for 4-1 BB.

[0120] In one embodiment, the binding affinity of a 4-1 BBL ECD mutein provided herein for 4-1 BB is determined by SPR, e.g. as described in the Examples herein. In one embodiment, the binding affinity of a 4-1 BBL ECD mutein provided herein for 4-1 BB, is thus determined when the 4-1 BBL ECD mutein is present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises IL-21 .

[0121] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits a binding affinity for human 4-1 BB expressed as pKo, that is at least 0.4 lower than the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB. pKo is understood herein to be -logio(Ko). In one embodiment, the 4-1 BBL ECD mutein having at least a 0.4 lower pKo for human 4-1 BB, relative to the affinity of a wild-type 4- 1 BBL ECD for human 4-1 BB, is a 4-1 BBL ECD mutein comprising at least one substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; V100Q; V100T; and A116D; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; V100Q; V100T; and A116D.

[0122] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits a binding affinity for human 4-1 BB expressed as pKo, that is at least 0.5 lower than the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB. In one embodiment, the 4-1 BBL ECD mutein having at least a 0.5 lower pKo for human 4-1 BB, relative to the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB, is a 4-1 BBL ECD mutein comprising at least one substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; V100Q; and V100T; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; V100Q; and V100T.

[0123] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits a in binding affinity for human 4-1 BB expressed as pKo, that is at least 1 .0 lower than the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB. In one embodiment, the 4-1 BBL ECD mutein having at least a 1 .0 lower pKo for human 4-1 BB, relative to the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB, is a 4-1 BBL ECD mutein comprising at least one substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; and V100Q; or the

[0124] 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; and V100Q.

[0125] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits a reduction in activity as measured by a suitable assay for measuring changes in a 4-1 BB signaling pathway as described above, relative to the activity of wild-type 4-1 BBL ECD under corresponding conditions. In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, 5000-fold or 10.000-fold reduction in activity as measured by a 4-1 BB signaling assay, relative to the activity of wild-type 4- 1 BBL ECD under corresponding conditions.

[0126] In one embodiment, a 4-1 BBL ECD mutein provided herein exhibits at least about a 2-fold,

[0127] 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 y6 T cell proliferation assay, relative to the activity of wild-type 4-1 BBL ECD under corresponding conditions. In one embodiment, the NK cell or y6 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 y6 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.

[0128] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with a monoclonal antibody against a TAA (e.g. trastuzumab), which conjugate further comprises IL-21 , exhibits a pECso 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. SKOV-3 cells), that is not more than 0.25 log less than the pECso of a corresponding control conjugate comprising wild-type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101 N; Y110Q; and V100Q; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101 N; Y110Q; and V100Q.

[0129] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , exhibits a pECso 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 0.10 log less than the pECso of a corresponding control conjugate comprising wild-type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; and V100Q; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; and V100Q.

[0130] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , exhibits a pECso 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 0.05 log less than the pECso of a corresponding control conjugate comprising wild-type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; and V100Q; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; and V100Q.

[0131] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , 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 70%, 75%, 80%, 85%, 90% or 95% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and a trimer of wild type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; Q227R; L101 N; Y110Q; Q230K; and V100Q; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; Q227E; Q227R; L101 N; Y110Q; Q230K; and V100Q.

[0132] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , 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 75% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and a trimer of wild type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; and V100Q; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; Q230K; and V100Q.

[0133] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , 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 80% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and a trimer of wild type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; and Q230K; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; Q227E; L101 N; Y110Q; and Q230K.

[0134] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , 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 85% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and a trimer of wild type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D; A154E; V153Q; and Q227E; or the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; A154E; a combination of A154D and G155Q; V153Q; and Q227E.

[0135] In one embodiment, a 4-1 BBL ECD mutein provided herein, when present in a homotrimeric fusion protein comprising three identical 4-1 BBL ECD mutein monomers connected through (GGGGS)4 polypeptide linkers, which homotrimer is present in a conjugate with trastuzumab, which conjugate further comprises IL-21 , 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 95% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and a trimer of wild type 4-1 BBL ECD in the same assay. Hence, in one embodiment, the 4-1 BBL ECD mutein comprises at least one amino acid substitution selected from the group consisting of: A154D and A154E; a combination of A154D and G155Q; and the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one amino acid substitution selected from the group consisting of: A154D; a combination of A154D and G155Q; and A154E.

[0136] In one embodiment, there is provided a 4-1 BBL ECD 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 4-1 BBL ECD mutein for 4-1 BB, produces reduced (little or no) agonist activity at a 4-1 BB 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 4-1 BBL ECD mutein in the conjugate shows significant agonist activity. This activity is the result of the 4-1 BBL ECD mutein being present in high local density on the surface of the target cells, which leads to an enhanced apparent affinity forthe 4-1 BB on local NK cells (see Figure 1) or y6 T cell, primarily through the mechanism of avidity. As such, a 4-1 BBL ECD 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 4-1 BBL ECD. 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 4-1 BBL ECD 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 cell, 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.

[0137] Thus, in one embodiment, there is provided an 4-1 BBL ECD mutein as described herein, wherein the 4-1 BBL ECD mutein, when present in a conjugate with an antigen binding protein that specifically binds an antigen, wherein the conjugate optionally further comprises IL-21 , 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 or y6 T cell proliferation assay in the absence of the antigen or cells expressing the antigen.

[0138] In one embodiment, the difference in induction of NK cell or y6 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: 11), a second trastuzumab heavy chain fused to wild type IL-21 (SEQ ID NO: 12) and trastuzumab light chains (SEQ ID NO: 2), wherein the constant regions of the first and second heavy chains are distinguished using knob-in-hole technology (see WO2024 / 056862). The wild type 4-1 BBL ECD amino acid sequence in the first heavy chain amino acid sequence of SEQ ID NO: 11 can be replaced by an amino acid sequence of an 4-1 BBL ECD 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.

[0139] In one embodiment, there is provided an 4-1 BBL ECD mutein as described herein, wherein the 4-1 BBL ECD 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: 1 1 , wherein the wild type 4- 1 BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1 BBL ECD mutein; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, 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 10, 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.

[0140] In one embodiment, there is provided an 4-1 BBL ECD mutein as described herein, wherein the 4-1 BBL ECD 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: 1 1 , wherein the wild type 4- 1 BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1 BBL ECD mutein; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, produces a fold-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: 11 ; a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2.

[0141] In the above embodiments, the NK cell or y6 T cell proliferation assays are preferably performed using NK 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 cells 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.

[0142] The reduced affinity of the 4-1 BBL ECD 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 4-1 BBL ECD, while at the same time the ability of a conjugate with an 4-1 BBL ECD mutein to 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.

[0143] Hence, in one embodiment, there is provided an 4-1 BBL ECD mutein as described herein, wherein the 4-1 BBL ECD 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: 11 , wherein the wild type 4-1 BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1 BBL ECD mutein; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, 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: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, in the same assay. The NK cell or y6 T cell cytotoxicity assay in the presence of SK-OV-3 cells preferably performed using NK cells or y6 T cells isolated from healthy donors. Preferably, the EC50 values in the NK cell or y6 T cell cytotoxicity assays are determined on the basis of the average values using NK cells or y6 T cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or y6 T cell cytotoxicity assays are preferably performed essentially as described in the examples herein.

[0144] Also the ability of a conjugate between an 4-1 BBL ECD mutein as described herein and an antigen binding protein to support long-term expansion of NK cells or y6 T cells in the presence of cells carrying the antigen that is bound by the antigen binding protein, preferably remains essentially unaffected.

[0145] Hence, in one embodiment, there is provided an 4-1 BBL ECD mutein as described herein, wherein the 4-1 BBL ECD 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: 11 , wherein the wild type 4-1 BBL ECD amino acid sequence is replaced by the amino acid sequence of the 4-1 BBL ECD mutein; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, induces a fold expansion of NK cells in the presence of SK-OV-3 cells in an NK cell or y6 T cell expansion assay, that is, with increasing preference, 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: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, in the same assay. The NK cell or y6 T cell expansion assay in the presence of SK-OV-3 cells preferably performed using NK cells or y6 T cells isolated from healthy donors. Preferably, the fold expansion of NK cells or y6 T cells in the assays is determined on the basis of the average values using NK cells or y6 T cells isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or y6 T cell expansion assays are preferably performed essentially as described in the examples herein.

[0146] A further advantage of the 4-1 BBL ECD muteins as described herein is that their reduced affinity for 4-1 BB the improves pharmaco-kinetics of therapeutics comprising the 4-1 BBL ECD muteins. In the body many cells, including T-, B- or NK cells, are present that express 4-1 BB molecules at their surfaces. These 4-1 BB molecules act as a sink for therapeutics comprising a moiety with affinity for 4-1 BB such as a 4-1 BBL ECD mutein. Upon binding to a surface expressed 4-1 BB, the therapeutic comprising the 4-1 BBL-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 4-1 BB of the 4-1 BBL ECD muteins as described herein reduces or prevents their disappearance in this sink and thereby improves pharmaco-kinetics of therapeutics comprising the 4-1 BBL ECD muteins.

[0147] In a further aspect, the present disclosure provides a trimeric fusion protein comprising three 4-1 BBL ECD monomers, wherein one, two, or three of the monomers are a 4-1 BBL ECD mutein as described herein above, fused together in a single polypeptide chain, as e.g. described in Fellermeier et al. (2016, supra). In one embodiment, the three 4-1 BBL ECD monomers are connected by polypeptide linkers. In one embodiment, the three 4-1 BBL ECD monomers are connected by polypeptide linkers selected from the group consisting of (GGGGS)i, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, GGGSGGG, GGSGGGGSGG and G, of which (GGGGS)4is preferred. Other suitable flexible polypeptide linker(s) are described herein below. In one embodiment, two or three of the 4-1 BBL ECD mutein monomers in the trimeric fusion protein are identical muteins. In one embodiment, two or three monomers of the 4-1 BBL ECD mutein in the trimeric fusion protein are different muteins. In the trimeric fusion protein, the 4-1 BBL ECD monomer that is not a 4-1 BBL ECD mutein as described herein above, can be a wild type 4-1 BBL ECD monomer, or a 4-1 BBL ECD mutein that is not described herein.

[0148] In one embodiment, the present disclosure provides a trimeric fusion protein comprising three monomers of a 4-1 BBL ECD mutein as described herein above, fused together in a single polypeptide chain, as e.g. described in Fellermeier et al. (2016, supra). In one embodiment, the three 4-1 BBL ECD monomers are connected by polypeptide linkers. In one embodiment, the three 4-1 BBL ECD monomers are connected by polypeptide linkers selected from the group consisting of (GGGGS)i, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)S, GGGSGGG, GGSGGGGSGG and G, ofwhich (GGGGS)4is preferred. Other suitable flexible polypeptide linker(s) are described herein below. In one embodiment, the three monomers of the 4-1 BBL ECD mutein in the trimeric fusion protein are identical muteins. In one embodiment, at least two of the three monomers of the 4-1 BBL ECD mutein in the trimeric fusion protein are different muteins.

[0149] In one embodiment, a fusion protein comprising three monomers of a 4-1 BBL ECD mutein, preferably a 4-1 BBL ECD mutein as described herein, fused together in a single polypeptide chain comprise three identical monomers of the 4-1 BBL ECD mutein. In another embodiment of the fusion protein at least one of the monomers differs from the other two, or all three monomers differ from each other.

[0150] It is to be understood herein that when reference is made to a 4-1 BBL ECD mutein as described herein this can also refer to a trimeric fusion protein comprising three monomers of such 4-1 BBL ECD muteins.

[0151] Conjugates comprising a 4-1 BBL ECD mutein

[0152] In a second aspect, the present disclosure provides a conjugate comprising one or more of the 4-1 BBL ECD muteins as described herein conjugated to a heterologous moiety. It is understood herein that the term “a conjugate comprising a 4-1 BBL ECD mutein as described herein” also includes a conjugate comprising a trimeric fusion protein comprising three 4-1 BBL ECD muteins as described herein. As used herein, the term "heterologous moiety" is synonymous with the term "conjugate moiety" and refers to any molecule (chemical or biochemical, naturally-occurring or noncoded) which is different from the 4-1 BBL ECD muteins described herein. Exemplary conjugate moieties that can be linked to any of the 4-1 BBL ECD muteins described herein include but are not limited to a heterologous peptide or polypeptide (including for example, an immunoglobulin or portion thereof (e.g., variable region, CDR, or Fc region)), a targeting agent, a diagnostic label such as a radioisotope, fluorophore or enzymatic label, a polymer including water soluble polymers, or other therapeutic or diagnostic agents. In some embodiments, a conjugate is provided comprising a 4-1 BBL ECD mutein of the present disclosure and an immunoglobulin. The conjugate in some embodiments comprises one or more of the 4-1 BBL ECD muteins described herein and one or more of: a peptide or polypeptide (which is distinct from the 4-1 BBL ECD muteins described herein), a nucleic acid molecule, an antibody or fragment thereof, a polymer, a quantum dot, a small molecule, a toxin, a diagnostic agent, a carbohydrate, an amino acid.

[0153] In one embodiment, there is provided a conjugate wherein the heterologous moiety is attached via non-covalent or covalent bonding to the 4-1 BBL ECD mutein as described herein. In exemplary embodiments, the linkage between the 4-1 BBL ECD mutein and the heterologous moiety is achieved via covalent chemical bonds, e.g., peptide bonds, disulfide bonds, and the like, or via physical forces, such as electrostatic, hydrogen, ionic, van der Waals, or hydrophobic or hydrophilic interactions. A variety of non-covalent coupling systems may be used, including, e.g., biotin-avidin, ligand / receptor, enzyme / substrate, nucleic acid / nucleic acid binding protein, lipid / lipid binding protein, cellular adhesion molecule partners; or any binding partners or fragments thereof which have affinity for each other.

[0154] In one embodiment, there is provided a conjugate wherein the 4-1 BBL ECD mutein as described herein is linked to a conjugate moiety via direct covalent linkage by reacting targeted amino acid residues of the 4-1 BBL ECD mutein with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues of these targeted amino acids. Reactive groups on the 4-1 BBL ECD mutein or conjugate moiety include, e.g., an aldehyde, amino, ester, thiol, a-haloacetyl, maleimido or hydrazino group. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N- hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride or other agents known in the art. Alternatively, the conjugate moieties can be linked to the 4-1 BBL ECD mutein indirectly through intermediate carriers, such as polysaccharide or polypeptide carriers. Examples of polysaccharide carriers include aminodextran. Examples of suitable polypeptide carriers include polylysine, polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixed polymers of these amino acids and others, e.g., serines, to confer desirable solubility properties on the resultant loaded carrier.

[0155] Cysteinyl residues are most commonly reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid, chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, alphabromo- p-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7- nitrobenzo-2-oxa-l ,3-diazole.

[0156] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

[0157] Lysinyl and amino-terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reaction with glyoxylate.

[0158] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1 ,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.

[0159] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O- acetyl tyrosyl species and 3-nitro derivatives, respectively.

[0160] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R-N=C=N-R'), where R and R are different alkyl groups, such as l-cyclohexyl-3-(2- morpholinyl-4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.

[0161] Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0162] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), deamidation of asparagine or glutamine, acetylation of the N- terminal amine, and / or amidation or esterification of the C-terminal carboxylic acid group.

[0163] Another type of covalent modification involves chemically or enzymatically coupling glycosides to the 4-1 BBL ECD mutein. Sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in W087 / 05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259- 306 (1981). In one embodiment, the heterologous moiety is attached to the 4-1 BBL ECD mutein as described herein via a linker. In some aspects, the linker comprises a chain of atoms from 1 to about 60, or 1 to 30 atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20 atoms long. In some embodiments, the chain atoms are all carbon atoms. In some embodiments, the chain atoms in the backbone of the linker are selected from the group consisting of C, O, N, and S. Chain atoms and linkers may be selected according to their expected solubility (hydrophilicity) so as to provide a more soluble conjugate. In some embodiments, the linker provides a functional group that is subject to cleavage by an enzyme or other catalyst or hydrolytic conditions found in the target tissue or organ or cell. In some embodiments, the length of the linker is long enough to reduce the potential for steric hindrance. If the linker is a covalent bond or a peptidyl bond and the conjugate is a polypeptide, the entire conjugate can be a fusion protein. Such peptidyl linkers may be any length. Exemplary peptidyl linkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5 to 10, 5 to 15, or 10 to 30 amino acids in length, and are flexible or rigid. Flexible linkers are usually applied when the joined domains require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g. Gly) or polar (e.g. Ser or Thr) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduces the unfavorable interaction between the linker and the protein moieties. Preferred flexible linkers have sequences consisting primarily of stretches of Gly and Ser residues (“GS” linker). An example of preferred (and widely used) flexible linker has the sequence of (GGGGS)n(SEQ ID NO: 30). By adjusting the copy number “n”, the length of this GS linker can be optimized to achieve appropriate separation of the functional domains, or to maintain necessary inter-domain interactions. The copy number “n” of this GS linker can e.g. be 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10. Specific examples of GS linkers include (GGGGS)4 (SEQ ID NO: 31), GGGSGGG (SEQ ID NO: 32), GGSGGGGSGG (SEQ ID NO: 33) and G. Besides the GS linkers, many other flexible linkers have been designed for recombinant fusion proteins. These flexible linkers are also rich in small or polar amino acids such as Gly and Ser, but can contain additional amino acids such as Thr and Ala to maintain flexibility, as well as polar amino acids such as Lys and Glu to improve solubility, such as e.g. the flexible linkers KESGSVSSEQLAQFRSLD (SEQ ID NO: 34) and EGKSSGSGSESKST (SEQ ID NO: 35), that have been applied for the construction of a bioactive scFvs.

[0164] In one embodiment, the conjugate is a conjugate that has a 4-1 BBL ECD mutein-valency that is higher than one that is higher than one, which understood to mean that the conjugate comprises more than one, e.g. two, three, four or five 4-1 BBL ECD mutein-moieties. Preferably, more than one 4-1 BBL ECD mutein-moieties are identical 4-1 BBL ECD mutein-moieties.

[0165] In one embodiment, the conjugate is a conjugate that has a valency of trimeric 4-1 BBL ECD mutein-fusion proteins that is higher than one that is higher than one, which understood to mean that the conjugate comprises more than one, e.g. two, three, four or five of such trimeric 4-1 BBL ECD mutein-fusion proteins. Preferably, more than one trimeric 4-1 BBL ECD mutein-fusion proteins are identical trimeric fusion proteins. Thus, in one embodiment, there is provided a conjugate wherein the heterologous moiety comprises a polypeptide. The polypeptide comprised in the heterologous moiety preferably is a polypeptide distinct from any of the 4-1 BBL ECD muteins described herein. In one embodiment, the conjugate is a fusion polypeptide, fusion protein, a chimeric protein or chimeric polypeptide comprising a 4-1 BBL ECD mutein or a trimeric 4-1 BBL ECD mutein-fusion protein as described herein and an heterologous moiety comprises a polypeptide fused in a single polypeptide chain. Additional descriptions of such conjugates as fusion proteins are provided hereinbelow.

[0166] In one embodiment, there is provided a conjugate wherein the heterologous moiety comprises a polypeptide that is an antigen-binding protein or a polypeptide chain of an antigenbinding protein.

[0167] In one embodiment, there is provided a conjugate wherein the heterologous moiety comprises an antigen-binding protein or a polypeptide chain of an antigen-binding protein, which antigen-binding protein comprises at least one of: a) at least one of: i) a first antigen-binding region that specifically binds a tumor associated antigen (TAA), that specifically binds an NK cell activating receptor or that specifically binds an epitope of a y6 T cell receptor (TCR), and ii) a second antigenbinding region that specifically binds a TAA, that specifically binds an NK cell activating receptor or that specifically binds an epitope of a y6 TCR; and, b) a third antigen-binding region that has or can have affinity for a surface antigen expressed on natural killer (NK) cells.

[0168] An antigen-binding region as used in an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein, can be derived from any of a variety of immunoglobulin or nonimmunoglobulin 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.

[0169] In a preferred embodiment, an antigen-binding region as used in an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as 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 antigenbinding 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as herein defined above. Antigen-binding regions that specifically bind tumor associated antigens

[0170] Thus, in one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one of i) a first antigen-binding region that specifically binds a TAA; and, ii) a second antigen-binding region that specifically binds a TAA. In one embodiment, the antigen-binding region that binds a TAA is an antigen-binding 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 10-4M, as may be determined as herein described above.

[0171] 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.

[0172] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises two antigen-binding regions that specifically bind a TAA, i.e. a first and a second antigen-binding region. In an 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, they can bind at least two different TAAs, or they can bind at least two different epitopes on the same TAA. In one embodiment of an 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, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein can be a homodimeric or a heterodimeric antigen binding protein.

[0173] 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 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.

[0174] 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.

[0175] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds to a TAA selected from the group consisting of: 5T4, ADAM9, ADAM10, ADAM12, ALK, ALPP, ALPP2, ALPPL2, AMHR2, ANGPT2, AXL, Angiopoietin-2, Apelin receptor, B7-H3, B7-H4, B7-H6, B7.1 , B7.2, BCMA, BTLA, CA125, CA9, CAIX, CCL2, CCR2, CCR4, CCR5, CCR6, CCR7, CD123, CD133, CD138, CD142, CD147, CD166, CD171 , CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD248, CD25, CD27, CD276, CD3, CD30, CD317, CD33, CD37, CD38, CD3E, CD4, CD40, CD44, CD44v6, CD45, CD46, CD47, CD52, CD56, CD70, CD71 , CD73, CD74, CD79, CD79B, CD80, CD80 / CD86, CDCP1 , CDH3, CDH6, CDK4, CEA, CEACAM5, CLDN18, CLEC14A, CLEC4, CSF1 R, CSF2, CSPG4, CT-7, CTGF, CTLA4, Cadherin 17, Cadherin 6, CanAg, Claudin 18.2, Claudin 6, Connexin 37, Cripto-1 , Crypto, DC3, DKK1 , DLK1 , DLL3, DLL4, DR5, E-cadherin, E- selectin, EBV-encoded nuclear antigen (EBNA)-I, EDA, EDB, EDNRB, EGF, EGFL7, EGFR, EGFRvlll, ENG, EPCAM, EPHA4, EphAIO, EphA2, EphA3, EphB2, EphB4, ExtradomainB (EDB) fibronectin, F3, FAP, FCGR1 , FGFR2, FGFR2b, FGFR3, FGFR4, FLT1 , FN, FOLH1 , FOLR1 , FRa, FSHR, FcRL5 / FcRH5, Fibronectin extra-domain B, Flt3, Fucosyl, GFRa4, GM3, GPCR5D, GPNMB, GPRC5D, GRP78, GUCY2C, Glycoprotein NMB, Glypican 1 , Glypican 2, Glypican 3, GnT-V, HAVCR2, HER-3 / ERBB3, HER-4 / ERBB4, HER2, HER3, HGF, HLA-G, HSP70, ICAM-1 , ICOS, IFNG, IGF-1 R, IGF1 , IGF1 R, IGF2, IL-1 accessory protein, IL-6 receptor, IL-8 receptor, IL13Ra2, IL17A, IL1 A, IL1 B, IL1 RAP, IL2RA, IL3RA, IL6, IL6R, ITGAV, ITGB6, Ig-idiotype, Integrin beta 6, KAAG-1 , KDR, KIRD2, KIT, KLK2, KLRC1 , Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), L1-CAM, L1 CAM, LAG3, LAGE-1 , LGR5, LIF, LIV-1 , LOXL2, LRRC32, Lewis-Y, MART-1 / Melan-A, MET, MIC-A / B, MICB, MIF, MISIIR, MMP2, MS4A1 , MSLN, MST1 R, MSTN, MUC1 , MUC1-C, MUC16, MUM-1 , Melanotransferrin, Mesothelin, Mud 6, NAG, NCAM1 , NKG2D, NOTCH1 , NOTCH2, NOTCH3, NRP1 , NT5E, NTRKR1 (EC 2.7.10.1), NaPi2b, Nectin-4, OLR1 , 0X40, P-cadherin, P1A, PCSK9, PD-L1 , PD1 , PDGF, PDGF alpha receptor, PDGF beta receptor, PDGFR, PDGFRA, PLAUR, PMEL, PRAME, PSCA, PSMA, PTK7, PTPRC, PVRL4, Plexin-A1 , RAGE, ROBO1 , ROR1 , ROR2, RSPO3, SCP-1 , SEZ6, SIRPA, SLAMF7, SLC34A2, SLC3A2, SSTR2, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, STEAP1 , STEAP2, Severe, T-cell receptor / CD3-zeta chain, TACSTD2, TGF-alpha, TGFB1 , TGFB2, TGFB3, TIGIT, Tissue factor / TF, TLR2, TM4SF1 , TMEFF2, TNFRSF10B, TNFRSF17, TNFRSF18, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF11 , TNFSF13B, TPBG, TRAILR1 , TRAILR2, TROP2, TSHR, TYRP1 , VEGF, VEGFA, VEGFR1 , VEGFR2, VH1 / VL1 , VH2A / L2, VH3A / L3, a GAGE-tumor antigen, a GD2 ganglioside, a GM2 ganglioside, a RAET1 protein, a UL16-binding protein (ULBP), a heterodimeric receptor comprised of at least one HER subunit, a human papillomavirus protein, a5p1 integrins, a5p3 integrins, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), anti-Mullerian hormone Type II receptor, avB3 integrin, avB6 integrin, avp6 integrins, bivalent, brain glycogen phosphorylase, c-erbB-2, cMET, colorectal associated antigen (CRC)- C017-1A / GA733, gastrin releasing peptide receptor antigen, gp100, gp75, gpA33, hCG, human papillomavirus protein, integrin receptors, mmp9, muc17, p15, phosphatidylserine, prostate specific antigen (PSA), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), sVE- cadherin, scatter factor receptor kinase, trivalent, a-catenin, a-fetoprotein, allbp3-integrins, p- catenin, and y-catenin, although this is not intended to be exhaustive.

[0176] In one embodiment therefore, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein 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 (SEQ ID NO: 24), CDR-H2 (SEQ ID NO: 25) and CDR-H3 (SEQ ID NO: 26) sequences as comprised in SEQ ID NO: 1 , and the CDR-L1 (SEQ ID NO: 27), CDR-L2 (SEQ ID NO: 28) and CDR-L3 (SEQ ID NO: 29) sequences as comprised in SEQ ID NO: 2 (trastuzumab); b) the CDR-H1 (SEQ ID NO: 150), CDR-H2 (SEQ ID NO: 151) and CDR-H3 (SEQ ID NO: 152) sequences as comprised in SEQ ID NO: 59, and the CDR-L1 (SEQ ID NO: 153), CDR- L2 (SEQ ID NO: 154) and CDR-L3 (SEQ ID NO: 155) sequences as comprised in SEQ ID NO: 60 (atezolizumab); c) the CDR-H1 (SEQ ID NO: 156), CDR-H2 (SEQ ID NO: 157) and CDR-H3 (SEQ ID NO: 158) sequences as comprised in SEQ ID NO: 9, and the CDR-L1 (SEQ ID NO: 159), CDR- L2 (SEQ ID NO: 160) and CDR-L3 (SEQ ID NO: 161) sequences as comprised in SEQ ID NO: 10 (avelumab); d) the CDR-H1 (SEQ ID NO: 162), CDR-H2 (SEQ ID NO: 163) and CDR-H3 (SEQ ID NO: 165) sequences as comprised in SEQ ID NO: 61 , and the CDR-L1 (SEQ ID NO: 165), CDR- L2 (SEQ ID NO: 166) and CDR-L3 (SEQ ID NO: 167) sequences as comprised in SEQ ID NO: 62 (durvalumab); e) 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); f) 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); g) 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); h) 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 (cosibelimab); i) 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 (margetuximab); j) 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 (pertuzumab); k) 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 (enoblituzumab); I) 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 (necitumumab); m) 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 (panitumumab); n) 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 (amivantamab EGFR-binding); o) 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 (amivantamab cMet-binding); p) 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 (zolbetuximab); q) 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 (dinutuximab); r) 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 (naxitamab); s) 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 (enfortumab); t) 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 (farletuzumab); u) 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 (tisotumab); v) 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 (mirvetuximab); w) 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 (sacituzumab); x) 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 (vobramitamab); y) 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 (Onartuzumab); z) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 144, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 145 (sibrotuzumab) aa) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 100, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 101 (olaratumab); ab) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 102, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 103 (rovalpituzumab); ac) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 238, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 239 (adebrelimab); ad) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 240, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 241 (alemtuzumab); ae) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 242, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 243 (belantamab); at) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 244, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 245 (Bevacizumab); ag) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 246, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 247 (brentuximab); ah) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 248, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 249 (camrelizumab); ai) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 250, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 251 (cemiplimab); aj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 252, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 253 (dostarlimab); ak) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 254, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 255 (emapalumab); al) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 256, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 257 (enlonstobart); am) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 258, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 259 (gemtuzumab); an) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 260, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 261 (ibritumomab); ao) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 262, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 263 (inotuzumab); ap) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 264, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 265 (ipilimumab); aq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 266, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 267 (isatuximab); ar) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 268, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 269 (loncastuximab); as) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 270, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 271 (mogamulizumab); at) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 272, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 273 (moxetumomab); au) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 274, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 275 (nimotuzumab); av) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 276, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 277 (nivolumab); aw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 278, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 279 (obinutuzumab); ax) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 280, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 281 (ofatumumab); ay) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 282, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 283 (pembrolizumab); az) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 284, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 285 (Penpulimab); ba) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 286, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 287 (polatuzumab); bb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 288, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 289 (prolgolimab); be) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 290, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 291 (pucotenlimab); bd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 292, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 293 (racotumomab) be) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 294, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 295 (ramucirumab); bf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 296, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 297 (relatlimab); bg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 298, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 299 (retifanlimab); bh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 300, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 301 (ripertamab); bi) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 302, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 303 (serplulimab); bj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 304, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 305 (sintilimab); bk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 306, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 307 (socazolimab); bl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 308, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 309 (sugemalimab); bm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 310, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 311 (tafasitamab); bn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 312, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 313 (tagitanlimab); bo) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 314, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 315 (tebentafusp); bp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 316, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 317 (Tislelizumab); bq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 318, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 319 (Toripalimab); br) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 320, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 321 (zuberitamab); bs) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 322, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 323 (benmelstobart); bt) bu) bv) bw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 324, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 325 (iparomlimab); bx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 326, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 327 (tuvonralimab); by) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 328, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 329 (anvatabart); bz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 330, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 331 (apamistamab); ca) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 332, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 333 (bemarituzumab); cb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 334, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 335 (cetrelimab); cd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 336, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 337 (cobolimab); ce) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 338, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 339 (datopotamab); cf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 340, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 341 (domvanalimab); eg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 342, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 343 (emactuzumab); ch) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 344, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 345 (favezelimab); ci) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 346, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 347 (felzartamab); cj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 348, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 349 (fianlimab); ck) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 350, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 351 (finotonlimab); cl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 352, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 353 (geptanolimab); cm) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 354, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 355 (gotistobart); cn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 356, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 357 (ivuxolimab); co) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 358, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 359 (lemzoparlimab); cp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 360, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 361 (luveltamab); cq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 362, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 363 (magrolimab); cr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 364, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 365 (meebotamab); cs) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 366, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 367 (monalizumab); ct) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 368, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 369 (nofazinlimab); cu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 370, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 371 (nurulimab); cv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 372, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 373 (ociperlimab); cw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 374, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 375 (oleclumab); ex) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 376, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 377 (onfekafusp); cy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 378, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 379 (patritumab); cz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 380, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 381 (pivekimab); da) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 382, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 383 (quavonlimab); db) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 384, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 385 (retlirafusp); de) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 386, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 387 (rosopatamab); dd) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 388, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 389 (rulonilimab); de) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 390, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 391 (sabatolimab); df) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 392, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 393 (sasanlimab); dg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 394, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 395 (telisotuzumab); dh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 396, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 397 (tiragolumab); di) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 398, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 399 (tusamitamab); dj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 400, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 401 (vibostolimab); dk) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 402, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 403 (vobramitamab); dl) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 404, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 405 (zilovertamab); dm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 406, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 407 (suvemcitug); dn) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 408, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 409 (becotatug); do) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 410, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 411 (tifcemalimab); dq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 412, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 413 (blinatumomab - CD19); dr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 414, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 415 (blinatumomab - CD3); ds) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 416, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 417 (cadonilimab - PD-1); dt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 418, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 419 (cadonilimab - CTLA4); du) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 420, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 421 (disitamab); dv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 422, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 423 (edrecolomab); dw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 424, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 425 (elranatamab - BCMA); dx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 426, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 427 (elranatamab - DC3); dy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 428, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 429 (epcoritamab - CD20); dz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 430, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 431 (epcoritamab - CD3); ea) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 432, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 433 (glofitamab - VH1 / VL1); eb); the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 434, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 435 (glofitamab - VH2A / L2); ec) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 436, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 437 (glofitamab - VH3 / VL3); ed) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 438, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 439 (mosunetuzumab - CD20); ef) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 440, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 441 (mosunetuzumab - CD3); eg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 442, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 443 (talquetamab - GPCR5D); eh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 444, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 445 (talquetamab - CD3); ei) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 446, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 447 (teclistamab - BCMA); ej) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 448, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 449 (teclistamab - CD3); ek) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 450, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 451 (tositumomab); el) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 452, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 453 (tremelimumab); em) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 454, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 455 (zimberelimab); en) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 456, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 457 (odronextamab - CD20); eo) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 458, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 459 (odronextamab - CD3); ep) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 460, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 461 (ivonescimab - PD-1); eq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 462, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 463 (ivonescimab - VEGF); er) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 464, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 465 (anbenitamab - VH1 / VL1); es) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 466, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 467 (anbenitamab - VH2 / VL2); et) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 468, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 469 (izalontamab - EGFR); eu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 470, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 471 (izalontamab - HER3); ev) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 472, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 473 (linvoseltamab - BCMA); ew) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 474, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 475 (linvoseltamab - CD3); ex) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 476, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 477 (tarlatamab - DLL3); ey) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 478, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 479 (tarlatamab - CD3); ez) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 480, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 481 (zanidatamab - VH1 / VL1); fa) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 482, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 483 (zanidatamab - VH2 / VL2); fb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 484, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 485 (volrustomig - PD-1); fc) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 486, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 487 (volrustomig - CTLA-4); fd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 488, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 489 (zenocutuzumab - HER3); fe) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 490, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 491 (zenocutuzumab - HER2); ff) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 492, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 493 (botensilimab - VH1 / VL1); fg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 494, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 495 (botensilimab - VH2 / VL2); th) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 496, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 497 (izalontamab - EGFR); fi) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 498, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 499 (izalontamab - HER3); fj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 500, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 501 (rilvegostomig - TIGIT); fk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 502, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 503 (rilvegostomig - PD-1); fl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 701 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 700 (abagovomab); fm) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 703, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 702 (abituzumab); fn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 705, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 704 (acasunlimab); fo) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 707, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 706 (alnuctamab); fp) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 709, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 708 (alomfilimab); fq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 711 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 710 (amatuximab); fr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 713, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 712 (anetumab); fs) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 715, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 714 (aplitabart); ft) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 717, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 716 (atigotatug); fu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 719, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 718 (balstilimab); fv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 721 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 720 (bavituximab); fw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 723, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 722 (bavunalimab); fx) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 725, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 724 (belrestotug); fy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 727, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 726 (bermekimab); fz) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 729, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 728 (bifikafusp); ga) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 731 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 730 (bintrafusp); gb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 733, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 732 (brenetafusp); gc) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 735, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 734 (briquilimab); gd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 737, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 736 (brontictuzumab); ge) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 739, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 738 (budigalimab); gf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 741 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 740 (cabiralizumab); gg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 743, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 742 (canakinumab); gh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 745, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 744 (cantuzumab); gi) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 747, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 746 (carlumab); gj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 749, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 748 (carotuximab); gk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 751 , and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 750 (caxmotabart); gl) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 753, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 752 (cergutuzumab); gm) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 755, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 754 (cibisatamab); gn) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 757, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 756 (cinrebafusp); go) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 759, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 758 (cixutumumab); gp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 761 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 760 (clazakizumab); gq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 763, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 762 (clivatuzumab); gr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 765, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 764 (cofetuzumab); gs) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 767, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 766 (coltuximab); gt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 769, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 768 (conatumumab); gu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 771 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 770 (dacetuzumab); gv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 773, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 772 (dalutrafusp); gw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 775, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 774 (danburstotug); gx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 777, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 776 (daratumumab); gy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 779, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 778 (demcizumab); gz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 781 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 780 (denintuzumab); ha) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 783, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 782 (denosumab); hb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 785, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 784 (depatuxizumab); he) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 787, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 786 (drozitumab); hd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 789, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 788 (duligotuzumab); he) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 791 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 790 (dusigitumab); hf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 793, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 792 (duvortuxizumab); hg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 795, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 794 (elotuzumab); hh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 797, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 796 (eluvixtamab); hi) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 799, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 798 (enapotamab); hj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 801 , and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 800 (enoticumab); hk) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 803, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 802 (epacmarstobart); hl) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 805, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 804 (etentamig); hm) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 807, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 806 (falbikitug); hn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 809, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 808 (faricimab); ho) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 811 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 810 (feladilimab); hp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 813, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 812 (ficerafusp); hq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 815, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 814 (ficlatuzumab); hr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 817, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 816 (figitumumab); hs) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 819, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 818 (flanvotumab); ht) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 821 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 820 (flotetuzumab); hu) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 823, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 822 (forimtamig); hv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 825, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 824 (futuximab); hw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 827, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 826 (ganitumab); hx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 829, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 828 (gevokizumab); hy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 831 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 830 (girentuximab); hz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 833, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 832 (glembatumumab); ia) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 835, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 834 (icrucumab); ib) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 837, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 836 (ifinatamab); ic) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 839, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 838 (iladatuzumab); id) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 841 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 840 (imalumab); ie) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 843, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 842 (imgatuzumab); if) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 845, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 844 (indusatumab); ig) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 847, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 846 (inebilizumab); ih) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 849, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 848 (ispectamab); ii) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 851 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 850 (istiratumab); ij) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 853, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 852 (izeltabart); ik) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 855, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 854 (izuralimab); il) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 857, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 856 (landogrozumab); im) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 859, and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 858 (laprituximab); in) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 861 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 860 (lenzilumab); io) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 863, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 862 (leronlimab); ip) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 865, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 864 (lifastuzumab); iq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 867, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 866 (ligufalimab); ir) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 869, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 868 (lilotomab); is) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 871 , and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 870 (lintuzumab); it) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 873, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 872 (lirilumab); iu) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 875, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 874 (livmoniplimab); iv) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 877, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 876 (lorvotuzumab); iw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 879, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 878 (lucatumumab); ix) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 881 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 880 (lumretuzumab); iy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 883, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 882 (matuzumab); iz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 885, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 884 (mipasetamab); ja) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 887, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 886 (modakafusp); jb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 889, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 888 (modotuximab); jc) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 891 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 890 (murlentamab); jd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 893, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 892 (nadunolimab); je) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 895, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 894 (naptumomab); jf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 897, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 896 (narlumosbart); jg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 899, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 898 (narnatumab); jh) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 901 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 900 (navicixizumab); ji) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 903, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 902 (nesvacumab); jj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 905, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 904 (nisevokitug); jk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 907, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 906 (omburtamab); jl) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 909, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 908 (ontuxizumab); jm) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 911 , and the CDR-L1 , CDR-L2 and CDR- L3 sequences as comprised in SEQ ID NO: 910 (otlertuzumab); jn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 913, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 912 (pamrevlumab); jo) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 915, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 914 (parsatuzumab); jp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 917, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 916 (pavurutamab); jq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 919, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 918 (pemivibart); jr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 921 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 920 (petosemtamab); js) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 923, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 922 (pimivalimab); jt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 925, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 924 (pinatuzumab); ju) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 927, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 926 (plozalizumab); jv) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 929, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 928 (pulocimab); jw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 931 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 930 (ragifilimab); jx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 933, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 932 (raludotatug); jy) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 935, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 934 (rilotumumab); jz) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 937, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 936 (rosmantuzumab); ka) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 939, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 938 (runimotamab); kb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 941 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 940 (sabestomig); kc) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 943, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 942 (selicrelumab); kd) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 945, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 944 (seribantumab); ke) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 947, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 946 (sigvotatug); kf) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 949, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 948 (simlukafusp); kg) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 951 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 950 (simtuzumab); kh) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 953, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 952 (sirexatamab); ki) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 955, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 954 (sofituzumab); kj) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 957, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 956 (spartalizumab); kk) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 959, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 958 (surzebiclimab); kl) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 961 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 960 (tabalumab); km) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 963, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 962 (tafolecimab); kn) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 965, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 964 (talacotuzumab); ko) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 967, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 966 (tarextumab); kp) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 969, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 968 (tavolimab); kq) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 971 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 970 (tebotelimab); kr) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 973, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 972 (teprotumumab); ks) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 975, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 974 (tidutamab); kt) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 977, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 976 (tigatuzumab); ku) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 979, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 978 (tilvestamab); kv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 981 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 980 (tobemstomig); kw) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 983, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 982 (tocilizumab); kx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 985, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 984 (tomaralimab); ky) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 987, and the CDR- L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 986 (tovecimig); kz) the CDR- H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 989, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 988 (tovetumab); la) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 991 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 990 (tucotuzumab); lb) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 993, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 992 (tuparstobart); Ic) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 995, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 994 (upifitamab); Id) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 997, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 996 (urabrelimab); le) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 999, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 998 (utomilumab); If) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1001 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1000 (vadastuximab); Ig) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1003, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1002 (vandortuzumab); Ih) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1005, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1004 (vanucizumab); li) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1007, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1006 (veligrotug); Ij) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1009, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1008 (verzistobart); Ik) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1011 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1010 (vesencumab); II) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1013, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1012 (vofatamab); Im) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1015, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1014 (vonlerolizumab); In) the CDR-H1 , CDR- H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1017, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1016 (vopikitug); Io) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1019, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1018 (vorsetuzumab); Ip) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1021 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1020 (xaluritamig); Iq) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1023, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1022 (zalutumumab); Ir) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1025, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1024 (zanolimumab); Is) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1027, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1026 (ivuxolimab-alt); It) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1029, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1028 (inotuzumab-alt); lu) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1031 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1030 (moxetumomab-alt); Iv) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1033, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1032 (luveltamab-alt); lw) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1035, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1034 (ibritumomab-alt); lx) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1037, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1036 (pivekimab-alt); ly) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1039, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1038 (avelumab-alt); Iz) the CDR-H1 , CDR-H2 and CDR- H3 sequences as comprised in SEQ ID NO: 1041 , and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1040 (sugemalimab-alt); ma) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1043, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1042 (nimotuzumab-alt); mb) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 1045, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 1044 (panitumumab-alt); me) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 540, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 541 (AR46A6); md) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 542, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 543 (KM4097); and me) the CDR-H1 , CDR-H2 and CDR-H3 sequences as comprised in SEQ ID NO: 544, and the CDR-L1 , CDR-L2 and CDR-L3 sequences as comprised in SEQ ID NO: 545 (K5-70).

[0177] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds to TROP2. TROP2 is a transmembrane glycoprotein encoded by the human Tacstd2 gene. The 323 amino acid sequence of human TROP2 described in NCBI accession number NP_002344, the disclosure of which is incorporated herein by reference. The human TROP2 mRNA sequence is described in NCBI accession number NM_002353, the disclosure of which is incorporated herein by reference. TROP2 is an intracellular calcium signal transducer that is differentially expressed in many cancers. TROP2 plays a role in tumor progression by actively interacting with several key molecular signaling pathways traditionally associated with cancer development and progression. Aberrant overexpression of TROP2 has been described in several solid cancers. TROP2 causes cancer cell growth, proliferation, invasion, migration, and survival of cancer cells, which leads to TROP2 being associated with tumor aggressiveness and poor prognosis. These facts make TROP2 a possible prognostic biomarker to identify high-risk patients, as well as an attractive therapeutic target for (late-stage) diseases.

[0178] In one embodiment therefore, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds TROP2, comprising 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) a CDR-H1 comprising the sequence of SEQ ID NO: 552, a CDR-H2 comprising the sequence of SEQ ID NO: 553, a CDR-H3 comprising the sequence of SEQ ID NO: 554, a CDR-L1 comprising the sequence of SEQ ID NO: 555, a CDR-L2 comprising the sequence of SEQ ID NO: 556, and a CDR-L3 comprising the sequence of SEQ ID NO: 557 (sacituzumab); b) a CDR-H1 comprising the sequence of SEQ ID NO: 558, a CDR-H2 comprising the sequence of SEQ ID NO: 559, a CDR-H3 comprising the sequence of SEQ ID NO: 560, a CDR-L1 comprising the sequence of SEQ ID NO: 561 , a CDR- L2 comprising the sequence of SEQ ID NO: 562, and a CDR-L3 comprising the sequence of SEQ ID NO: 563 (datopotamab); c) a CDR-H1 comprising the sequence of SEQ ID NO: 564, a CDR-H2 comprising the sequence of SEQ ID NO: 565, a CDR-H3 comprising the sequence of SEQ ID NO: 566, a CDR-L1 comprising the sequence of SEQ ID NO: 567, a CDR-L2 comprising the sequence of SEQ ID NO: 568, and a CDR-L3 comprising the sequence of SEQ ID NO: 569 (KM4097); d) a CDR-H1 comprising the sequence of SEQ ID NO: 570, a CDR-H2 comprising the sequence of SEQ ID NO: 571 , a CDR-H3 comprising the sequence of SEQ ID NO: 572, a CDR-L1 comprising the sequence of SEQ ID NO: 573, a CDR-L2 comprising the sequence of SEQ ID NO: 574, and a CDR- L3 comprising the sequence of SEQ ID NO: 575 (AR47A6.4.2); and, e) a CDR-H1 comprising the sequence of SEQ ID NO: 576, a CDR-H2 comprising the sequence of SEQ ID NO: 577, a CDR-H3 comprising the sequence of SEQ ID NO: 578, a CDR-L1 comprising the sequence of SEQ ID NO: 579, a CDR-L2 comprising the sequence of SEQ ID NO: 580, and a CDR-L3 comprising the sequence of SEQ ID NO: 581 (K5-70).

[0179] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds to Her2 (Erb2 / Neu). The human epidermal growth factor receptor-2 (HER2) receptor (previously called HER2 / Neu) is a transmembrane glycoprotein with tyrosine kinase activity is encoded by the ERBB2 gene and that belongs to the epidermal growth factor receptor family. Amino acid sequences of human HER2 are described in NCBI accession numbers NP_001005862, NP_001276865, NP_001276866, NP_001276867 and NP_004439 NP_002344, the disclosure of which is incorporated herein by reference. Human HER2 mRNA sequences are described in NCBI accession numbers NM_001005862, NM_001289936, NM_001289937, NM_001289938 and NM_004448, the disclosure of which is incorporated herein by reference. Receptors of the epidermal growth factor receptor family are essential in controlling epithelial cell growth and differentiation. Aberrant HER2 protein overexpression has associations with adenocarcinomas, including breast, ovary, endometrium, cervix, as well as lung, esophageal, gastroesophageal junction, gastric, and bladder cancers. HER2 amplification or overexpression occurs in approximately 20% to 30% of human breast cancers. This protein is strongly associated with increased disease recurrence and is a poor prognostic factor for survival. For this reason, HER2 is an essential target for the therapy of various types of cancer.

[0180] In one embodiment therefore, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds HER2, comprising 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) a CDR-H1 comprising the sequence of SEQ ID NO: 24, a CDR-H2 comprising the sequence of SEQ ID NO: 25, a CDR-H3 comprising the sequence of SEQ ID NO: 26, a CDR-L1 comprising the sequence of SEQ ID NO: 27, a CDR-L2 comprising the sequence of SEQ ID NO: 28, and a CDR-L3 comprising the sequence of SEQ ID NO: 29 (trastuzumab); b) a CDR-H1 comprising the sequence of SEQ ID NO: 594, a CDR-H2 comprising the sequence of SEQ ID NO: 595, a CDR-H3 comprising the sequence of SEQ ID NO: 596, a CDR-L1 comprising the sequence of SEQ ID NO: 597, a CDR-L2 comprising the sequence of SEQ ID NO: 598, and a CDR-L3 comprising the sequence of SEQ ID NO: 599 (margetuximab); c) a CDR-H1 comprising the sequence of SEQ ID NO: 600, a CDR-H2 comprising the sequence of SEQ ID NO: 601 , a CDR-H3 comprising the sequence of SEQ ID NO: 602, a CDR-L1 comprising the sequence of SEQ ID NO: 603, a CDR-L2 comprising the sequence of SEQ ID NO: 604, and a CDR-L3 comprising the sequence of SEQ ID NO: 605 (pertuzumab); d) a CDR-H1 comprising the sequence of SEQ ID NO: 606, a CDR-H2 comprising the sequence of SEQ ID NO: 607, a CDR-H3 comprising the sequence of SEQ ID NO: 608, a CDR-L1 comprising the sequence of SEQ ID NO: 609, a CDR-L2 comprising the sequence of SEQ ID NO: 610, and a CDR-L3 comprising the sequence of SEQ ID NO: 611 (disitamab); e) a CDR-H1 comprising the sequence of SEQ ID NO: 600, a CDR-H2 comprising the sequence of SEQ ID NO: 601 , a CDR-H3 comprising the sequence of SEQ ID NO: 602,, a CDR-L1 comprising the sequence of SEQ ID NO: 612, a CDR-L2 comprising the sequence of SEQ ID NO: 613, and a CDR-L3 comprising the sequence of SEQ ID NO: 614 (anbenitamab2); f) a CDR-H1 comprising the sequence of SEQ ID NO: 615, a CDR-H2 comprising the sequence of SEQ ID NO: 616, a CDR-H3 comprising the sequence of SEQ ID NO: 617,, a CDR-L1 comprising the sequence of SEQ ID NO: 618, a CDR-L2 comprising the sequence of SEQ ID NO: 619, and a CDR-L3 comprising the sequence of SEQ ID NO: 620 (zanidatamab2); and, g) a CDR-H1 comprising the sequence of SEQ ID NO: 621 , a CDR- H2 comprising the sequence of SEQ ID NO: 622, a CDR-H3 comprising the sequence of SEQ ID NO: 623,, a CDR-L1 comprising the sequence of SEQ ID NO: 624, a CDR-L2 comprising the sequence of SEQ ID NO: 625, and a CDR-L3 comprising the sequence of SEQ ID NO: 626 (zenocutuzumab).

[0181] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds to EGF receptor (EGFR, ERBB1). The human epidermal growth factor receptor-1 (EGFR; ErbB-1 ; HER1 in humans) is a transmembrane glycoprotein with tyrosine kinase activity is encoded by the ERBB1 gene and that belongs to the epidermal growth factor receptor family. Amino acid sequences of human EGFR are described in NCBI accession numbers NP_001333826, NP_001333827, NP_001333828, and NP_001333829, the disclosure of which is incorporated herein by reference. Human EGFR mRNA sequences are described in NCBI accession numbers NM_001346897, NM_001346898, NM_001346899, NM_001346900, and NM_001346941 , the disclosure of which is incorporated herein by reference. Receptors of the epidermal growth factor receptor family are essential in controlling epithelial cell growth and differentiation. Aberrant EGFR protein overexpression has associations with a wide variety of tumors. Interruption of EGFR signalling, either by blocking EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumors and improve the patient's condition. For this reason, EGFR is an essential target for the therapy of various types of cancer.

[0182] In one embodiment therefore, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that specifically binds EGFR, comprising 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) a CDR-H1 comprising the sequence of SEQ ID NO: 627, a CDR-H2 comprising the sequence of SEQ ID NO: 628, a CDR-H3 comprising the sequence of SEQ ID NO: 629, a CDR-L1 comprising the sequence of SEQ ID NO: 630, a CDR-L2 comprising the sequence of SEQ ID NO: 631 , and a CDR-L3 comprising the sequence of SEQ ID NO: 632 (cetuximab); b) a CDR-H1 comprising the sequence of SEQ ID NO: 633, a CDR-H2 comprising the sequence of SEQ ID NO: 634, a CDR-H3 comprising the sequence of SEQ ID NO: 635, a CDR-L1 comprising the sequence of SEQ ID NO: 636, a CDR- L2 comprising the sequence of SEQ ID NO: 637, and a CDR-L3 comprising the sequence of SEQ ID NO: 638 (necitumumab); c) a CDR-H1 comprising the sequence of SEQ ID NO: 639, a CDR-H2 comprising the sequence of SEQ ID NO: 640, a CDR-H3 comprising the sequence of SEQ ID NO: 641 , a CDR-L1 comprising the sequence of SEQ ID NO: 642, a CDR-L2 comprising the sequence of SEQ ID NO: 643, and a CDR-L3 comprising the sequence of SEQ ID NO: 644 (panitumumab); d) a CDR-H1 comprising the sequence of SEQ ID NO: 645, a CDR-H2 comprising the sequence of SEQ ID NO: 646, a CDR-H3 comprising the sequence of SEQ ID NO: 647, a CDR-L1 comprising the sequence of SEQ ID NO: 648, a CDR-L2 comprising the sequence of SEQ ID NO: 649, and a CDR-L3 comprising the sequence of SEQ ID NO: 650 (nimotuzumab); e) a CDR-H1 comprising the sequence of SEQ ID NO: 651 , a CDR-H2 comprising the sequence of SEQ ID NO: 652, a CDR-H3 comprising the sequence of SEQ ID NO: 653,, a CDR-L1 comprising the sequence of SEQ ID NO: 654, a CDR-L2 comprising the sequence of SEQ ID NO: 655, and a CDR-L3 comprising the sequence of SEQ ID NO: 656 (becotatug); f) a CDR-H1 comprising the sequence of SEQ ID NO: 657, a CDR-H2 comprising the sequence of SEQ ID NO: 658, a CDR-H3 comprising the sequence of SEQ ID NO: 659,, a CDR-L1 comprising the sequence of SEQ ID NO: 660, a CDR-L2 comprising the sequence of SEQ ID NO: 661 , and a CDR-L3 comprising the sequence of SEQ ID NO: 662 (amivantamab); and, g) a CDR-H1 comprising the sequence of SEQ ID NO: 663, a CDR-H2 comprising the sequence of SEQ ID NO: 664, a CDR-H3 comprising the sequence of SEQ ID NO: 665,, a CDR-L1 comprising the sequence of SEQ ID NO: 666, a CDR-L2 comprising the sequence of SEQ ID NO: 667, and a CDR-L3 comprising the sequence of SEQ ID NO: 668 (izalontamab). In one embodiment therefore, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises an immunoglobulin single variable domain (ISVD) comprising a combination of complementarity-determining regions (CDRs) CDR1 , CDR2, and CDR3 selected from the group consisting of: a) the CDR1 , CDR2, and CDR3 sequences as comprised in SEQ ID NO: 504 (envafolimab); b) the CDR1 , CDR2, and CDR3 sequences as comprised in SEQ ID NO: 505 (erfonrilimab - PD-L1); c) the CDR1 , CDR2, and CDR3 sequences as comprised in SEQ ID NO: 506 (erfonrilimab - CTLA-4); and, d) the CDR1 , CDR2, and CDR3 sequences as comprised in SEQ ID NO: 507 (ozekibart).

[0183] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises a combination of variable heavy (VH) and variable light (VL) domains selected from the group consisting of: a) the VH sequence as comprised in SEQ ID NO: 39 and the VL sequence as comprised in SEQ ID NO: 40 (trastuzumab); b) the VH sequence as comprised in SEQ ID NO: 41 and the VL sequence as comprised in SEQ ID NO: 42 (cetuximab); c) the VH sequence as comprised in SEQ ID NO: 43 and the VL sequence as comprised in SEQ ID NO: 44 (rituximab); d) the VH sequence as comprised in SEQ ID NO: 45 and the VL sequence as comprised in SEQ ID NO: 46 (daratumumab); e) the VH sequence as comprised in SEQ ID NO: 47 and the VL sequence as comprised in SEQ ID NO: 48 (avelumab); f) the VH sequence as comprised in SEQ ID NO: 104 and the VL sequence as comprised in SEQ ID NO: 105 (atezolizumab); g) the VH sequence as comprised in SEQ ID NO: 106 and the VL sequence as comprised in SEQ ID NO: 107 (durvalumab); h) the VH sequence as comprised in SEQ ID NO: 108 and the VL sequence as comprised in SEQ ID NO: 109 (cosibelimab); i) the VH sequence as comprised in SEQ ID NO: 1 10 and the VL sequence as comprised in SEQ ID NO: 111 (margetuximab); j) the VH sequence as comprised in SEQ ID NO: 112 and the VL sequence as comprised in SEQ ID NO: 113 (pertuzumab); k) the VH sequence as comprised in SEQ ID NO: 114 and the VL sequence as comprised in SEQ ID NO: 115 (enoblituzumab); I) the VH sequence as comprised in SEQ ID NO: 116 and the VL sequence as comprised in SEQ ID NO: 117 (necitumumab); m) the VH sequence as comprised in SEQ ID NO: 118 and the VL sequence as comprised in SEQ ID NO: 1 19 (panitumumab); n) the VH sequence as comprised in SEQ ID NO: 120 and the VL sequence as comprised in SEQ ID NO: 121 (amivantamab EGFR-binding); o) the VH sequence as comprised in SEQ ID NO: 122 and the VL sequence as comprised in SEQ ID NO: 123 (amivantamab cMet-binding); p) the VH sequence as comprised in SEQ ID NO: 124 and the VL sequence as comprised in SEQ ID NO: 125 (zolbetuximab); q) the VH sequence as comprised in SEQ ID NO: 126 and the VL sequence as comprised in SEQ ID NO: 127 (dinutuximab); r) the VH sequence as comprised in SEQ ID NO: 128 and the VL sequence as comprised in SEQ ID NO: 129 (naxitamab); s) the VH sequence as comprised in SEQ ID NO: 130 and the VL sequence as comprised in SEQ ID NO: 131 (enfortumab); t) the VH sequence as comprised in SEQ ID NO: 132 and the VL sequence as comprised in SEQ ID NO: 133 (farletuzumab); u) the VH sequence as comprised in SEQ ID NO: 134 and the VL sequence as comprised in SEQ ID NO: 135 (tisotumab); v) the VH sequence as comprised in SEQ ID NO: 136 and the VL sequence as comprised in SEQ ID NO: 137 (mirvetuximab); w) the VH sequence as comprised in SEQ ID NO: 138 and the VL sequence as comprised in SEQ ID NO: 139 (sacituzumab); x) the VH sequence as comprised in SEQ ID NO: 140 and the VL sequence as comprised in SEQ ID NO: 141 (vobramitamab); y) the VH sequence as comprised in SEQ ID NO: 142 and the VL sequence as comprised in SEQ ID NO: 143 (onartuzumab); z) the VH sequence as comprised in SEQ ID NO: 144 and the VL sequence as comprised in SEQ ID NO: 145 (sibrotuzumab); aa) the VH sequence as comprised in SEQ ID NO: 146 and the VL sequence as comprised in SEQ ID NO: 147 (olaratumab); ab) the VH sequence as comprised in SEQ ID NO: 148 and the VL sequence as comprised in SEQ ID NO: 149 (rovalpituzumab); ac) the VH sequence as comprised in SEQ ID NO: 238, and the VL sequence as comprised in SEQ ID NO: 239 (adebrelimab); ad) the VH sequence as comprised in SEQ ID NO: 240, and the VL sequence as comprised in SEQ ID NO: 241 (alemtuzumab); ae) the VH sequence as comprised in SEQ ID NO: 242, and the VL sequence as comprised in SEQ ID NO: 243 (belantamab); at) the VH sequence as comprised in SEQ ID NO: 244, and the VL sequence as comprised in SEQ ID NO: 245 (Bevacizumab); ag) the VH sequence as comprised in SEQ ID NO: 246, and the VL sequence as comprised in SEQ ID NO: 247 (brentuximab); ah) the VH sequence as comprised in SEQ ID NO: 248, and the VL sequence as comprised in SEQ ID NO: 249 (camrelizumab); ai) the VH sequence as comprised in SEQ ID NO: 250, and the VL sequence as comprised in SEQ ID NO: 251 (cemiplimab); aj) the VH sequence as comprised in SEQ ID NO: 252, and the VL sequence as comprised in SEQ ID NO: 253 (dostarlimab); ak) the VH sequence as comprised in SEQ ID NO: 254, and the VL sequence as comprised in SEQ ID NO: 255 (emapalumab); al) the VH sequence as comprised in SEQ ID NO: 256, and the VL sequence as comprised in SEQ ID NO: 257 (enlonstobart); am) the VH sequence as comprised in SEQ ID NO: 258, and the VL sequence as comprised in SEQ ID NO: 259 (gemtuzumab); an) the VH sequence as comprised in SEQ ID NO: 260, and the VL sequence as comprised in SEQ ID NO: 261 (ibritumomab); ao) the VH sequence as comprised in SEQ ID NO: 262, and the VL sequence as comprised in SEQ ID NO: 263 (inotuzumab); ap) the VH sequence as comprised in SEQ ID NO: 264, and the VL sequence as comprised in SEQ ID NO: 265 (ipilimumab); aq) the VH sequence as comprised in SEQ ID NO: 266, and the VL sequence as comprised in SEQ ID NO: 267 (isatuximab); ar) the VH sequence as comprised in SEQ ID NO: 268, and the VL sequence as comprised in SEQ ID NO: 269 (loncastuximab); as) the VH sequence as comprised in SEQ ID NO: 270, and the VL sequence as comprised in SEQ ID NO: 271 (mogamulizumab); at) the VH sequence as comprised in SEQ ID NO: 272, and the VL sequence as comprised in SEQ ID NO: 273 (moxetumomab); au) the VH sequence as comprised in SEQ ID NO: 274, and the VL sequence as comprised in SEQ ID NO: 275 (nimotuzumab); av) the VH sequence as comprised in SEQ ID NO: 276, and the VL sequence as comprised in SEQ ID NO: 277 (nivolumab); aw) the VH sequence as comprised in SEQ ID NO: 278, and the VL sequence as comprised in SEQ ID NO: 279 (obinutuzumab); ax) the VH sequence as comprised in SEQ ID NO: 280, and the VL sequence as comprised in SEQ ID NO: 281 (ofatumumab); ay) the VH sequence as comprised in SEQ ID NO: 282, and the VL sequence as comprised in SEQ ID NO: 283 (pembrolizumab); az) the VH sequence as comprised in SEQ ID NO: 284, and the VL sequence as comprised in SEQ ID NO: 285 (Penpulimab); ba) the VH sequence as comprised in SEQ ID NO: 286, and the VL sequence as comprised in SEQ ID NO: 287 (polatuzumab); bb) the VH sequence as comprised in SEQ ID NO: 288, and the VL sequence as comprised in SEQ ID NO: 289 (prolgolimab); be) the VH sequence as comprised in SEQ ID NO: 290, and the VL sequence as comprised in SEQ ID NO: 291 (pucotenlimab); bd) the VH sequence as comprised in SEQ ID NO: 292, and the VL sequence as comprised in SEQ ID NO: 293 (racotumomab) be) the VH sequence as comprised in SEQ ID NO: 294, and the VL sequence as comprised in SEQ ID NO: 295 (ramucirumab); bf) the VH sequence as comprised in SEQ ID NO: 296, and the VL sequence as comprised in SEQ ID NO: 297 (relatlimab); bg) the VH sequence as comprised in SEQ ID NO: 298, and the VL sequence as comprised in SEQ ID NO: 299 (retifanlimab); bh) the VH sequence as comprised in SEQ ID NO: 300, and the VL sequence as comprised in SEQ ID NO: 301 (ripertamab); bi) the VH sequence as comprised in SEQ ID NO: 302, and the VL sequence as comprised in SEQ ID NO: 303 (serplulimab); bj) the VH sequence as comprised in SEQ ID NO: 304, and the VL sequence as comprised in SEQ ID NO: 305 (sintilimab); bk) the VH sequence as comprised in SEQ ID NO: 306, and the VL sequence as comprised in SEQ ID NO: 307 (socazolimab); bl) the VH sequence as comprised in SEQ ID NO: 308, and the VL sequence as comprised in SEQ ID NO: 309 (sugemalimab); bm) the VH sequence as comprised in SEQ ID NO: 310, and the VL sequence as comprised in SEQ ID NO: 311 (tafasitamab); bn) the VH sequence as comprised in SEQ ID NO: 312, and the VL sequence as comprised in SEQ ID NO: 313 (tagitanlimab); bo) the VH sequence as comprised in SEQ ID NO: 314, and the VL sequence as comprised in SEQ ID NO: 315 (tebentafusp); bp) the VH sequence as comprised in SEQ ID NO: 316, and the VL sequence as comprised in SEQ ID NO: 317 (Tislelizumab); bq) the VH sequence as comprised in SEQ ID NO: 318, and the VL sequence as comprised in SEQ ID NO: 319 (Toripalimab); br) the VH sequence as comprised in SEQ ID NO: 320, and the VL sequence as comprised in SEQ ID NO: 321 (zuberitamab); bs) the VH sequence as comprised in SEQ ID NO: 322, and the VL sequence as comprised in SEQ ID NO: 323 (benmelstobart); bt) bu) bv) bw) the VH sequence as comprised in SEQ ID NO: 324, and the VL sequence as comprised in SEQ ID NO: 325 (iparomlimab); bx) the VH sequence as comprised in SEQ ID NO: 326, and the VL sequence as comprised in SEQ ID NO: 327 (tuvonralimab); by) the VH sequence as comprised in SEQ ID NO: 328, and the VL sequence as comprised in SEQ ID NO: 329 (anvatabart); bz) the VH sequence as comprised in SEQ ID NO: 330, and the VL sequence as comprised in SEQ ID NO: 331 (apamistamab); ca) the VH sequence as comprised in SEQ ID NO: 332, and the VL sequence as comprised in SEQ ID NO: 333 (bemarituzumab); cb) the VH sequence as comprised in SEQ ID NO: 334, and the VL sequence as comprised in SEQ ID NO: 335 (cetrelimab); cd) the VH sequence as comprised in SEQ ID NO: 336, and the VL sequence as comprised in SEQ ID NO: 337 (cobolimab); ce) the VH sequence as comprised in SEQ ID NO: 338, and the VL sequence as comprised in SEQ ID NO: 339 (datopotamab); cf) the VH sequence as comprised in SEQ ID NO: 340, and the VL sequence as comprised in SEQ ID NO: 341 (domvanalimab); eg) the VH sequence as comprised in SEQ ID NO: 342, and the VL sequence as comprised in SEQ ID NO: 343 (emactuzumab); ch) the VH sequence as comprised in SEQ ID NO: 344, and the VL sequence as comprised in SEQ ID NO: 345 (favezelimab); ci) the VH sequence as comprised in SEQ ID NO: 346, and the VL sequence as comprised in SEQ ID NO: 347 (felzartamab); cj) the VH sequence as comprised in SEQ ID NO: 348, and the VL sequence as comprised in SEQ ID NO: 349 (fianlimab); ck) the VH sequence as comprised in SEQ ID NO: 350, and the VL sequence as comprised in SEQ ID NO: 351 (finotonlimab); cl) the VH sequence as comprised in SEQ ID NO: 352, and the VL sequence as comprised in SEQ ID NO: 353 (geptanolimab); cm) the VH sequence as comprised in SEQ ID NO: 354, and the VL sequence as comprised in SEQ ID NO: 355 (gotistobart); cn) the VH sequence as comprised in SEQ ID NO: 356, and the VL sequence as comprised in SEQ ID NO: 357 (ivuxolimab); co) the VH sequence as comprised in SEQ ID NO: 358, and the VL sequence as comprised in SEQ ID NO: 359 (lemzoparlimab); cp) the VH sequence as comprised in SEQ ID NO: 360, and the VL sequence as comprised in SEQ ID NO: 361 (luveltamab); cq) the VH sequence as comprised in SEQ ID NO: 362, and the VL sequence as comprised in SEQ ID NO: 363 (magrolimab); cr) the VH sequence as comprised in SEQ ID NO: 364, and the VL sequence as comprised in SEQ ID NO: 365 (mecbotamab); cs) the VH sequence as comprised in SEQ ID NO: 366, and the VL sequence as comprised in SEQ ID NO: 367 (monalizumab); ct) the VH sequence as comprised in SEQ ID NO: 368, and the VL sequence as comprised in SEQ ID NO: 369 (nofazinlimab); cu) the VH sequence as comprised in SEQ ID NO: 370, and the VL sequence as comprised in SEQ ID NO: 371 (nurulimab); cv) the VH sequence as comprised in SEQ ID NO: 372, and the VL sequence as comprised in SEQ ID NO: 373 (ociperlimab); cw) the VH sequence as comprised in SEQ ID NO: 374, and the VL sequence as comprised in SEQ ID NO: 375 (oleclumab); ex) the VH sequence as comprised in SEQ ID NO: 376, and the VL sequence as comprised in SEQ ID NO: 377 (onfekafusp); cy) the VH sequence as comprised in SEQ ID NO: 378, and the VL sequence as comprised in SEQ ID NO: 379 (patritumab); cz) the VH sequence as comprised in SEQ ID NO: 380, and the VL sequence as comprised in SEQ ID NO: 381 (pivekimab); da) the VH sequence as comprised in SEQ ID NO: 382, and the VL sequence as comprised in SEQ ID NO: 383 (quavonlimab); db) the VH sequence as comprised in SEQ ID NO: 384, and the VL sequence as comprised in SEQ ID NO: 385 (retlirafusp); de) the VH sequence as comprised in SEQ ID NO: 386, and the VL sequence as comprised in SEQ ID NO: 387 (rosopatamab); dd) the VH sequence as comprised in SEQ ID NO: 388, and the VL sequence as comprised in SEQ ID NO: 389 (rulonilimab); de) the VH sequence as comprised in SEQ ID NO: 390, and the VL sequence as comprised in SEQ ID NO: 391 (sabatolimab); df) the VH sequence as comprised in SEQ ID NO: 392, and the VL sequence as comprised in SEQ ID NO: 393 (sasanlimab); dg) the VH sequence as comprised in SEQ ID NO: 394, and the VL sequence as comprised in SEQ ID NO: 395 (telisotuzumab); dh) the VH sequence as comprised in SEQ ID NO: 396, and the VL sequence as comprised in SEQ ID NO: 397 (tiragolumab); di) the VH sequence as comprised in SEQ ID NO: 398, and the VL sequence as comprised in SEQ ID NO: 399 (tusamitamab); dj) the VH sequence as comprised in SEQ ID NO: 400, and the VL sequence as comprised in SEQ ID NO: 401 (vibostolimab); dk) the VH sequence as comprised in SEQ ID NO: 402, and the VL sequence as comprised in SEQ ID NO: 403 (vobramitamab); dl) the VH sequence as comprised in SEQ ID NO: 404, and the VL sequence as comprised in SEQ ID NO: 405 (zilovertamab); dm) the VH sequence as comprised in SEQ ID NO: 406, and the VL sequence as comprised in SEQ ID NO: 407 (suvemcitug); dn) the VH sequence as comprised in SEQ ID NO: 408, and the VL sequence as comprised in SEQ ID NO: 409 (becotatug); do) the VH sequence as comprised in SEQ ID NO: 410, and the VL sequence as comprised in SEQ ID NO: 411 (tifcemalimab); dq) the VH sequence as comprised in SEQ ID NO: 412, and the VL sequence as comprised in SEQ ID NO: 413 (blinatumomab - CD19); dr) the VH sequence as comprised in SEQ ID NO: 414, and the VL sequence as comprised in SEQ ID NO: 415 (blinatumomab - CD3); ds) the VH sequence as comprised in SEQ ID NO: 416, and the VL sequence as comprised in SEQ ID NO: 417 (cadonilimab - PD-1); dt) the VH sequence as comprised in SEQ ID NO: 418, and the VL sequence as comprised in SEQ ID NO: 419 (cadonilimab - CTLA4); du) the VH sequence as comprised in SEQ ID NO: 420, and the VL sequence as comprised in SEQ ID NO: 421 (disitamab); dv) the VH sequence as comprised in SEQ ID NO: 422, and the VL sequence as comprised in SEQ ID NO: 423 (edrecolomab); dw) the VH sequence as comprised in SEQ ID NO: 424, and the VL sequence as comprised in SEQ ID NO: 425 (elranatamab - BCMA); dx) the VH sequence as comprised in SEQ ID NO: 426, and the VL sequence as comprised in SEQ ID NO: 427 (elranatamab - DC3); dy) the VH sequence as comprised in SEQ ID NO: 428, and the VL sequence as comprised in SEQ ID NO: 429 (epcoritamab - CD20); dz) the VH sequence as comprised in SEQ ID NO: 430, and the VL sequence as comprised in SEQ ID NO: 431 (epcoritamab

[0184] - CD3); ea) the VH sequence as comprised in SEQ ID NO: 432, and the VL sequence as comprised in SEQ ID NO: 433 (glofitamab - VH1 / VL1); eb); the VH sequence as comprised in SEQ ID NO: 434, and the VL sequence as comprised in SEQ ID NO: 435 (glofitamab - VH2 / VL2); ec) the VH sequence as comprised in SEQ ID NO: 436, and the VL sequence as comprised in SEQ ID NO: 437 (glofitamab - VH3 / VL3); ed) the VH sequence as comprised in SEQ ID NO: 438, and the VL sequence as comprised in SEQ ID NO: 439 (mosunetuzumab - CD20); ef) the VH sequence as comprised in SEQ ID NO: 440, and the VL sequence as comprised in SEQ ID NO: 441 (mosunetuzumab - CD3); eg) the VH sequence as comprised in SEQ ID NO: 442, and the VL sequence as comprised in SEQ ID NO: 443 (talquetamab - GPCR5D); eh) the VH sequence as comprised in SEQ ID NO: 444, and the VL sequence as comprised in SEQ ID NO: 445 (talquetamab

[0185] - CD3); ei) the VH sequence as comprised in SEQ ID NO: 446, and the VL sequence as comprised in SEQ ID NO: 447 (teclistamab - BCMA); ej) the VH sequence as comprised in SEQ ID NO: 448, and the VL sequence as comprised in SEQ ID NO: 449 (teclistamab - CD3); ek) the VH sequence as comprised in SEQ ID NO: 450, and the VL sequence as comprised in SEQ ID NO: 451 (tositumomab); el) the VH sequence as comprised in SEQ ID NO: 452, and the VL sequence as comprised in SEQ ID NO: 453 (tremelimumab); em) the VH sequence as comprised in SEQ ID NO: 454, and the VL sequence as comprised in SEQ ID NO: 455 (zimberelimab); en) the VH sequence as comprised in SEQ ID NO: 456, and the VL sequence as comprised in SEQ ID NO: 457 (odronextamab - CD20); eo) the VH sequence as comprised in SEQ ID NO: 458, and the VL sequence as comprised in SEQ ID NO: 459 (odronextamab - CD3); ep) the VH sequence as comprised in SEQ ID NO: 460, and the VL sequence as comprised in SEQ ID NO: 461 (ivonescimab

[0186] - PD-1); eq) the VH sequence as comprised in SEQ ID NO: 462, and the VL sequence as comprised in SEQ ID NO: 463 (ivonescimab - VEGF); er) the VH sequence as comprised in SEQ ID NO: 464, and the VL sequence as comprised in SEQ ID NO: 465 (anbenitamab - VH1 / VL1); es) the VH sequence as comprised in SEQ ID NO: 466, and the VL sequence as comprised in SEQ ID NO: 467 (anbenitamab - VH2 / VL2); et) the VH sequence as comprised in SEQ ID NO: 468, and the VL sequence as comprised in SEQ ID NO: 469 (izalontamab - EGFR); eu) the VH sequence as comprised in SEQ ID NO: 470, and the VL sequence as comprised in SEQ ID NO: 471 (izalontamab

[0187] - HER3); ev) the VH sequence as comprised in SEQ ID NO: 472, and the VL sequence as comprised in SEQ ID NO: 473 (linvoseltamab - BCMA); ew) the VH sequence as comprised in SEQ ID NO: 474, and the VL sequence as comprised in SEQ ID NO: 475 (linvoseltamab - CD3); ex) the VH sequence as comprised in SEQ ID NO: 476, and the VL sequence as comprised in SEQ ID NO: 477 (tarlatamab - DLL3); ey) the VH sequence as comprised in SEQ ID NO: 478, and the VL sequence as comprised in SEQ ID NO: 479 (tarlatamab - CD3); ez) the VH sequence as comprised in SEQ ID NO: 480, and the VL sequence as comprised in SEQ ID NO: 481 (zanidatamab - VH1 / VL1); fa) the VH sequence as comprised in SEQ ID NO: 482, and the VL sequence as comprised in SEQ ID NO: 483 (zanidatamab - VH2 / VL2); fb) the VH sequence as comprised in SEQ ID NO: 484, and the VL sequence as comprised in SEQ ID NO: 485 (volrustomig - PD-1); fc) the VH sequence as comprised in SEQ ID NO: 486, and the VL sequence as comprised in SEQ ID NO: 487 (volrustomig - CTLA-4); fd) the VH sequence as comprised in SEQ ID NO: 488, and the VL sequence as comprised in SEQ ID NO: 489 (zenocutuzumab - HER3); fe) the VH sequence as comprised in SEQ ID NO: 490, and the VL sequence as comprised in SEQ ID NO: 491 (zenocutuzumab - HER2); ft) the VH sequence as comprised in SEQ ID NO: 492, and the VL sequence as comprised in SEQ ID NO: 493 (botensilimab - VH1 / VL1); fg) the VH sequence as comprised in SEQ ID NO: 494, and the VL sequence as comprised in SEQ ID NO: 495 (botensilimab

[0188] - VH2 / VL2); fh) the VH sequence as comprised in SEQ ID NO: 496, and the VL sequence as comprised in SEQ ID NO: 497 (izalontamab - EGFR); fi) the VH sequence as comprised in SEQ ID NO: 498, and the VL sequence as comprised in SEQ ID NO: 499 (izalontamab - HER3); fj) the VH sequence as comprised in SEQ ID NO: 500, and the VL sequence as comprised in SEQ ID NO: 501 (rilvegostomig - TIGIT); fk) the VH sequence as comprised in SEQ ID NO: 502, and the VL sequence as comprised in SEQ ID NO: 503 (rilvegostomig - PD-1); fl) the VH sequence as comprised in SEQ ID NO: 701 , and the VL sequence as comprised in SEQ ID NO: 700 (abagovomab); fm) the VH sequence as comprised in SEQ ID NO: 703, and the VL sequence as comprised in SEQ ID NO: 702 (abituzumab); fn) the VH sequence as comprised in SEQ ID NO: 705, and the VL sequence as comprised in SEQ ID NO: 704 (acasunlimab); fo) the VH sequence as comprised in SEQ ID NO: 707, and the VL sequence as comprised in SEQ ID NO: 706 (alnuctamab); fp) the VH sequence as comprised in SEQ ID NO: 709, and the VL sequence as comprised in SEQ ID NO: 708 (alomfilimab); fq) the VH sequence as comprised in SEQ ID NO: 711 , and the VL sequence as comprised in SEQ ID NO: 710 (amatuximab); fr) the VH sequence as comprised in SEQ ID NO: 713, and the VL sequence as comprised in SEQ ID NO: 712 (anetumab); fs) the VH sequence as comprised in SEQ ID NO: 715, and the VL sequence as comprised in SEQ ID NO: 714 (aplitabart); ft) the VH sequence as comprised in SEQ ID NO: 717, and the VL sequence as comprised in SEQ ID NO: 716 (atigotatug); fu) the VH sequence as comprised in SEQ ID NO: 719, and the VL sequence as comprised in SEQ ID NO: 718 (balstilimab); fv) the VH sequence as comprised in SEQ ID NO: 721 , and the VL sequence as comprised in SEQ ID NO: 720 (bavituximab); fw) the VH sequence as comprised in SEQ ID NO: 723, and the VL sequence as comprised in SEQ ID NO: 722 (bavunalimab); fx) the VH sequence as comprised in SEQ ID NO: 725, and the VL sequence as comprised in SEQ ID NO: 724 (belrestotug); fy) the VH sequence as comprised in SEQ ID NO: 727, and the VL sequence as comprised in SEQ ID NO: 726 (bermekimab); fz) the VH sequence as comprised in SEQ ID NO: 729, and the VL sequence as comprised in SEQ ID NO: 728 (bifikafusp); ga) the VH sequence as comprised in SEQ ID NO: 731 , and the VL sequence as comprised in SEQ ID NO: 730 (bintrafusp); gb) the VH sequence as comprised in SEQ ID NO: 733, and the VL sequence as comprised in SEQ ID NO: 732 (brenetafusp); gc) the VH sequence as comprised in SEQ ID NO: 735, and the VL sequence as comprised in SEQ ID NO: 734 (briquilimab); gd) the VH sequence as comprised in SEQ ID NO: 737, and the VL sequence as comprised in SEQ ID NO: 736 (brontictuzumab); ge) the VH sequence as comprised in SEQ ID NO: 739, and the VL sequence as comprised in SEQ ID NO: 738 (budigalimab); gf) the VH sequence as comprised in SEQ ID NO: 741 , and the VL sequence as comprised in SEQ ID NO: 740 (cabiralizumab); gg) the VH sequence as comprised in SEQ ID NO: 743, and the VL sequence as comprised in SEQ ID NO: 742 (canakinumab); gh) the VH sequence as comprised in SEQ ID NO: 745, and the VL sequence as comprised in SEQ ID NO: 744 (cantuzumab); gi) the VH sequence as comprised in SEQ ID NO: 747, and the VL sequence as comprised in SEQ ID NO: 746 (carlumab); gj) the VH sequence as comprised in SEQ ID NO: 749, and the VL sequence as comprised in SEQ ID NO: 748 (carotuximab); gk) the VH sequence as comprised in SEQ ID NO: 751 , and the VL sequence as comprised in SEQ ID NO: 750 (caxmotabart); gl) the VH sequence as comprised in SEQ ID NO: 753, and the VL sequence as comprised in SEQ ID NO: 752 (cergutuzumab); gm) the VH sequence as comprised in SEQ ID NO: 755, and the VL sequence as comprised in SEQ ID NO: 754 (cibisatamab); gn) the VH sequence as comprised in SEQ ID NO: 757, and the VL sequence as comprised in SEQ ID NO: 756 (cinrebafusp); go) the VH sequence as comprised in SEQ ID NO: 759, and the VL sequence as comprised in SEQ ID NO: 758 (cixutumumab); gp) the VH sequence as comprised in SEQ ID NO: 761 , and the VL sequence as comprised in SEQ ID NO: 760 (clazakizumab); gq) the VH sequence as comprised in SEQ ID NO: 763, and the VL sequence as comprised in SEQ ID NO: 762 (clivatuzumab); gr) the VH sequence as comprised in SEQ ID NO: 765, and the VL sequence as comprised in SEQ ID NO: 764 (cofetuzumab); gs) the VH sequence as comprised in SEQ ID NO: 767, and the VL sequence as comprised in SEQ ID NO: 766 (coltuximab); gt) the VH sequence as comprised in SEQ ID NO: 769, and the VL sequence as comprised in SEQ ID NO: 768 (conatumumab); gu) the VH sequence as comprised in SEQ ID NO: 771 , and the VL sequence as comprised in SEQ ID NO: 770 (dacetuzumab); gv) the VH sequence as comprised in SEQ ID NO: 773, and the VL sequence as comprised in SEQ ID NO: 772 (dalutrafusp); gw) the VH sequence as comprised in SEQ ID NO: 775, and the VL sequence as comprised in SEQ ID NO: 774 (danburstotug); gx) the VH sequence as comprised in SEQ ID NO: 777, and the VL sequence as comprised in SEQ ID NO: 776 (daratumumab); gy) the VH sequence as comprised in SEQ ID NO: 779, and the VL sequence as comprised in SEQ ID NO: 778 (demcizumab); gz) the VH sequence as comprised in SEQ ID NO: 781 , and the VL sequence as comprised in SEQ ID NO: 780 (denintuzumab); ha) the VH sequence as comprised in SEQ ID NO: 783, and the VL sequence as comprised in SEQ ID NO: 782 (denosumab); hb) the VH sequence as comprised in SEQ ID NO: 785, and the VL sequence as comprised in SEQ ID NO: 784 (depatuxizumab); he) the VH sequence as comprised in SEQ ID NO: 787, and the VL sequence as comprised in SEQ ID NO: 786 (drozitumab); hd) the VH sequence as comprised in SEQ ID NO: 789, and the VL sequence as comprised in SEQ ID NO: 788 (duligotuzumab); he) the VH sequence as comprised in SEQ ID NO: 791 , and the VL sequence as comprised in SEQ ID NO: 790 (dusigitumab); hf) the VH sequence as comprised in SEQ ID NO: 793, and the VL sequence as comprised in SEQ ID NO: 792 (duvortuxizumab); hg) the VH sequence as comprised in SEQ ID NO: 795, and the VL sequence as comprised in SEQ ID NO: 794 (elotuzumab); hh) the VH sequence as comprised in SEQ ID NO: 797, and the VL sequence as comprised in SEQ ID NO: 796 (eluvixtamab); hi) the VH sequence as comprised in SEQ ID NO: 799, and the VL sequence as comprised in SEQ ID NO: 798 (enapotamab); hj) the VH sequence as comprised in SEQ ID NO: 801 , and the VL sequence as comprised in SEQ ID NO: 800 (enoticumab); hk) the VH sequence as comprised in SEQ ID NO: 803, and the VL sequence as comprised in SEQ ID NO: 802 (epacmarstobart); hl) the VH sequence as comprised in SEQ ID NO: 805, and the VL sequence as comprised in SEQ ID NO: 804 (etentamig); hm) the VH sequence as comprised in SEQ ID NO: 807, and the VL sequence as comprised in SEQ ID NO: 806 (falbikitug); hn) the VH sequence as comprised in SEQ ID NO: 809, and the VL sequence as comprised in SEQ ID NO: 808 (faricimab); ho) the VH sequence as comprised in SEQ ID NO: 811 , and the VL sequence as comprised in SEQ ID NO: 810 (feladilimab); hp) the VH sequence as comprised in SEQ ID NO: 813, and the VL sequence as comprised in SEQ ID NO: 812 (ficerafusp); hq) the VH sequence as comprised in SEQ ID NO: 815, and the VL sequence as comprised in SEQ ID NO: 814 (ficlatuzumab); hr) the VH sequence as comprised in SEQ ID NO: 817, and the VL sequence as comprised in SEQ ID NO: 816 (figitumumab); hs) the VH sequence as comprised in SEQ ID NO: 819, and the VL sequence as comprised in SEQ ID NO: 818 (flanvotumab); ht) the VH sequence as comprised in SEQ ID NO: 821 , and the VL sequence as comprised in SEQ ID NO: 820 (flotetuzumab); hu) the VH sequence as comprised in SEQ ID NO: 823, and the VL sequence as comprised in SEQ ID NO: 822 (forimtamig); hv) the VH sequence as comprised in SEQ ID NO: 825, and the VL sequence as comprised in SEQ ID NO: 824 (futuximab); hw) the VH sequence as comprised in SEQ ID NO: 827, and the VL sequence as comprised in SEQ ID NO: 826 (ganitumab); hx) the VH sequence as comprised in SEQ ID NO: 829, and the VL sequence as comprised in SEQ ID NO: 828 (gevokizumab); hy) the VH sequence as comprised in SEQ ID NO: 831 , and the VL sequence as comprised in SEQ ID NO: 830 (girentuximab); hz) the VH sequence as comprised in SEQ ID NO: 833, and the VL sequence as comprised in SEQ ID NO: 832 (glembatumumab); ia) the VH sequence as comprised in SEQ ID NO: 835, and the VL sequence as comprised in SEQ ID NO: 834 (icrucumab); ib) the VH sequence as comprised in SEQ ID NO: 837, and the VL sequence as comprised in SEQ ID NO: 836 (ifinatamab); ic) the VH sequence as comprised in SEQ ID NO: 839, and the VL sequence as comprised in SEQ ID NO: 838 (iladatuzumab); id) the VH sequence as comprised in SEQ ID NO: 841 , and the VL sequence as comprised in SEQ ID NO: 840 (imalumab); ie) the VH sequence as comprised in SEQ ID NO: 843, and the VL sequence as comprised in SEQ ID NO: 842 (imgatuzumab); if) the VH sequence as comprised in SEQ ID NO: 845, and the VL sequence as comprised in SEQ ID NO: 844 (indusatumab); ig) the VH sequence as comprised in SEQ ID NO: 847, and the VL sequence as comprised in SEQ ID NO: 846 (inebilizumab); ih) the VH sequence as comprised in SEQ ID NO: 849, and the VL sequence as comprised in SEQ ID NO: 848 (ispectamab); ii) the VH sequence as comprised in SEQ ID NO: 851 , and the VL sequence as comprised in SEQ ID NO: 850 (istiratumab); ij) the VH sequence as comprised in SEQ ID NO: 853, and the VL sequence as comprised in SEQ ID NO: 852 (izeltabart); ik) the VH sequence as comprised in SEQ ID NO: 855, and the VL sequence as comprised in SEQ ID NO: 854 (izuralimab); il) the VH sequence as comprised in SEQ ID NO: 857, and the VL sequence as comprised in SEQ ID NO: 856 (landogrozumab); im) the VH sequence as comprised in SEQ ID NO: 859, and the VL sequence as comprised in SEQ ID NO: 858 (laprituximab); in) the VH sequence as comprised in SEQ ID NO: 861 , and the VL sequence as comprised in SEQ ID NO: 860 (lenzilumab); io) the VH sequence as comprised in SEQ ID NO: 863, and the VL sequence as comprised in SEQ ID NO: 862 (leronlimab); ip) the VH sequence as comprised in SEQ ID NO: 865, and the VL sequence as comprised in SEQ ID NO: 864 (lifastuzumab); iq) the VH sequence as comprised in SEQ ID NO: 867, and the VL sequence as comprised in SEQ ID NO: 866 (ligufalimab); ir) the VH sequence as comprised in SEQ ID NO: 869, and the VL sequence as comprised in SEQ ID NO: 868 (lilotomab); is) the VH sequence as comprised in SEQ ID NO: 871 , and the VL sequence as comprised in SEQ ID NO: 870 (lintuzumab); it) the VH sequence as comprised in SEQ ID NO: 873, and the VL sequence as comprised in SEQ ID NO: 872 (lirilumab); iu) the VH sequence as comprised in SEQ ID NO: 875, and the VL sequence as comprised in SEQ ID NO: 874 (livmoniplimab); iv) the VH sequence as comprised in SEQ ID NO: 877, and the VL sequence as comprised in SEQ ID NO: 876 (lorvotuzumab); iw) the VH sequence as comprised in SEQ ID NO: 879, and the VL sequence as comprised in SEQ ID NO: 878 (lucatumumab); ix) the VH sequence as comprised in SEQ ID NO: 881 , and the VL sequence as comprised in SEQ ID NO: 880 (lumretuzumab); iy) the VH sequence as comprised in SEQ ID NO: 883, and the VL sequence as comprised in SEQ ID NO: 882 (matuzumab); iz) the VH sequence as comprised in SEQ ID NO: 885, and the VL sequence as comprised in SEQ ID NO: 884 (mipasetamab); ja) the VH sequence as comprised in SEQ ID NO: 887, and the VL sequence as comprised in SEQ ID NO: 886 (modakafusp); jb) the VH sequence as comprised in SEQ ID NO: 889, and the VL sequence as comprised in SEQ ID NO: 888 (modotuximab); jc) the VH sequence as comprised in SEQ ID NO: 891 , and the VL sequence as comprised in SEQ ID NO: 890 (murlentamab); jd) the VH sequence as comprised in SEQ ID NO: 893, and the VL sequence as comprised in SEQ ID NO: 892 (nadunolimab); je) the VH sequence as comprised in SEQ ID NO: 895, and the VL sequence as comprised in SEQ ID NO: 894 (naptumomab); jf) the VH sequence as comprised in SEQ ID NO: 897, and the VL sequence as comprised in SEQ ID NO: 896 (narlumosbart); jg) the VH sequence as comprised in SEQ ID NO: 899, and the VL sequence as comprised in SEQ ID NO: 898 (narnatumab); jh) the VH sequence as comprised in SEQ ID NO: 901 , and the VL sequence as comprised in SEQ ID NO: 900 (navicixizumab); ji) the VH sequence as comprised in SEQ ID NO: 903, and the VL sequence as comprised in SEQ ID NO: 902 (nesvacumab); jj) the VH sequence as comprised in SEQ ID NO: 905, and the VL sequence as comprised in SEQ ID NO: 904 (nisevokitug); jk) the VH sequence as comprised in SEQ ID NO: 907, and the VL sequence as comprised in SEQ ID NO: 906 (omburtamab); jl) the VH sequence as comprised in SEQ ID NO: 909, and the VL sequence as comprised in SEQ ID NO: 908 (ontuxizumab); jm) the VH sequence as comprised in SEQ ID NO: 91 1 , and the VL sequence as comprised in SEQ ID NO: 910 (otlertuzumab); jn) the VH sequence as comprised in SEQ ID NO: 913, and the VL sequence as comprised in SEQ ID NO: 912 (pamrevlumab); jo) the VH sequence as comprised in SEQ ID NO: 915, and the VL sequence as comprised in SEQ ID NO: 914 (parsatuzumab); jp) the VH sequence as comprised in SEQ ID NO: 917, and the VL sequence as comprised in SEQ ID NO: 916 (pavurutamab); jq) the VH sequence as comprised in SEQ ID NO: 919, and the VL sequence as comprised in SEQ ID NO: 918 (pemivibart); jr) the VH sequence as comprised in SEQ ID NO: 921 , and the VL sequence as comprised in SEQ ID NO: 920 (petosemtamab); js) the VH sequence as comprised in SEQ ID NO: 923, and the VL sequence as comprised in SEQ ID NO: 922 (pimivalimab); jt) the VH sequence as comprised in SEQ ID NO: 925, and the VL sequence as comprised in SEQ ID NO: 924 (pinatuzumab); ju) the VH sequence as comprised in SEQ ID NO: 927, and the VL sequence as comprised in SEQ ID NO: 926 (plozalizumab); jv) the VH sequence as comprised in SEQ ID NO: 929, and the VL sequence as comprised in SEQ ID NO: 928 (pulocimab); jw) the VH sequence as comprised in SEQ ID NO: 931 , and the VL sequence as comprised in SEQ ID NO: 930 (ragifilimab); jx) the VH sequence as comprised in SEQ ID NO: 933, and the VL sequence as comprised in SEQ ID NO: 932 (raludotatug); jy) the VH sequence as comprised in SEQ ID NO: 935, and the VL sequence as comprised in SEQ ID NO: 934 (rilotumumab); jz) the VH sequence as comprised in SEQ ID NO: 937, and the VL sequence as comprised in SEQ ID NO: 936 (rosmantuzumab); ka) the VH sequence as comprised in SEQ ID NO: 939, and the VL sequence as comprised in SEQ ID NO: 938 (runimotamab); kb) the VH sequence as comprised in SEQ ID NO: 941 , and the VL sequence as comprised in SEQ ID NO: 940 (sabestomig); kc) the VH sequence as comprised in SEQ ID NO: 943, and the VL sequence as comprised in SEQ ID NO: 942 (selicrelumab); kd) the VH sequence as comprised in SEQ ID NO: 945, and the VL sequence as comprised in SEQ ID NO: 944 (seribantumab); ke) the VH sequence as comprised in SEQ ID NO: 947, and the VL sequence as comprised in SEQ ID NO: 946 (sigvotatug); kf) the VH sequence as comprised in SEQ ID NO: 949, and the VL sequence as comprised in SEQ ID NO: 948 (simlukafusp); kg) the VH sequence as comprised in SEQ ID NO: 951 , and the VL sequence as comprised in SEQ ID NO: 950 (simtuzumab); kh) the VH sequence as comprised in SEQ ID NO: 953, and the VL sequence as comprised in SEQ ID NO: 952 (sirexatamab); ki) the VH sequence as comprised in SEQ ID NO: 955, and the VL sequence as comprised in SEQ ID NO: 954 (sofituzumab); kj) the VH sequence as comprised in SEQ ID NO: 957, and the VL sequence as comprised in SEQ ID NO: 956 (spartalizumab); kk) the VH sequence as comprised in SEQ ID NO: 959, and the VL sequence as comprised in SEQ ID NO: 958 (surzebiclimab); kl) the VH sequence as comprised in SEQ ID NO: 961 , and the VL sequence as comprised in SEQ ID NO: 960 (tabalumab); km) the VH sequence as comprised in SEQ ID NO: 963, and the VL sequence as comprised in SEQ ID NO: 962 (tafolecimab); kn) the VH sequence as comprised in SEQ ID NO: 965, and the VL sequence as comprised in SEQ ID NO: 964 (talacotuzumab); ko) the VH sequence as comprised in SEQ ID NO: 967, and the VL sequence as comprised in SEQ ID NO: 966 (tarextumab); kp) the VH sequence as comprised in SEQ ID NO: 969, and the VL sequence as comprised in SEQ ID NO: 968 (tavolimab); kq) the VH sequence as comprised in SEQ ID NO: 971 , and the VL sequence as comprised in SEQ ID NO: 970 (tebotelimab); kr) the VH sequence as comprised in SEQ ID NO: 973, and the VL sequence as comprised in SEQ ID NO: 972 (teprotumumab); ks) the VH sequence as comprised in SEQ ID NO: 975, and the VL sequence as comprised in SEQ ID NO: 974 (tidutamab); kt) the VH sequence as comprised in SEQ ID NO: 977, and the VL sequence as comprised in SEQ ID NO: 976 (tigatuzumab); ku) the VH sequence as comprised in SEQ ID NO: 979, and the VL sequence as comprised in SEQ ID NO: 978 (tilvestamab); kv) the VH sequence as comprised in SEQ ID NO: 981 , and the VL sequence as comprised in SEQ ID NO: 980 (tobemstomig); kw) the VH sequence as comprised in SEQ ID NO: 983, and the VL sequence as comprised in SEQ ID NO: 982 (tocilizumab); kx) the VH sequence as comprised in SEQ ID NO: 985, and the VL sequence as comprised in SEQ ID NO: 984 (tomaralimab); ky) the VH sequence as comprised in SEQ ID NO: 987, and the VL sequence as comprised in SEQ ID NO: 986 (tovecimig); kz) the VH sequence as comprised in SEQ ID NO: 989, and the VL sequence as comprised in SEQ ID NO: 988 (tovetumab); la) the VH sequence as comprised in SEQ ID NO: 991 , and the VL sequence as comprised in SEQ ID NO: 990 (tucotuzumab); lb) the VH sequence as comprised in SEQ ID NO: 993, and the VL sequence as comprised in SEQ ID NO: 992 (tuparstobart); Ic) the VH sequence as comprised in SEQ ID NO: 995, and the VL sequence as comprised in SEQ ID NO: 994 (upifitamab); Id) the VH sequence as comprised in SEQ ID NO: 997, and the VL sequence as comprised in SEQ ID NO: 996 (urabrelimab); le) the VH sequence as comprised in SEQ ID NO: 999, and the VL sequence as comprised in SEQ ID NO: 998 (utomilumab); If) the VH sequence as comprised in SEQ ID NO: 1001 , and the VL sequence as comprised in SEQ ID NO: 1000 (vadastuximab); Ig) the VH sequence as comprised in SEQ ID NO: 1003, and the VL sequence as comprised in SEQ ID NO: 1002 (vandortuzumab); Ih) the VH sequence as comprised in SEQ ID NO: 1005, and the VL sequence as comprised in SEQ ID NO: 1004 (vanucizumab); li) the VH sequence as comprised in SEQ ID NO: 1007, and the VL sequence as comprised in SEQ ID NO: 1006 (veligrotug); Ij) the VH sequence as comprised in SEQ ID NO: 1009, and the VL sequence as comprised in SEQ ID NO: 1008 (verzistobart); Ik) the VH sequence as comprised in SEQ ID NO: 1011 , and the VL sequence as comprised in SEQ ID NO: 1010 (vesencumab); II) the VH sequence as comprised in SEQ ID NO: 1013, and the VL sequence as comprised in SEQ ID NO: 1012 (vofatamab); Im) the VH sequence as comprised in SEQ ID NO: 1015, and the VL sequence as comprised in SEQ ID NO: 1014 (vonlerolizumab); In) the VH sequence as comprised in SEQ ID NO: 1017, and the VL sequence as comprised in SEQ ID NO: 1016 (vopikitug); Io) the VH sequence as comprised in SEQ ID NO: 1019, and the VL sequence as comprised in SEQ ID NO: 1018 (vorsetuzumab); Ip) the VH sequence as comprised in SEQ ID NO: 1021 , and the VL sequence as comprised in SEQ ID NO: 1020 (xaluritamig); Iq) the VH sequence as comprised in SEQ ID NO: 1023, and the VL sequence as comprised in SEQ ID NO: 1022 (zalutumumab); Ir) the VH sequence as comprised in SEQ ID NO: 1025, and the VL sequence as comprised in SEQ ID NO: 1024 (zanolimumab); Is) the VH sequence as comprised in SEQ ID NO: 1027, and the VL sequence as comprised in SEQ ID NO: 1026 (ivuxolimab-alt); It) the VH sequence as comprised in SEQ ID NO: 1029, and the VL sequence as comprised in SEQ ID NO: 1028 (inotuzumab-alt); lu) the VH sequence as comprised in SEQ ID NO: 1031 , and the VL sequence as comprised in SEQ ID NO: 1030 (moxetumomab-alt); Iv) the VH sequence as comprised in SEQ ID NO: 1033, and the VL sequence as comprised in SEQ ID NO: 1032 (luveltamab-alt); Iw) the VH sequence as comprised in SEQ ID NO: 1035, and the VL sequence as comprised in SEQ ID NO: 1034 (ibritumomab-alt); lx) the VH sequence as comprised in SEQ ID NO: 1037, and the VL sequence as comprised in SEQ ID NO: 1036 (pivekimab-alt); ly) the VH sequence as comprised in SEQ ID NO: 1039, and the VL sequence as comprised in SEQ ID NO: 1038 (avelumab-alt); Iz) the VH sequence as comprised in SEQ ID NO: 1041 , and the VL sequence as comprised in SEQ ID NO: 1040 (sugemalimab-alt); ma) the VH sequence as comprised in SEQ ID NO: 1043, and the VL sequence as comprised in SEQ ID NO: 1042 (nimotuzumab-alt); mb) the VH sequence as comprised in SEQ ID NO: 1045, and the VL sequence as comprised in SEQ ID NO: 1044 (panitumumab-alt); me) the VH sequence as comprised in SEQ ID NO: 540, and the VL sequence as comprised in SEQ ID NO: 541 (AR46A6); md) the VH sequence as comprised in SEQ ID NO: 542, and the VL sequence as comprised in SEQ ID NO: 543 (KM4097); and me) the VH sequence as comprised in SEQ ID NO: 544, and the VL sequence as comprised in SEQ ID NO: 545 (K5-70).

[0189] In one embodiment therefore, an antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an immunoglobulin single variable domain (ISVD) comprising a VHH domain selected from the group consisting of: a) the VHH sequence as comprised in SEQ ID NO: 504 (envafolimab); b) the VHH sequence as comprised in SEQ ID NO: 505 (erfonrilimab - PD-L1); c) the VHH sequence as comprised in SEQ ID NO: 506 (erfonrilimab - CTLA-4); and, d) the VHH sequence as comprised in SEQ ID NO: 507 (ozekibart).

[0190] Antigen-binding regions that specifically bind an NK cell activating receptor

[0191] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one or a second antigen-binding region that specifically binds an NK cell activating receptor. In one embodiment, the antigen-binding region that specifically binds an NK cell activating receptor is an agonistic antigen-binding region that activates the NK cell receptor. Preferably, the antigen-binding region comprises at least one immunoglobulin variable region, more preferably, the immunoglobulin variable region comprises or consists of a Fab or an immunoglobulin single variable domain (ISVD). In one embodiment, the antigen-binding region is a human or humanized antigen-binding region.

[0192] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises two antigen-binding regions that specifically bind an NK cell activating receptor. The two antigen-binding regions can bind the same NK cell activating receptor orthey can bind at least two different NK cell activating receptors. In one embodiment, the two antigen binding regions are identical. In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein is an antigen binding protein wherein the NK cell activating receptor (that is bound by the antigen-binding region) is selected from the group consisting of: NKp46, NKp30, NKG2D, CD16A, SLAMF7, NKp44, CD94-NKG2C / E, KIR2DS1 , KIR2DS3, KIR2DS4, KIR2DS5, KIR2DS2, KIR2DL4, KIR3DS1 , CD160, NKp80, DNAM1 , 2B4, CRACC, 4-BB, 0X40, CRTAM, CD27, PSGL1 , CD96, CD100, CEACAM1 , CD59, PD-L1 , Tim3 and NTB-A, of which NKp46, NKp30, NKG2D, CD16A, and SLAMF7 are preferred.

[0193] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises the antigen-binding region that specifically binds an NK cell activating receptor that is a natural cytotoxicity receptor (NCR). Natural cytotoxicity receptors are type 1 transmembrane proteins of the immunoglobulin superfamily, which upon stimulation mediate NK killing and release of IFNy. They bind viral ligands such as hemagglutinins and hemagglutinin neuraminidases, some bacterial ligands and cellular ligands related to tumor growth such as PCNA. Natural Cytotoxicity Receptors include NKp46, NKp44, and NKp30. In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises the antigen-binding region that specifically binds an NK cell activating receptor that is an NCR selected from the group consisting of NKp46, NKp44 and NKp30.

[0194] “NKp46” refers to a protein or polypeptide encoded by an Ncr1 gene or by a cDNA prepared from such a gene. NKp46 has also been designated as NCR1 , CD335 (cluster of differentiation, NKP46, NK-p46, and LY94. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp46 polypeptide (e.g., an NKp46 polypeptide that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 50, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 304 amino acid residue sequence of human NKp46 (isoform a) is shown in in SEQ ID NO: 50, which corresponds to NCBI accession number NP_004820, the disclosure of which is incorporated herein by reference. The human NKp46 mRNA sequence is described in NCBI accession number NM_004829, the disclosure of which is incorporated herein by reference.

[0195] “NKp44” refers to a protein or polypeptide encoded by an Ncr2 gene or by a cDNA prepared from such a gene. NKp44 has also been designated as NCR2, CD336 (cluster of differentiation

[0196] 336), NKP44, NK-p44, LY95, and dJ149M18.1. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp44 polypeptide (e.g., an NKp44 polypeptide that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 51 , or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 276 amino acid residue sequence of human NKp46 is shown in SEQ ID NO: 51 , which corresponds to NCBI accession number NP_004819, the disclosure of which is incorporated herein by reference. The human NKp46 mRNA sequence is described in NCBI accession number NM_004828, the disclosure of which is incorporated herein by reference.

[0197] “NKp30” refers to a protein or polypeptide encoded by an Ncr3 gene or by a cDNA prepared from such a gene. NKp30 has also been designated as NCR3 and CD337 (cluster of differentiation

[0198] 337). Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKp30 polypeptide (e.g., an NKp30 polypeptide that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO:52, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 201 amino acid residue sequence of human NKp30 is shown in below in SEQ ID NO: 52, which corresponds to NCBI accession number NP_667341 , the disclosure of which is incorporated herein by reference. The human NKp30 mRNA sequence is described in NCBI accession number NM_147130, the disclosure of which is incorporated herein by reference.

[0199] NKG2D is an activating receptor (transmembrane protein) belonging to the NKG2 family of C-type lectin-like receptors. NKG2D is encoded by KLRK1 gene in humans. NKG2D recognizes induced-self proteins from MIC and RAET1 / ULBP families which appear on the surface of stressed, malignant transformed, and infected cells. “NKG2D” refers to a protein or polypeptide encoded by a KLRK1 gene or by a cDNA prepared from such a gene. NKG2D has also been designated as KLRK1 , CD314 (cluster of differentiation 314), D12S2489E, KLR, NKG2-D, natural killer group 2D, killer cell lectin-like receptor K1 , killer cell lectin like receptor K1 . Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term NKG2D polypeptide (e.g., an NKG2D polypeptide that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 53, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 216 amino acid residue sequence of human NKG2D is shown in SEQ ID NO: 53, which corresponds to NCBI accession number NP_001186734, the disclosure of which is incorporated herein by reference. The human NKG2D mRNA sequence is described in NCBI accession number NM_007360, the disclosure of which is incorporated herein by reference.

[0200] DNAM-1 is a ~65 kDa glycoprotein expressed on the surface of amongst others NK cells. It is a member of the immunoglobulin superfamily containing 2 Ig-like domains of the V-set. DNAM-1 mediates cellular adhesion to other cells bearing its ligands, CD1 12 and CD155, and cross-linking DNAM-1 with antibodies causes cellular activation. “DNAM-1 ” refers to a protein or polypeptide encoded by a KLRK1 gene or by a cDNA prepared from such a gene. DNAM-1 has also been designated as CD226 (cluster of differentiation 226), DNAM-1 , DNAM1 , PTA1 and TLiSAI . Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term DNAM-1 polypeptide (e.g., an DNAM-1 polypeptide that is at least 90%, at least 95%, at least 98% or 99% identical to SEQ ID NO: 54, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 336 amino acid residue sequence of human DNAM-1 is shown in SEQ ID NO: 54, which corresponds to NCBI accession number NP_006557, the disclosure of which is incorporated herein by reference. The human DNAM-1 mRNA sequence is described in NCBI accession number NM_006566, the disclosure of which is incorporated herein by reference.

[0201] As indicated above, CD16A is an immunoglobulin gamma Fc region receptor (FcyRllla) that is expressed on NK cells and through which NK cells recognize IgG that is bound to the surface of a pathogen-infected or TAA-expressing target cell. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term CD16A polypeptide (e.g., an CD16A polypeptide that is at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 55, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 254 amino acid residue sequence of human CD16A is shown in SEQ ID NO: 55, which corresponds to UniProt accession no. P08637, the disclosure of which is incorporated herein by reference.

[0202] “SLAMF7” is a protein that in humans is encoded by the human SLAMF7 gene. Isoform 1 SLAMF7 mediates NK cell activation through a SH2D1A-independent extracellular signal-regulated ERK-mediated pathway. SLAMF7 has also been designated as CD319 (cluster of differentiation 319), 19A, CRACC, and CS1. Any naturally occurring isoform, allele, ortholog or variant is encompassed by the term SLAMF7 polypeptide (e.g., a SLAMF7 polypeptide that is at least 90%, at least 95%, at least 98%, at least or 99% or 100% identical to SEQ ID NO: 56, or a contiguous sequence of at least 20, at least 30, at least 50, at least 100 or at least 200 amino acid residues thereof). The 335 amino acid residue sequence of human SLAMF7 is shown in SEQ ID NO: 56, which corresponds to UniProt accession no. Q9NQ25-1 , the disclosure of which is incorporated herein by reference.

[0203] In one embodiment of an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein, the antigen-binding region that specifically binds an NK cell activating receptor is an agonistic antigen-binding region that activates the NK cell receptor. As used herein, an antigen-binding region that has “agonist” activity at an NK cell activating receptor is an agent that can cause or increase "signaling by the NK cell activating receptor". "Signaling by the NK cell activating receptor" refers to an ability of an NK cell activating receptor to activate or transduce an intracellular signaling pathway. Changes in NK cell activating receptor-signaling activity can be measured, for example, by assays designed to measure changes in NK cell activating receptorsignaling 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 NK cell activating receptorsensitive promoters and enhancers, or indirectly by a downstream effect mediated by the NK cell activating receptor polypeptide (e.g. activation of specific cytolytic machinery in NK cells). Reporter genes can be naturally occurring genes (e.g. monitoring cytokine production) or they can be genes artificially introduced into a cell. Other genes can be placed under the control of such regulatory elements and thus serve to report the level of NK cell activating receptor-signaling activity.

[0204] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises at least one antigen-binding region that is obtained / obtainable from a monoclonal antibody against NK cell activating receptor 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 NK cell activating receptor 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 that are obtained / obtainable from a monoclonal antibody against NK cell activating receptor as is known in the art. Many examples of monoclonal antibodies against NK cell activating receptor have been described in the art. Anti- NKp46 monoclonal antibodies are described WO 201 1 / 086179, WO 2016 / 209021 and in Gauthier et al. (2019, Cell 177, 1701-1713) or in WO 2016 / 207278, such as NKp46-1 , -2, -3, -4, -6 or -9. Anti-NKG2D monoclonal antibodies described WO 2009 / 077483, WO 2018 / 148447, WO 2019 / 157366, WO 2018 / 148445, WO 2018 / 152518 and WO 2019 / 195409, which include the heavy and light chain sequences of SEQ ID NO.’s: 16 and 20, respectively. Monoclonal antibodies against NKG2A are e.g. described in WO 2008 / 009545, WO 2009 / 092805, WO 2016 / 032334, WO 2020 / 094071 and WO 2020 / 102501 . Monoclonal antibodies against NKp30 are e.g. described in WO 2020 / 172605. Monoclonal antibodies against DNAM-1 are e.g. described in WO 2013 / 140787. Examples of anti-SLAMF7 monoclonal antibodies include Elotuzumab and others described in US2018208653. Monoclonal antibodies against 4-1 BB (CD137) are e.g. described in WO 2005 / 035584, WO 2006 / 088464 and US2006188439. Monoclonal antibodies against 0X40 are e.g. described in WO 2007 / 062245, US2010136030, US2019100596, WO 2013 / 008171 and WO 2013 / 028231 . Monoclonal antibodies against CD96 are e.g. described in WO 2019 / 091449. Monoclonal antibodies against CD160 are e.g. described in US2012003224 and US2013122006. Monoclonal antibodies against KIR2DS1-5 are e.g. described in WO 2016 / 031936.

[0205] Antigen binding regions binding to y<5 T cell receptors

[0206] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein, can further comprise a third antigen-binding region, which is an antigen-binding region specifically binds an epitope of a y6 T cell receptor (TCR).

[0207] The third antigen-binding region as used in an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as herein defined above.

[0208] 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 10-3M or 10-4M, as may be determined as herein described above.

[0209] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as 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.

[0210] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises a third antigen-binding region that specifically binds to a y6 TCR, preferably to any y6 TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / de rived 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: IM1571 U, Beckman Coulter Life Sci.), and clone 11 F2 (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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein.

[0211] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises a third antigen-binding region that specifically binds to a V62 TCR, which antigen-binding region comprises CDR amino acid sequences that are obtained / de rived 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein.

[0212] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein 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 / de rived 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein.

[0213] In a preferred embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as 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 antigenbinding 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.

[0214] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein 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 / de rived 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 an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein.

[0215] In one embodiment, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprises a third antigen-binding region that specifically binds an activating epitope of a y6 T cell. In a preferred embodiment, the third antigen-binding region specifically binds an activating epitope in the V region of a V61 chain of a y6 TCR.

[0216] 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 y6 T cell population, preferably the V61 + T cell population. Accordingly, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprising a third antigen-binding region that specifically binds an activating epitope of a y6 T cell, can be used to modulate y6 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 y6 TCR. In an additional or alternative embodiment, binding of the activating epitope by the third antigen-binding region activates degranulation of the y6 T cell. In a further additional or alternative embodiment, binding of the activating epitope by the third antigen-binding region promotes y6 T cell mediated killing of cells expressing the antigen (e.g. TAA) targeted by the antigen binding protein.

[0217] In one embodiment, an activating epitope of TRDV1 is one that, upon being bound by a third antigen-binding region in an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein, results in down-regulation of the receptor and optionally activates the V61 cell. In some embodiments said down-regulation 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 V61 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 V61 cell, for example CD107a 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.

[0218] 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 V61 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 V61 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, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein 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. V61 cells) represent only a small number of the total T-cell population, an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein can be used to selectively (and indirectly) downregulate proteins in the TCR complex, such as CD3, by only downregulating them in V61 cells.

[0219] 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 down regulating CD3 molecules not associated with said TRDV1 TCR complex.

[0220] 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 V61 chain of a y6 TCR.

[0221] 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 V61 chain of the y6 TCR, in particular not in the CDR3 of the V61 chain. In a preferred embodiment, a third antigen-binding region binds an epitope that is located within the non-variable region of the V61 chain of the y6 TCR. It will be appreciated that such binding allows for the unique recognition of the V61 chain without the restriction to the sequences of the TCR which are highly variable (in particular CDR3). Various y6 TCR complexes which recognize antigen may be recognized in this way, solely by presence of the V61 chain. As such, it will be appreciated that any V61 chain-comprising y6 TCR may be recognized using an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein, irrespective of the specificity of the y6 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: 510, e.g. the portions of the V61 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: 510. In some embodiments, the third antigen-binding region binds an the epitope that comprises amino acids in the TRDV-1 CDR2 sequence.

[0222] In a similar manner to the well characterized ap T cells, y6 T cells utilize a distinct set of somatically rearranged variable (V), diversity (D), joining (J), and constant (C) genes, although y6 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 V61 chain or in the C-region of the V61 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 V61 chain. The third antigen-binding region may therefore only bind in the V region of the V61 chain. Thus, in one embodiment, the epitope consists of an epitope in the V region of the y6 TCR (e.g. amino acid residues 1-90 of SEQ ID NO: 510).

[0223] Reference to the epitope is made in relation to the V61 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: 510. SEQ ID NO: 510 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: 510, CDR2 is defined as amino acid residues 50-54 of SEQ ID NO: 510, and CDR3 is defined as amino acid residues 93-104 of SEQ ID NO: 510 (Xu et al., PNAS USA 108(6):2414-2419 (201 1)).

[0224] Therefore, in one embodiment, there is provided an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprising a third antigen-binding region that binds the epitope of a y6 TCR with a binding affinity (KD) as measured by surface plasmon resonance of less than 1.5 x 10-7M (i.e.150 nM). In one embodiment, the KD of the third antigen-binding region for the epitope of a y6 TCR is (with increasing preference) 1 .5 x 10-7M (i.e.150 nM) or less, 1 .3 x

[0225] 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

[0226] 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

[0227] (i.e. 2 nM) or less, or 1 x 10-9M (i.e. 1 nM) or less.

[0228] In one embodiment, there is provided an antigen binding protein in a conjugate with a 4-1 BBL ECD mutein as described herein comprising a third antigen-binding region that binds the epitope of a y6 TCR with a binding affinity (KD) as measured by surface plasmon resonance of higher than 1 .5 x 10-7M (i.e.150 nM). In one embodiment, the KD of the third antigen-binding region for the epitope of a y6 TCR is (with increasing preference) 1 .5 x 10-7M (i.e.150 nM) ...

Claims

Claims1 . A mutein of a 4-1 BB ligand (4-1 BBL) extracellular domain (ECD), wherein the 4-1 BBL ECD mutein exhibits a binding affinity for human 4-1 BB, expressed in pKo, that is at least 1 .0 lower than the pKo of a wild-type 4-1 BBL ECD for human 4-1 BB, wherein the 4-1 BBL ECD mutein, when present as part of a homotrimer of the 4-1 BBL ECD mutein in a conjugate with an antibody that specifically binds a tumor-associated antigen (TAA), which conjugate further comprises a human IL-21 , 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 tumor cells expressing the TAA, which proliferation is not less than 75% of the proliferation induced by a corresponding control conjugate comprising a trimer of wild type 4-1 BBL ECD in the same assay, wherein the amino acid sequence of the 4-1 BBL ECD mutein differs from a wild type human 4-1 BBL ECD amino acid sequence of SEQ ID NO: 37 in that the 4-1 BBL ECD mutein comprises at least one substitution selected from the group consisting of: A154D, A154E, a combination of A154D and G155Q, V153Q, Q227E, L101 N, Y110Q, Q230K, and V100Q.

2. A 4-1 BBL ECD mutein according to claim 1 , wherein the 4-1 BBL ECD mutein comprises no other amino acid sequence modification than the at least one substitution selected from the group consisting of: A154D, A154E, a combination of A154D and G155Q, V153Q, Q227E, L101 N, Y110Q, Q230K, and V100Q.

3. A fusion protein comprising three 4-1 BBL ECD monomers, wherein one, two or three of the monomers are a 4-1 BBL ECD mutein according to claim 1 or 2, wherein the three 4-1 BBL ECD monomers are fused together in a single polypeptide chain, and wherein, optionally, the three 4-1 BBL ECD monomers are connected by polypeptide linkers.

4. A conjugate comprising: i) a 4-1 BBL ECD mutein according to claim 1 or 2, or a fusion protein according to claim 3; and ii) a heterologous moiety, wherein preferably, the heterologous moiety comprises a polypeptide, and wherein more preferably, the polypeptide is an antigenbinding protein or a polypeptide chain of an antigen-binding protein.

5. A conjugate according to claim 4, wherein the antigen-binding protein comprises at least one of: a) at least one of: i) a first antigen-binding region that specifically binds a TAA, that specifically binds an NK cell activating receptor or that specifically binds an epitope of a y6 T cell receptor (TCR), and ii) a second antigen-binding region that specifically binds a TAA, that specifically binds an NK cell activating receptor or that specifically binds an epitope of a y6 TCR; and,b) a dimeric Fc region that binds to CD16A, or wherein the dimeric Fc region is modified to reduce affinity for CD16A, relative to a corresponding wild-type Fc region.

6. A conjugate according to claim 5, wherein the TAA is selected from the group consisting of: 5T4, ADAM9, ADAM10, ADAM12, ALK, ALPP, ALPP2, ALPPL2, AMHR2, ANGPT2, AXL, Angiopoietin-2, Apelin receptor, B7-H3, B7-H4, B7-H6, B7.1 , B7.2, BCMA, BTLA, CA125, CA9, CAIX, CCL2, CCR2, CCR4, CCR5, CCR6, CCR7, CD123, CD133, CD138, CD142, CD147, CD166, CD171 , CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD248, CD25, CD27, CD276, CD3, CD30, CD317, CD33, CD37, CD38, CD3E, CD4, CD40, CD44, CD44v6, CD45, CD46, CD47, CD52, CD56, CD70, CD71 , CD73, CD74, CD79, CD79B, CD80, CD80 / CD86, CDCP1 , CDH3, CDH6, CDK4, CEA, CEACAM5, CLDN18, CLEC14A, CLEC4, CSF1 R, CSF2, CSPG4, CT-7, CTGF, CTLA4, Cadherin 17, Cadherin 6, CanAg, Claudin 18.2, Claudin 6, Connexin 37, Cripto-1 , Crypto, DC3, DKK1 , DLK1 , DLL3, DLL4, DR5, E- cadherin, E-selectin, EBV-encoded nuclear antigen (EBNA)-I, EDA, EDB, EDNRB, EGF, EGFL7, EGFR, EGFRvlll, ENG, EPCAM, EPHA4, EphAI O, EphA2, EphA3, EphB2, EphB4, ExtradomainB (EDB) fibronectin, F3, FAP, FCGR1 , FGFR2, FGFR2b, FGFR3, FGFR4, FLT1 , FN, FOLH1 , FOLR1 , FRa, FSHR, FcRL5 / FcRH5, Fibronectin extra-domain B, Flt3, Fucosyl, GFRa4, GM3, GPCR5D, GPNMB, GPRC5D, GRP78, GUCY2C, Glycoprotein NMB, Glypican 1 , Glypican 2, Glypican 3, GnT-V, HAVCR2, HER-3 / ERBB3, HER-4 / ERBB4, HER2, HER3, HGF, HLA-G, HSP70, ICAM-1 , ICOS, IFNG, IGF-1 R, IGF1 , IGF1 R, IGF2, IL-1 accessory protein, IL-6 receptor, IL-8 receptor, IL13Ra2, IL17A, IL1 A, IL1 B, IL1 RAP, IL2RA, IL3RA, IL6, IL6R, ITGAV, ITGB6, Ig-idiotype, Integrin beta 6, KAAG-1 , KDR, KIRD2, KIT, KLK2, KLRC1 , Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), L1-CAM, L1 CAM, LAG3, LAGE-1 , LGR5, LIF, LIV-1 , LOXL2, LRRC32, Lewis-Y, MART-1 / Melan-A, MET, MIC-A / B, MICB, MIF, MISIIR, MMP2, MS4A1 , MSLN, MST1 R, MSTN, MUC1 , MUC1- C, MUC16, MUM-1 , Melanotransferrin, Mesothelin, Mud 6, NAG, NCAM1 , NKG2D, NOTCH1 , NOTCH2, NOTCH3, NRP1 , NT5E, NTRKR1 (EC 2.7.10.1), NaPi2b, Nectin-4, OLR1 , 0X40, P-cadherin, P1A, PCSK9, PD-L1 , PD1 , PDGF, PDGF alpha receptor, PDGF beta receptor, PDGFR, PDGFRA, PLAUR, PMEL, PRAME, PSCA, PSMA, PTK7, PTPRC, PVRL4, Plexin-A1 , RAGE, ROBO1 , ROR1 , ROR2, RSPO3, SCP-1 , SEZ6, SIRPA, SLAMF7, SLC34A2, SLC3A2, SSTR2, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, STEAP1 , STEAP2, Severe, T-cell receptor / CD3-zeta chain, TACSTD2, TGF-alpha, TGFB1 , TGFB2, TGFB3, TIGIT, Tissue factor / TF, TLR2, TM4SF1 , TMEFF2, TNFRSF10B, TNFRSF17, TNFRSF18, TNFRSF4, TNFRSF8, TNFRSF9, TNFSF11 , TNFSF13B, TPBG, TRAILR1 , TRAILR2, TROP2, TSHR, TYRP1 , VEGF, VEGFA, VEGFR1 , VEGFR2, VH1 / VL1 , VH2A / L2, VH3 / VL3, a GAGE-tumor antigen, a GD2 ganglioside, a GM2 ganglioside, a RAET1 protein, a UL16-binding protein (ULBP), a heterodimeric receptor comprised of at least one HER subunit, a human papillomavirus protein, a5p1 integrins, a5p3 integrins, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), anti-Mullerian hormone Type II receptor, avB3 integrin, avB6 integrin, avp6 integrins, bivalent, brainglycogen phosphorylase, c-erbB-2, cMET, colorectal associated antigen (CRC)-C017- 1A / GA733, gastrin releasing peptide receptor antigen, gp100, gp75, gpA33, hCG, human papillomavirus protein, integrin receptors, mmp9, muc17, p15, phosphatidylserine, prostate specific antigen (PSA), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), sVE-cadherin, scatter factor receptor kinase, trivalent, a-catenin, a-fetoprotein, allbp3-integrins, p-catenin, and y-catenin.

7. A conjugate according to claim 5 or 6, wherein the dimeric Fc region is modified to reduce affinity for CD16A, relative to a corresponding wild-type Fc region and wherein the antigenbinding region that specifically binds an epitope of a y6 TCR, binds an epitope in the V region of a V61 chain, a V62 chain or a Vy9 chain of a y6 TCR.

8. A conjugate according to any one of claims 4 - 8, comprising, in addition to the 4-1 BBL ECD mutein or the fusion protein, at least one further agonist that is at least one of: i) a further NK cell activating cytokine selected from the group consisting of: an IL-21 receptor agonist, an IL-15 receptor agonist, a type I interferon (IFN-1) receptor agonist, an IL-2 receptor agonist, an IL-12 receptor agonist and an IL-18 receptor agonist; ii) a y6 T cell-activating agonist selected from the group consisting of: an IL-21 receptor agonist, an IL-15 receptor agonist, a type I interferon (IFN-1) receptor agonist, an IL-2 receptor agonist, an IL-12 receptor agonist and an IL-18 receptor agonist; and, iii) a further y6 T cell co-stimulatory agonist selected from the group consisting of: a CD27 agonist and a GITR agonist, wherein preferably: i) the IL-21 R agonist comprises or consist of an IL-21 polypeptide or an agonistic antigenbinding region that specifically binds IL-21 R; or, ii) the IL-15R agonist comprises or consist of an IL-15 polypeptide or an agonistic antigenbinding region that specifically binds IL-15R; iii) the type I interferon (IFN-1) receptor agonist comprises or consists of an IFN-1 polypeptide or an agonistic antigen-binding region that specifically binds the IFN-a receptor; iv) the IL-2R agonist comprises or consists of an IL-2 polypeptide or an agonistic antigenbinding region that specifically binds IL-2R; v) the IL-12R agonist comprises or consists of an IL-12 polypeptide or an agonistic antigen-binding region that specifically binds IL-12R; vi) the IL-18R agonist comprises or consists of an IL-18 polypeptide or an agonistic antigen-binding region that specifically binds IL-18R; vii) the CD27 agonist comprises or consists of at least one CD70 extracellular domain (ECD) or agonistic antigen-binding region that specifically binds CD27; and, viii) 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.

9. A conjugate according to claim 8, wherein the IL-21 polypeptide is an IL-21 mutein comprises at least one amino acid substitution, deletion or insertion selected from the group consisting of: (N82- A83- G84- R85- R86- Q87- K88-), 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, R1 1 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, K21 H, K21 N, K21 Q, K21 E, K21 D, I67T, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74E, L74K, L74R, L74N, L74Q, L74S, L74P, K75E, K75Q, K75N, K75S, K75-, K76-, K112H, K112N, K112Q, K112E, K112D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, 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).

10. A conjugate according to claim 9, wherein the conjugate comprises a combination of a 4- 1 BBL ECD mutein and an IL-21 mutein, wherein the 4-1 BBL ECD mutein is selected from the group consisting of: 4-1 BBL mutein A154D; 4-1 BBL mutein A154E; 4-1 BBL mutein V153Q; 4-1 BBL mutein Q227E; 4-1 BBL mutein L101 N; 4-1 BBL mutein Y110Q; 4-1 BBL mutein Q230K; and 4-1 BBL mutein V100Q; and wherein the IL-21 mutein is selected from the group consisting of: IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); IL-21 mutein L20W; IL-21 mutein L74D; IL-21 mutein L20N; IL-21 mutein I67N; IL-21 mutein L20S; IL-21 mutein L13E; IL-21 mutein I8H; IL-21 mutein (N63- E64- R65- I66-); and IL-21 mutein L74F, and wherein preferably, the 4-1 BBL ECD mutein and the IL-21 mutein are each conjugated to an antigen binding protein, whereby more preferably, the 4-1 BBL ECD mutein is present as a hetero- or homotrimer of 4-1 BBL ECDs connected through polypeptide linkers.

11. A conjugate according to claim 10, wherein the conjugate 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; 4-1 BBL mutein A154E and IL-21 mutein I8H; 4-1 BBL mutein A154E and IL-21 mutein (N63- E64- R65- I66-); 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- 166-); 4-1 BBL mutein A154D + G155Q and IL-21 mutein L74F; 4-1 BBL 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- 166-); 4-1 BBL 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 L101 N and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein L101 N and IL-21 mutein L20W; 4-1 BBL mutein L101 N 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 L101 N and IL-21 mutein L20S; 4-1 BBL mutein L101 N and IL-21 mutein L13E; 4-1 BBL mutein L101 N and IL-21 mutein I8H; 4-1 BBL mutein L101 N and IL-21 mutein (N63- E64- R65- I66-); 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- I66-); 4-1 BBL mutein Y110Q and IL-21 mutein L74F; 4- 1 BBL 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- 166-); 4-1 BBL 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 V100Q and IL-21 mutein L20W; 4-1 BBL mutein V100Q 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- I66-); and,4-1 BBL mutein V100Q and IL-21 mutein L74F; of which the combinations: 4-1 BBL mutein A154D and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); and, 4-1 BBL mutein A154D and IL-21 mutein L20W, are preferred.

12. A conjugate according to any one of claims 4 - 11 , wherein the first antigen-binding region that specifically binds a TAA and the second antigen-binding region that specifically binds a TAA form an immunoglobulin structure with the dimeric Fc region, and wherein at least one of: i) the 4-1 BBL ECD mutein or the fusion protein; and ii) the further agonist, is present on at least one or on both sides of the immunoglobulin structure.

13. A pharmaceutical composition comprising a 4-1 BBL ECD mutein according to claim 1 or 2, a fusion protein according to claim 3, or a conjugate according to any one of claims 4 - 12, and a pharmaceutically acceptable carrier.

14. A 4-1 BBL ECD mutein according to claim 1 or 2, a fusion protein according to claim 3, a conjugate according to any one of claims 4 - 12, or a composition according to claim 13, for use in the treatment of a cancer, preferably a cancer comprising tumor cells expressing the TAA, wherein optionally, the 4-1 BBL ECD mutein, the conjugate or the composition is used in combination with an adoptive transfer of immune cells, wherein preferably the immune cells are selected from T cells and NK cells.

15. A 4-1 BBL ECD mutein according to claim 1 or 2, a fusion protein according to claim 3, a conjugate according to any one of claims 4 - 12 or a composition according to claim 13, for a use according to claim 14, wherein at least one of: a) the 4-1 BBL ECD mutein, the conjugate or the composition 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 4-1 BBL ECD mutein, the conjugate or the composition 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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