IL-21 MUTEINS, FUSION PROTEINS COMPRISING THE SAME AND USES THEREOF
Field of the invention
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 IL- 21 muteins, preferably IL-21 muteins having an altered affinity, relative to the affinity of wild-type IL- 21 for the IL-21 receptor. The invention further relates to fusion proteins comprising such IL-21 muteins and the use of such IL-21 muteins and fusion proteins in medical treatments of e.g. cancer.
Background of the invention
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.
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.
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.
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.
More recently, WO2024/056862 and WO2024/056861 disclose such multifunctional antigenbinding proteins comprising as NK cell-activating cytokine an IL-21 R agonist, such as interleukin- 21 (IL-21). Co-pending applications EP24208721.1 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, function and location of the NK cells and/or y6 T cells. However, the specificity of the IL21 R agonist in these proteins is not yet specific for function in the tumor only, potentially causing side effects in the periphery.
IL-21 is a T cell derived pleiotropic cytokine that regulates the activity of both innate and adaptive immune cells. The IL-21 receptor (IL-21 R) is broadly expressed in the hematopoietic system on T and B lymphocytes, natural killer (NK) cells as well as myeloid cells. IL-21 can augment NK cell survival and effector function. Because IL-21 plays a key role in anti-tumor and anti-viral responses, in addition to exerting major effects on inflammatory responses that lead to the development of autoimmune diseases and inflammatory diseases, IL-21 has been an attractive target for therapy. However, the biological effects of IL-21 are highly context-dependent, with IL-21 capable of exerting either immuno-stimulatory or immuno-regulatory effects, which has rendered the development of IL-21 -based therapies complex. For instance, while capable of directly activating T cells, IL-21 stimulation can induce apoptosis in dendritic cells, limiting antigen presentation and subsequent T cell responses. In addition, IL-21 can induce proliferation or apoptosis in B cells depending on the presence of other stimulatory signals. Clinical studies treating cancer patients with recombinant IL-21 have found IL-21 to be generally well-tolerated at low doses but to reach dose-limiting toxicities between 30-100 ug/kg/day, including hepatotoxicity, lymphopenias, thrombocytopenias, and hypersensitivity reactions, which may limit efficacy (Thompson et al. J Clin Oncol. 2008;26(12):2034-2039; Davis et al. Clin Cancer Res. 2009;15(6):2123-2129; Timmerman et al. Clin Cancer Res. 2012;18(20):5752-5760; Bhatia S et al. J Immunother Cancer. 2014;2:2; Steele et al. Br J Cancer. 2012;106(5):793-798). Accordingly, care must be taken to avoid broad IL-21 R activation in leukocytes and the potential toxicity thereof. Furthermore, due to the high affinity of IL-21 for its cognate receptor and broad distribution of the IL-21 R in the hematopoietic compartment, fusion of wild type IL-21 to a monoclonal antibody significantly lowers exposure and shortens half-life compared to the parent antibody (Shen et al. Front Immunol. 2020;11 :832). The therapeutic application of IL-21 signaling must therefore be balanced and targeted, such that the effects triggered by IL-21 are designed to occur at the right time and only at the right place in the body.
There is therefore a need in the art for IL-21 treatment modalities that address these issues. It is thus an object of the present invention to provide for such IL-21 treatment modalities.
Description of the invention
Definitions
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.
“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.
“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.
“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.
“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. Exemplary”: this term means “serving as an example, instance, or illustration,” and should not be construed as excluding other configurations disclosed herein.
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.
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.
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.
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.
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.
“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.
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. Alternative conservative amino acid residue substitution classes.
Alternative physical and functional classifications of amino acid residues.
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.
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.
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.
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.
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).
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.
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 conjugated 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.
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.
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.
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.
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).
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).
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.
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.
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).
"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).
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).
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.
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 .
Avimers are multidomain proteins derived from the A-domain scaffold family. The native domains of approximately 35 amino acids adopt a defined disulfide bonded structure. Diversity is generated by shuffling of the natural variation exhibited by the family of A-domains. For further details see Nature Biotechnology 23(12), 1556 - 1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).
A transferrin is a monomeric serum transport glycoprotein. Transferrins can be engineered to bind different target antigens by insertion of peptide sequences in a permissive surface loop. Examples of engineered transferrin scaffolds include the Trans-body. For further details see J. Biol. Chem 274, 24066-24073 (1999).
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 .
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.
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).
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.
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.
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 .
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.
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)).
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.
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.
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.
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 defintions1
Kabat et al. also defined a numbering system for variable region sequences that is applicable to any antibody. One of ordinary skill in the art can unambiguously assign this system of "Kabat numbering" to any variable region sequence, without reliance on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Unless otherwise specified, references to the numbering of specific amino acid residue positions in an antibody variable region are according to the Kabat numbering system.
With the exception of CDR1 in VH, CDRs generally comprise the amino acid residues that form the hypervariable loops. CDRs also comprise "specificity determining residues," or "SDRs," which are residues that contact antigen. SDRs are contained within regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1 , a-CDRL2, a-CDR-L3, a-CDR-H1 , a-CDR-H2, and a-CDR-H3) occur at amino acid residues 31-34 of L1 , 50-55 of L2, 89-96 of L3, 31- 35B of H1 , 50-58 of H2, and 95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619- 1633 (2008).) Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., supra.
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.
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.
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.
The term "specifically binds" refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding region or antigen-binding protein can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigenbinding protein (KD), is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein. Alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1 /KD. Affinity can be determined in a manner known per se, depending on the specific combination of antigen-binding protein and antigen of interest. Avidity is herein understood to refer to the strength of binding of a target molecule with multiple binding sites by a larger complex of binding agents, i.e. the strength of binding of multivalent binding. Avidity is related to both the affinity between an antigenic determinant and its antigen-binding site on the antigen-binding protein and the valency, i.e. the number of binding sites present on the antigen-binding protein. Affinity, on the other hand refers to simple monovalent receptor ligand systems.
Typically, an antigen-binding region of a conjugate of the invention thereof will specifically bind its target molecule (antigen) with a dissociation constant (KD) of about 10-6 to 10-12 M or less, and preferably 10-8 to 10-12 M or less, and/or with a binding affinity of at least 10-6 M or 10-7 M, preferably at least 10-8 M, more preferably at least 10-9 M, such as at least 1 O-10, 10’11, 10-12 M or less. Any KD value greater than 10-4 M (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 10-4 M, 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.
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 106 M-1 S-1 by 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.
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.
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.
"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.
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.
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.
Amino acid substitutions are herein indicated as AOXAS, wherein Ao indicates the original amino acid, X indicates the position of that original amino acid in the original amino acid sequence, and As indicates the substitute amino acid as present in that position in the modified amino acid sequence. For example, I8H denotes that the original amino acid isoleucine (I) in position 8 is changed to a histidine (H).
Amino acid deletions are herein indicated as A0X-, wherein Ao indicates 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 Ao is 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.
Amino acid insertion are herein indicated as A0X insAi-n, wherein Ao indicates 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.
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).
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.
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.
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.
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.
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.
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). The term “gene” means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5’ leader sequence, a coding region and a 3’ non-translated sequence (3’ end) comprising a polyadenylation site. “Expression of a gene” refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.
The term “homologous” when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. When used to indicate the relatedness of two nucleic acid sequences the term “homologous” means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.
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.
Detailed description of the invention
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 EP24208721.1 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 IL-21 , remains challenging because of the risks of toxicity and other undesired side-effects. There remains therefore a need in the art for IL-21 treatment modalities that reduce the systemic pleiotropic effect of IL-21 , while maintaining their potential when targeted to tumors or sites infected by pathogens. The present invention therefore provides novel IL-21 muteins having an altered affinity for IL-21 R. For example, IL-21 muteins described herein and having a reduced affinity for IL-21 R, and conjugates described herein comprising such IL-21 muteins, will have reduced pleiotropic- and undesired side-effects, when present in the bloodstream, while, when present at targeted sites, their avidity will ensure their local efficacy. Without being bound by a theory, the IL-21 -containing conjugates described herein are designed to utilize the immune potentiating activity of IL-21 (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.
IL-21 and IL-21 muteins
IL-21 is a protein that in humans is encoded by the IL-21 gene (Entrez Gene ID: 59067). IL- 21 is a cytokine that has potent regulatory effects on cells of the immune system, including natural killer (NK) cells and y6 T cells, and which induces cell division/proliferation in its target cells. Amino acid sequences for human IL-21 precursor (including its signal sequence) are described in NCBI accession numbers NP_001193935 and NP_068575, the disclosures of which are incorporated herein by reference. IL-21 (mature/processed) comprises amino acids 30 - 153 of NP_001193935 or amino acids 30 - 162 of NP_068575 (i.e. SEQ ID NO: 38).
IL-21 exerts its effects on target cells through the IL-21 receptor (IL-21 R) is expressed on the surface of immune cells, including T, B and NK cells. IL-21 R is similar in structure to the receptors for other type I cytokines like IL-2 or IL-15 and requires dimerization of the human IL-21 receptor alpha chain with the common gamma chain (yc) in order to bind IL-21 . IL-21 R is encoded in humans by the IL-21 R gene (Entrez Gene ID: 50615). Amino acid sequences for human IL-21 R are described in NCBI accession numbers NP_068570, NP_851564 and NP_851565, the disclosures of which are incorporated herein by reference. An example of an IL-21 R alpha chain is herein provided as SEQ ID NO: 173 and an example of an IL-21 R gamma chain is herein provided as SEQ ID NO: 174.
As used herein, an “IL-21 R agonist” is an agent that has “agonist” activity at the IL-21 receptor, which means that the agent that can cause or increase " IL-21 R signaling". “IL-21 R signaling” refers to an ability of IL-21 R, e.g. when expressed on the surface of T, B and NK cells and triggered by its natural ligand IL-21 , to activate or transduce an intracellular signaling pathway. The “natural ligand IL-21 ” is herein understood as a human wild type IL-21 comprising or consisting of an amino acid sequence as indicated above. When bound to IL-21 , the IL-21 receptor acts through the Jak/STAT pathway, utilizing Jak1 and Jak3 and a STAT3 homodimer to activate its target genes. IL-21 R agonist activity, i.e. changes in IL-21 R signaling activity, can be measured, for example, by assays designed to measure changes in the IL-21 R signaling pathways, e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or assays designed to measure expression of reporter genes under control of IL-21 R-sensitive promoters and enhancers, or indirectly by a downstream effect mediated by IL-21 R (e.g. activation of specific cytolytic machinery in NK cells or y6 T cells). A suitable cell-based assay for biological activity of an IL-21 R agonist, is e.g. described in Maurer et al. (mAbs. 2012; 4(1): 69-83.), wherein a murine pre-B-cell line is transfected with both the human IL-21 R and a STAT-responsive luciferase reporter gene. IL-21 R agonist activity can be determined using this cell line by measuring the level of STAT3 phosphorylation using anti-pSTAT3 antibody-conjugated beads and/or by detecting luciferase luminescence, upon contacting the cell line with an IL-21 R agonist. The natural ligand IL-21 can serve as a positive control in an assay for IL-21 R agonist activity and can also be used as a reference for the amount of IL-21 R agonist activity of a given non-natural IL-21 R agonist, such as a conjugate comprising an IL-21 mutein as described herein.
When IL-21 binds to IL-21 R, the Jak/STAT signaling pathway is activated to activate target genes. While IL-21-induced signaling may be therapeutically desirable, careful consideration of the timing and the location of the signaling is needed, given IL-21 R's broad expression profile and due to the fact that IL-21 has the ability to potentiate CD8 T cell responses as well as to suppress antigen presentation and T cell priming. The data presented herein for supports the use of carefully designed IL-21 muteins to achieve IL-21 R signaling at the appropriate time and place and to improve pharmacokinetics of therapeutics comprising such IL-21 muteins.
In a first aspect, the present disclosure provides IL-21 muteins comprising at least one amino acid substitution, deletion and/or insertion. Amino acid substitutions, deletions and insertions in an IL-21 mutein provided herein are indicated relative to the wild-type human IL-21 amino acid sequence, which is provided herein as SEQ ID NO: 38. Hence, to allow for allelic variation, a wildtype human IL-21 preferably comprises an amino acid sequence having, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 38. In one embodiment, there is provided an IL-21 mutein comprising at least one amino acid substitution, deletion or insertion selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R11 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, 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-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D). In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution, deletion or insertion selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R11 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, 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, K1 12D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D).
In one embodiment, there is provided an IL-21 mutein comprising a combination of at least two, three, four, five, six, seven or eight amino acid substitutions, deletions and/or insertions selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R11 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, 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-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66- ), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D). In one embodiment, the IL-21 mutein comprises no other modifications than the at least two, three, four, five, six, seven or eight amino acid substitutions, deletions and/or insertions selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R11 Q, R11 N, R11Y, Q12K, Q12R, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, 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, K1 12D, N59-, T60-, G61-, N62-, N63-, E64-, R65-, I66-, (N63-, E64-, R65- and I66-), (N59-, T60-, G61-, N62-, N63-, E64-, R65- and I66-), (K75- and R76-), (K75-, R76- and R77-), G84 insGGGG, and, K75 insX (wherein X is one, two or three amino acids selected from the group consisting of G, S and D).
In one embodiment, there is provided an IL-21 mutein comprising a deletion of at least one amino acid in the region of N59 to I66. In one embodiment, there is provided an IL-21 mutein comprising a deletion of at least two, three, four, five, six, seven or eight amino acids in the region of N59 to I66. In one embodiment, there is provided an IL-21 mutein comprising the deletions (N59- , T60-, G61-, N62-, N63-, E64-, R65- and I66-). In one embodiment, there is provided an IL-21 mutein comprising the deletions (N63-, E64-, R65- and I66-).
In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to an IL-21 receptor (IL-21 R), preferably an IL-21 R having an amino acid sequence of SEQ ID NO: 173. In another embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to an IL-21 R gamma chain, preferably an IL- 21 R gamma chain having an amino acid sequence of SEQ ID NO: 174.
In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the human IL-21 R with a reduced affinity, relative to the affinity of wildtype IL-21 for the human IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the alpha chain of the human IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the alpha chain of the human IL-21 R. In one embodiment, there is provided an IL-21 mutein as structurally defined above, which IL-21 mutein binds to the gamma chain of the human IL-21 R with a reduced affinity, relative to the affinity of wild-type IL-21 for the gamma chain of the human IL-21 R.
In one embodiment, the IL-21 mutein having a with a reduced affinity, relative to the affinity of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution or deletion selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; K75E; L13E; D4H; I8H; (N63- E64- R65- I66-); I67N; L74D; L74F; L20S; and L20N. In one embodiment, the IL- 21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; K75E; L13E; D4H; I8H; (N63- E64- R65- I66-); I67N; L74D; L74F; L20S; and L20N.
The IL-21 muteins provided herein bind to IL-21 R in a non-covalent and reversible manner. In one embodiment, the binding strength of an IL-21 mutein to IL-21 R may be described in terms of its affinity, a measure of the strength of interaction between the binding site of the mutein and the IL-21 R. In one embodiment, an IL-21 mutein provided herein has a low-affinity for IL-21 R and thus will bind a lesser amount of IL-21 R than a wild type IL-21. In one embodiment, an IL-21 mutein provided herein has an equilibrium association constant, KA, which is, with decreasing preference, at least 105 M-1, at least 106 M-1, at least 107 M-1, at least 108 M-1, at least 109 M-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. In one embodiment, the binding strength of an IL-21 mutein provided herein to IL-21 R may be described in terms of its affinity, i.e. KD. KD is the equilibrium dissociation constant, a ratio of koff/kon, between the IL-21 mutein and IL-21 R. KD and KA are inversely related. The KD value relates to the concentration of the mutein (the amount of mutein needed for a particular experiment or application) and so the lower the KD value (lower concentration needed) the higher the affinity of the mutein. In one embodiment, the binding strength of an IL-21 mutein provided herein to IL-21 R may be described in terms of KD. In one embodiment, the KD of an IL-21 mutein provided herein is about 10-4 M, about 10-5 M, about 10-6 M, or less. In one embodiment, the KD of an IL-21 mutein provided herein is micromolar, nanomolar, or picomolar. In one embodiment, the KD of an IL-21 mutein provided herein is within a range of about 10 to 10-6 M, or 10-7 to 10-9 M. In one embodiment, an IL-21 mutein provided herein binds to the human IL-21 R with a KD that is greater than or is about 0.04 nM. In one embodiment, an IL-21 mutein provided herein binds to the human IL-21 R with a KD of about 0.01 nM to about 20 nM, 0.02 nM to 20 nM, 0.05 nM to 20 nM, 0.05 nM to 15 nM, 0.1 nM to 15 nM, 0.1 nM to 10 nM, 1 nM to 10 nM, or 5 nM to 10 nM.
In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity forthe human IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000- fold or 1 ,000.000-fold reduction in binding affinity for IL-21 R, relative to, relative to the affinity of wild-type IL-21 for IL-21 R. In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity forthe alpha chain of IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in binding affinity for the alpha chain of IL- 21 R, relative to, relative to the affinity of wild-type IL-21 for the alpha chain of IL-21 R. In one embodiment, an IL-21 mutein provided herein exhibits a reduction in binding affinity for the gamma chain of IL-21 R. In one embodiment, the IL-21 mutein provided herein is a mutein that exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000- fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in binding affinity for the gamma chain of IL-21 R, relative to, relative to the affinity of wild-type IL-21 for the gamma chain of IL-21 R.
In one embodiment, the binding affinity of an IL-21 mutein provided herein is determined by SPR, e.g. as described in the Examples herein. In one embodiment, the binding affinity of an IL-21 mutein provided herein is thus determined when the IL-21 mutein is present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate can further comprise a trimer of a 4-1 BB ligand extracellular domain (4-1 BBL ECD).
In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 0.5 lower than the pKo of wild-type IL-21 for the IL-21 R. pKo is understood herein to be -logio(Ko). In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 0.5 lower than the pKo of wild-type IL-21 for the IL-21 R, is an IL- 21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8V; I8Q; Q12K; K73Y; L13E; D4H; I8H; I67N; L74D; L20S; and L20N.
In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1 .0 lower than the pKo of wild-type IL-21 for the IL-21 R, In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; I8Q; Q12K; K73Y; L13E; I67N; L74D; L20S; and L20N.
In one embodiment, an IL-21 mutein provided herein exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.6 lower than the pKo of wild-type IL-21 for the IL-21 R, In one embodiment, the IL-21 mutein having a pKo for the human IL-21 R that is at least 1.6 lower than the pKo of wild-type IL-21 for the IL-21 R, is an IL-21 mutein comprising at least one amino acid substitution selected from the group consisting of: L20W; R5H; Q12K; I67N; L74D; L20S; and L20N. In one embodiment, the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; R5H; Q12K; I67N; L74D; L20S; and L20N.ln one embodiment, an IL-21 mutein provided herein exhibits a reduction in activity as measured by an in vitro STAT3 phosphorylation assay, relative to the activity of wild-type IL-21 under corresponding conditions. In one embodiment, an IL-21 mutein provided herein exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000- fold, 5,000-fold, 10,000-fold, 20,000-fold, 50,000-fold, 100,000-fold, 200,000-fold, 500,000-fold or 1 ,000,000-fold reduction in activity as measured by a STAT3 phosphorylation assay, relative to the activity of wild-type IL-21 under corresponding conditions.
In one embodiment, an IL-21 mutein provided herein exhibits at least about a 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1 ,000-fold, 2,000-fold, 5,000-fold, 10,000-fold reduction in activity as measured by a NK cell or y6 T cell proliferation assay, relative to the activity of wild-type IL-21 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 ory6 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.
In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with a monoclonal antibody against a TAA (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of tumor cells expressing the TAA (e.g. SKOV3 cells), that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L13E; I8H; (N63- E64- R65- I66-); I67T; I67N; L74G; L74D; L74F; L20S; L20N; and G84insGGGGG, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L13E; I8H; (N63- E64- R65- I66-); I67T; I67N; L74G; L74D; L74F; L20S; L20N; and G84insGGGGG.
In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2 higher than the EC50 of a corresponding control conjugate comprising wildtype IL-21 in the same assay. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; I8H; (N63- E64- R65- I66-); I67T; I67N; L74G; L74D; L74F; and L20N, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; I8H; (N63- E64- R65- 166-); I67T; I67N; L74G; L74D; L74F; and L20N.
In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) has a reduced affinity, relative to the affinity of wild-type IL-21 forthe IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- I66-); and L74F, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- I66-); and L74F.
In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.0 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) has a reduced affinity, relative to the affinity of wild-type IL-21 forthe IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; I8H; (N63- E64- R65- 166-); and L74F, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; I8H; (N63- E64- R65- I66-); and L74F.
In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity, expressed in pKo, for the human IL-21 R that is at least 0.5 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; and I8H, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; and I8H.
In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; and L13E, or the IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; and L13E.
In one embodiment, an IL-21 mutein provided herein is an IL-21 mutein which: i) when present in a conjugate with a monoclonal antibody (e.g. trastuzumab), which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), exhibits an EC50 for induction of proliferation of NK cells in a 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not more than a factor 2.0 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay; and, ii) exhibits a binding affinity for the human IL-21 R, expressed in pKo, that is at least 1.0 lower than the pKo of wild-type IL-21 for the IL-21 R. Hence, in one embodiment, the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; and I67N, orthe IL-21 mutein comprises no other modifications than the at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; and I67N.
In one embodiment, an IL-21 mutein provided herein, when present in a conjugate with trastuzumab, which conjugate further comprises a trimer of an 4-1 BBL ECD mutein comprising a substitution A154D, induces a maximal proliferation of NK cells at a saturating concentration of 25 nM of the conjugate in a normalized 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, which proliferation is at least 65%, 70%, 75%, 80%, 85%, or 89% of the proliferation induced by a corresponding control conjugate comprising wild-type IL-21 and wild type 4-1 BBL in the same assay. Hence, in one embodiment, the IL-21 mutein comprises the amino acid substitution L20W, or the IL-21 mutein comprises no other modifications than the amino acid substitution L20W.
In one embodiment, there is provided an IL-21 mutein as described herein, which, when present in a conjugate with an antigen-binding protein that specifically binds a (target) antigen, as a result of the reduced affinity of the IL-21 mutein for the IL-21 R, produces reduced (little or no) agonist activity at an IL-21 R expressed at the surface of a cell, in the absence of the antigen or cells carrying the antigen. However, when the conjugate is bound to the antigen or to cells carrying the antigen, the IL-21 mutein in the conjugate manifests significant agonist activity. This activity is the result of the IL-21 mutein being present in high local density on the surface of the target cells, which leads to an enhanced apparent affinity for the IL-21 R on local NK cells (see Figure 1) or y6 T cells, primarily through the mechanism of avidity. As such, an IL-21 mutein as described herein in a conjugate with an antigen-binding protein broadens the therapeutic window as compared to a corresponding conjugate comprising a wild type IL-21. The term “therapeutic window” is herein understood as the ratio (or fold-difference) of the EC50 values obtained from a functional assay (e.g., a proliferation assay) comparing conditions in which cancer cells are absent to conditions in which they are present. A therapeutic window of 10 (or 1 log) would mean that the EC50 without cancer cells was 10 times higher ( a less potent effect) as compared to when the cancer cells were present. Upon systemic administration the conjugate comprising the IL-21 mutein will have little or no effect on cells in the periphery, including T-, B- or NK cells, while remaining effective in stimulating these immune cells, in particular NK cells or y6 T cells, at a tumor site or a site of infection or inflammation. This is because the target antigen bound by the antigen-binding protein is present at high local concentrations only in these specific areas, enabling the effect of avidity.
Thus, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a conjugate with an antigen binding protein that specifically binds an antigen, wherein the conjugate optionally further comprises a 4-1 BBL extracellular domain (ECD), has an EC50 in an NK cell or y6 T cell proliferation assay in the presence of the antigen or cells expressing the antigen that is, with increasing preference, at least a factor 2, 5, 10, 20, 50, 100, 200, 500, 1 ,000, 2,000, 5,000, 10,000, 20,000, 50,000 or 100,000 lower than the EC50 in a corresponding NK cell proliferation assay in the absence of the antigen or cells expressing the antigen.
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 IL-21 amino acid sequence in the second heavy chain amino acid sequence of SEQ ID NO: 12 can be replaced by an amino acid sequence of an IL-21 mutein to be assayed, e.g. for its ability to induce NK cell or y6 T cell proliferation in the presence and absence of tumor cells expressing HER2, such as the SK-OV-3 cells.
In one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL- 21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 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 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.
In one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL- 21 mutein, when present in a multispecific antigen binding proteins consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, produces a difference in EC50 in an NK cell or y6 T cell proliferation assay in the presence vs absence of SK-OV-3 cells that is, with increasing preference, at least a factor 2, 5, 10, 20, 50, 100, 200, 500, 1 ,000, 2,000 or 5,000 higher than the difference in EC50 in an NK cell or y6 T cell proliferation assay in the presence vs absence of SK-OV-3 cells as produced by a reference multispecific antigen binding proteins consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 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.
In the above embodiments, the NK cell or y6 T cell proliferation assays are preferably performed using NK cells or y6 T cells isolated from healthy donors. In the above embodiments, the EC50 values in the NK cell or y6 T cell proliferation assays are preferably determined on the basis of the average values using NK cells or y6 T cell isolated from at least 5 different healthy donors. In the above embodiments, the NK cell or y6 T cell proliferation assays are preferably performed essentially as described in the examples herein.
While the reduced affinity of the IL-21 muteins described herein when present in a conjugate with an antigen binding protein increases the therapeutic window as compared to a corresponding conjugate comprising a wild type IL-21 , at the same time the ability of a conjugate with an IL-21 muteinto induce NK cell or y6 T cell cytotoxicity against cells carrying the antigen that is bound by the antigen binding protein, preferably remains essentially unaffected.
Hence, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 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.
Also the ability of a conjugate between an IL-21 mutein as described herein and an antigen binding protein to support long-term expansion of NK cells or y6 T cell in the presence of cells carrying the antigen that is bound by the antigen binding protein, preferably remains essentially unaffected.
Hence, in one embodiment, there is provided an IL-21 mutein as described herein, wherein the IL-21 mutein, when present in a multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 11 ; ii) a second heavy chain comprising the amino acid sequence of SEQ ID NO: 12, wherein the wild type IL-21 amino acid sequence is replaced by the amino acid sequence of the IL-21 mutein; and iii) light chains comprising the amino acid sequence of SEQ ID NO: 2, induces a fold expansion of NK cells or y6 T cells in the presence of SK-OV-3 cells in an NK cell expansion assay, that is, with increasing preference, at least equal to, or at least 2-fold, at least 5-fold or at least 10-fold higher than the fold expansion induced by a reference multispecific antigen binding protein consisting of i) a first heavy chain comprising the amino acid sequence of SEQ ID NO: 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.
A further advantage of the IL-21 muteins as described herein is that their reduced affinity for IL-21 R the improves pharmaco-kinetics of therapeutics comprising the IL-21 muteins. In the body many cells, including T-, B- or NK cells, are present that express IL-21 R molecules at their surfaces. These IL-21 R molecules act as a sink for therapeutics comprising a moiety with affinity for IL-21 R such as an IL-21 mutein. Upon binding to a surface expressed IL-21 R, the therapeutic comprising the IL-21 -moiety will be internalized and will therefore no longer be available for exerting its therapeutic effect, e.g. in the tumor microenvironment. Hence, the reduced affinity for IL-21 R of the IL-21 muteins as described herein reduces or prevents their disappearance in this sink and thereby improves pharmaco-kinetics of therapeutics comprising the IL-21 muteins.
In addition, the reduced affinity IL-21 muteins as described herein for IL-21 R will diminish its potential apoptosis-inducing effect on dendritic cells, which can therefore remain active in antigen presentation and subsequent induction of T cell responses.
IL-21 mute in conjugates
In a second aspect, the present disclosure provides a conjugate comprising one or more of the IL-21 muteins as described herein conjugated to a heterologous moiety. 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 non-coded) which is different from the IL- 21 muteins described herein. Exemplary conjugate moieties that can be linked to any of the IL-21 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 an IL-21 mutein of the present disclosure and an immunoglobulin. The conjugate in some embodiments comprises one or more of the IL-21 muteins described herein and one or more of: a peptide or polypeptide (which is distinct from the IL-21 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.
In one embodiment, there is provided a conjugate wherein the heterologous moiety is attached via non-covalent or covalent bonding to the IL-21 mutein as described herein. In exemplary embodiments, the linkage between the IL- 21 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.
In one embodiment, there is provided a conjugate wherein the IL-21 mutein as described herein is linked to a conjugate moiety via direct covalent linkage by reacting targeted amino acid residues of the IL-21 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 IL-21 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 IL-21 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.
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.
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.
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.
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.
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.
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.
Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
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.
Another type of covalent modification involves chemically or enzymatically coupling glycosides to the IL-21 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 IL-21 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.
In one embodiment, the conjugate is a conjugate that has IL-21 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 IL-21 mutein-moieties. Preferably, more than one IL-21 mutein- moieties are identical IL-21 mutein-moieties. 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 IL-21 muteins described herein. In one embodiment, the conjugate is a fusion polypeptide, fusion protein, a chimeric protein or chimeric polypeptide comprising an IL-21 mutein 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.
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.
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.
An antigen-binding region as used in an antigen binding protein in a conjugate with an IL-21 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.
In a preferred embodiment, an antigen-binding region as used in an antigen binding protein in a conjugate with an IL-21 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 antigen-binding domain such as a scFv, a VH domain, a VL domain, or an immunoglobulin single variable domain (ISVD) such as a dAb, a V-NAR domain or a VHH domain. An immunoglobulin variable region to be used in an antigen binding protein in a conjugate with an IL-21 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 In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 10-4 M, as may be determined as herein described above.
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.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein can be a homodimeric or a heterodimeric antigen binding protein.
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. 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.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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, AFP, ALK, ALPP, ALPP2, ALPPL2, AXL, Angiopoietin-2, Apelin receptor, B7-H3, B7-H4, B7-H6, B7.1 , B7.2, BCMA, BTLA, CA125, CAIX, CCR4, CCR6, CCR7, CD123, CD133, CD138, CD142, CD147, CD166, CD171 , CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD25, CD27, CD276, CD3, CD30, CD317, CD33, CD38, CD3E, CD4, CD40, CD44v6, CD45, CD46, CD47, CD52, CD56, CD70, CD71 , CD73, CD74, CD79, CD79B, CD80, CD80/CD86, CDCP1 , CDH3, CDK4, CEA, CEACAM5, CLDN18, CLEC14A, CLEC4, CSF1 R, CSPG4, CT-7, CTLA4, Cadherin 17, Cadherin 6, CanAg, Claudin 18.2, Claudin 6, cMet, Connexin 37, Cripto-1 , Crypto, DC3, DLK1 , DLL3, DLL4, DR5, E-cadherin, E- selectin, EBV-encoded nuclear antigen (EBNA)-I, EDA, EDB, EDNRB, EGF, EGFR, EGFRvlll, EPCAM, EPHA4, EphAIO, EphA2, EphA3, EphB2, EphB4, ExtradomainB (EDB) fibronectin, F3, FAP, FGFR2, FGFR2b, FGFR4, FOLH1 , FOLR1 , FRa, FSHR, FcRL5/FcRH5, Fibronectin extradomain B, Flt3, GFRa4, GM3, GPCR5D, GPRC5D, GRP78, GUCY2C, Glycoprotein NMB, Glypican 1 , Glypican 2, Glypican 3, GnT-V, HAVCR2, HER-2/ERBB2, HER-3/ERBB3, HER-4/ERBB4, HER2, HER3, HER4, HLA-G, HSP70, ICAM-1 , IFNG, IGF-1 R, IL-1 accessory protein, IL-6 receptor, IL-8 receptor, IL13Ra2, IL3RA, Ig-idiotype, Integrin beta 6, KAAG-1 , KDR, KLK2, KLRC1 , Killer Ig- Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), L1-CAM, L1 CAM, LAG3, LAGE-1 , LGR5, LIV-1 , Lewis-Y, MART-1 /Melan-A, MET, MIC-A/B, MICB, MISIIR, MMP2, MS4A1 , MSLN, MUC1 , MUC1-C, MUC16, MUM-1 , Melanotransferrin, Mesothelin, Mud 6, NAG, NKG2D, NT5E, NTRKR1 (EC 2.7.10.1), NaPi2b, Nectin-4, OLR1 , 0X40, P-cadherin, P1A, PD-L1 , PD1 , PDGF, PDGF alpha receptor, PDGF beta receptor, PDGFR, PDGFRA, PLAUR, PRAME, PSCA, PSMA, PTK7, PTPRC, PVRL4, Plexin-A1 , RAGE, ROBO1 , ROR1 , ROR2, SCP-1 , SEZ6, SLAMF7, SLC3A2, SSTR2, SSX- 1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, STEAP1 , STEAP2, T-cell receptor/CD3-zeta chain, TACSTD2, TGF-alpha, TIGIT, Tissue factor/TF, TM4SF1 , TMEFF2, TNFRSF10B, TNFRSF17, TNFRSF4, TNFRSF8, 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, avp1 integrins, avp3 integrins, avp6 integrins, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), anti-Mullerian hormone Type II receptor, brain glycogen phosphorylase, c-erbB-2,, colorectal associated antigen (CRC)-C017-1A/GA733, gastrin releasing peptide receptor antigen, gp100, gp75, gpA33, hCG, human papillomavirus protein, integrin receptors, mmp9, muc17, p15, prostate specific antigen (PSA), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), sVE-cadherin, scatter factor receptor kinase, a-catenin, a-fetoprotein, allbp3-integrins, p-catenin, and y-catenin, although this is not intended to be exhaustive.
In one embodiment therefore, an antigen binding protein in a conjugate with an IL-21 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 - VH2/VL2); 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 /L1); 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 - VH2A/L2); fh) 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).
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.
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).
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.
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).
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.
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 an IL-21 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). In one embodiment, an antigen binding protein in a conjugate with an IL-21 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
- 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
- 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
- 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 - VH2A/L2); 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
- 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 - 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).
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).
Antigen-binding regions that specifically bind an NK cell activating receptor
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 or they 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 an IL-21 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. In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein comprises the antigenbinding region that specifically binds an NK cell activating receptor that is an NCR selected from the group consisting of NKp46, NKp44 and NKp30.
“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.
“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
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.
“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
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. 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.
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.
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.
“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.
In one embodiment of an antigen binding protein in a conjugate with an IL-21 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 antigenbinding 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 receptor-signaling pathways, e.g. by monitoring phosphorylation of signal transduction components, assays to measure the association of certain signal transduction components with other proteins or intracellular structures, or in the biochemical activity of components such as kinases, or assays designed to measure expression of reporter genes under control of 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.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 2011/086179, 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.
Antigen binding regions binding to yd T cell receptors
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 (TOR).
The third antigen-binding region as used in an antigen binding protein in a conjugate with an IL-21 mutein as described herein can be derived from any of a variety of immunoglobulin or nonimmunoglobulin 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 an IL-21 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 an IL-21 mutein as described herein can be a human or humanized immunoglobulin variable region or an immunoglobulin single variable domain as herein defined above.
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 10-4 M, as may be determined as herein described above.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein.
In a preferred embodiment, an antigen binding protein in a conjugate with an IL-21 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 antigen-binding region that specifically binds an epitope in the V region of a V61 chain of a y6 TCR, also does not bind or interact with other domains found within a y6 TCR, such as TRDJ, TRDC, TRGJ or TRGC. In one embodiment, an antigen binding protein in a conjugate with an IL-21 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 an IL-21 mutein as described herein.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 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.
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 an IL-21 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.
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 an IL-21 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. 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 an IL-21 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 an IL-21 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.
In one embodiment, a T-cell receptor complex activating epitope is one that upon activation, by being bound by a third antigen-binding region, downregulates the T-cell receptor complex, whilst not downregulating CD3 molecules not associated with said TRDV1 TCR complex.
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.
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 an IL-21 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.
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).
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)).
Therefore, in one embodiment, there is provided an antigen binding protein in a conjugate with an IL-21 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 10-7 M (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 10-7 M (i.e.150 nM) or less, 1 .3 x 10-7 M (i.e.130 nM) or less, 1 .0 x 10-7 M (i.e.100 nM) or less, 5.0 x 10-8 M (i.e.50 nM) or less, 2.0 x 10-8 M (i.e. 20 nM) or less, 1 .0 x 10-8 M (i.e. 10 nM) or less, 5 x 10-9 M (i.e. 5 nM) or less, 2 x 10-9 M (i.e. 2 nM) or less, or 1 x 10-9 M (i.e. 1 nM) or less.
In one embodiment, there is provided an antigen binding protein in a conjugate with an IL-21 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 10-7 M (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 10-7 M (i.e.150 nM) or more, 3 x 10-7 M (i.e. 300 nM) or more, 1.0 x 10-6 M (i.e.1 pM) or more, 2.0 x 10-6 M (i.e. 2 pM) or more, 5.0 x 10-6 M (i.e. 5 pM) or more, 1 .0 x 10-5 M (i.e. 10 pM) or more, 2 x 10-5 M (i.e. 20 pM) or more, 5 x 10-5 M (i.e. 50 pM) or more, or 1 x 10-4 M (i.e. 100 pM) or more.
In one embodiment, the binding affinity of the third antigen-binding region in an antigen binding protein in a conjugate with an IL-21 mutein as described herein, is established by coating the third antigen-binding region or the antigen binding protein comprising the third antigen-binding region directly or indirectly (e.g. by capture with an antibody) onto the surface of a sensor (e.g. an amine high capacity chip or equivalent), wherein the target bound by the third antigen-binding region (e.g. the V61 chain of a y6 TCR) is flowed over the chip to detect binding. Suitably, a MASS-2 instrument (which may also be referred to as Sierra SPR-32) is used at 25 °C in PBS + 0.02 % Tween 20 running buffer at 30 pl/min.
Unlike anti-V61 antibodies of the prior art which focus on depletion of V61 T-cells or attempt activation of V61 T-cells via the TCR without any appropriate co stimulation or synapse formation, the antigen binding proteins provided herein are useful for the activation of V61 T-cells via the TRDV1 -binding third antigen-binding region. Although they may cause downregulation of the TCRs on T-cells to which they bind, they do not cause V61 T-cell depletion, but rather they stimulate the T-cells and hence may be useful in therapeutic settings that would benefit from the activation of this compartment of T-cells. Activation of V61 T-cells is evident through TCR downregulation, CD3 downregulation, changes in activation markers such as CD25 and Ki67 and degranulation marker CD107a. Activation of V61 T-cell in turn triggers release of inflammatory cytokines such as INFy and TNFa to promote immune licensing.
In one embodiment, there is provided an antigen binding protein in a conjugate with an IL-21 mutein as described herein comprising a TRDV1-binding third antigen-binding region, characterized in that the antigen binding protein: a. does not exhibit CDC or ADCC; and b. does not deplete V61 T-cells.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 mutein as described herein also stimulates V61 T-cell proliferation. T-cell depletion is the process of T cell death, removal or reduction. References to an antigen binding protein in a conjugate with an IL-21 mutein as described herein not depleting the V61 T cells refers to a depletion of less than about 30% or less than about 20% (preferably less than about 10%) of the viable V61 T+ cell population when incubated by one or more of the antigen binding proteins as described herein, and as measured by any via suitable means in a controlled study (for example via controlled flow cytometry methodology or via other established controlled assays). ADCC and CDC are mechanisms by which T-cell depletion may occur. Reference to an antigen binding protein in a conjugate with an IL-21 mutein as described herein not causing ADCC or CDC refers to a depletion of less than about 30% or less than about 20% (preferably less than about 10%) of the viable V61 T+ cell population via ADCC and/or CDC when incubated by one or more of the antigen binding proteins as described herein, as measured by any via suitable means (for example via controlled flow cytometry methodology or via other established controlled assays).
In one embodiment, there is provided an antigen binding protein in a conjugate with an IL-21 mutein as described herein, characterized in that it does not induce secretion of IL-17A. IL-17A (lnterleukin-17A) is a pro-tumorigenic cytokine which is produced by activated T-cells. IL-17A can enhance tumor growth and dampen the anti- cancer immune response. Reference to an antigen binding protein in a conjugate with an IL-21 mutein as described herein not inducing secretion of IL-17A refers to inducing less than about 30%, or less than about 20%, or less than about 10% of the IL-17A secretion induced by equivalent anti-CD3 antigen binding proteins.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises a third antigen-binding region that specifically binds to a V61 TCR, which antigen-binding region comprises a combination of complementarity-determining regions (CDRs) CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 sequence of SEQ ID NO: 511 , the CDR-H2 sequence of SEQ ID NO: 512, CDR-H3 sequence of SEQ ID NO: 513, the CDR-L1 sequence of SEQ ID NO: 514, the CDR- L2 sequence of SEQ ID NO: 515 (amino acids YDS) and the CDR-L3 sequence of SEQ ID NO: 516; b) the CDR-H1 sequence of SEQ ID NO: 517, the CDR-H2 sequence of SEQ ID NO: 518, CDR-H3 sequence of SEQ ID NO: 519, the CDR-L1 sequence of SEQ ID NO: 520, the CDR-L2 sequence of SEQ ID NO: 521 (amino acids DAS) and the CDR-L3 sequence of SEQ ID NO: 522; c) the CDR-H1 sequence of SEQ ID NO: 523, the CDR-H2 sequence of SEQ ID NO: 524, CDR-H3 sequence of SEQ ID NO: 525, the CDR-L1 sequence of SEQ ID NO: 526, the CDR-L2 sequence of SEQ ID NO: 527 (amino acids VAS) and the CDR-L3 sequence of SEQ ID NO: 528.
In one embodiment, an antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises a third antigen-binding region that specifically binds to a V61 TOR, which antigen-binding region comprises a combination of variable heavy (VH) and variable light (VL) domain sequences that have, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the VH and VL sequence combinations selected from the group consisting of: a) the VH sequence as comprised in SEQ ID NO: 529, and the VL sequence as comprised in SEQ ID NO: 530; b) the VH sequence as comprised in SEQ ID NO: 531 , and the VL sequence as comprised in SEQ ID NO: 532; c) the VH sequence as comprised in SEQ ID NO: 533, and the VL sequence as comprised in SEQ ID NO: 534.
An antigen-binding region with affinity for a surface antigen expressed on NK cells
In one embodiment, an antigen binding protein in a conjugate with an IL-21 mutein as described herein, can further comprise a third antigen-binding region, which is an antigen-binding region that has or can have affinity for a surface antigen expressed on NK cells.
In one embodiment of the an antigen binding protein in a conjugate with an IL-21 mutein as described herein, the third antigen-binding region that has or can have affinity for a surface antigen expressed on NK cells comprises or consists of an immunoglobulin Fc region, or at least a portion thereof that binds the type III Fey receptor (FcyRllla) as expressed on (human) NK cells, also referred to herein as CD16A. In one embodiment, the immunoglobulin Fc region at least comprises at least one of a CH2 and CH3 domain. In one embodiment, the immunoglobulin Fc region at least comprises at least one of a CH2 and CH3 domain and a hinge region. In one embodiment, the immunoglobulin Fc region comprises or consists of a hinge region and a CH2 and CH3 domain. In one embodiment, the immunoglobulin Fc region is a dimeric Fc region or at least a portion thereof that binds CD16A.
In one embodiment, an Fc region or portion thereof that binds CD16A be a wild-type region or portion thereof.
In one embodiment, an Fc region or portion thereof that binds CD16A can be modified to enhance or reduce its binding affinity to CD16A. Within the Fc region, CD16A binding is mediated by the hinge region and the CH2 domain. For example, within human lgG1 , the interaction with CD16 is primarily focused on amino acid residues D265 - E269, N297 - T299, A327 - I332, L 234 - S239, and carbohydrate residue N-acetyl-D-glucosamine in the CH2 domain (see, Sondermann et al., 2000 Nature, 406(6793):267-273). Based on the known domains, mutations can be selected to enhance or reduce the binding affinity to CD16A, such as by using phage-displayed libraries or yeast surface-displayed cDNA libraries or can be designed based on the known three-dimensional structure of the interaction. In one embodiment, the Fc region or portion is lgG2. Thus, in one embodiment, where the antigen binding protein is intended to have increased affinity for CD16A, an Fc region or portion thereof that binds CD16A, can comprise a modification to increase affinity for CD16A. Thus, an Fc region or portion thereof that binds CD16A, can comprise one or more amino acid modifications (e.g. amino acid substitutions, deletions, insertions) which increase binding to (human) CD16A and optionally another receptor such as FcRn. Typical modifications include modified human lgG1 -derived constant regions comprising at least one amino acid modification (e.g. substitution, deletions, insertions), and/or altered types of glycosylation, e.g., hypofucosylation. A modification can, for example, increase binding of an Fc region to FcyRllla (CD16A) on NK cells. Examples of modifications are provided in US 10,577,419, the disclosure of which is incorporated herein by reference. Specific mutations (in lgG1 Fc regions) which enhance FcyRllla (CD16A) binding, include E333A, S239D/I332E and S239D/A330L/I332E.
In one embodiment, the antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an Fc region or portion thereof that binds CD16A comprising at least one amino acid modification (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications) relative to a wild-type Fc region, such that the molecule has enhanced binding affinity for (human) CD16A relative to a molecule comprising a wild-type Fc region, optionally wherein the variant Fc region comprises a substitution at any one or more of positions 239, 298, 330, 332, 333 and/or 334 (e.g. S239D, S298A, A330L, I332E, E333A and/or K334A substitutions), optionally wherein the variant Fc region comprises a substitution at residues S239 and I332, e.g. a S239D and I332E substitution (Kabat EU numbering).
In one embodiment, the antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an Fc region or portion thereof that binds CD16A comprising altered glycosylation patterns that increase binding affinity for (human) CD16A. Such carbohydrate modifications can be accomplished by, for example, by expressing a nucleic acid encoding the antigen binding protein in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery are known in the art and can be used as host cells in which to express recombinant antibodies to thereby produce an antibody with altered glycosylation. See, for example. Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1 , as well as, European Patent No: EP 1 ,176,195; WO 06/133148; WO 03/035835; WO 99/54342, each of which is incorporated herein by reference in its entirety. In one embodiment, the antigen binding protein comprises one or more hypofucosylated constant regions. Such an antigen binding protein can comprise an amino acid alteration or cannot comprise an amino acid alteration and/or may be expressed or synthesized or treated under conditions that result in hypofucosylation. In one embodiment, in a composition comprising the antigen binding protein in a conjugate with an IL-21 mutein as described herein, at least 20, at least 30, at least 40, at least 50, at least 60, at least 75, at least 85, at least 90, at least 95% or substantially all of the antigen binding proteins have a constant region comprising a core carbohydrate structure (e.g. complex, hybrid and high mannose structures) which lacks fucose. In one embodiment, there is provided is an antigen binding protein which is free of N-linked glycans comprising a core carbohydrate structure having fucose. The core carbohydrate will preferably be a sugar chain at Asn297. In one embodiment, the antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an Fc region or portion thereof that binds CD16A that is modified to have increased binding affinity for CD16A, has a binding affinity for human CD16A that is at least 1 , 2 or 3 log greater than that of a conventional or wild-type human IgG 1 antibody, e.g., as assessed by surface plasmon resonance.
In another embodiment, where an antigen binding protein is intended to have reduced affinity for CD16A, a CH2 and/or CH3 domain, an Fc region or portion thereof that binds CD16A, can comprise a modification to decrease affinity for CD16A. For example, CH2 mutations in a dimeric Fc region protein at reside N297 (Kabat numbering) can eliminate CD16A binding. Other modification in the Fc region that reduce or eliminate binding to CD16A include the L234A/L235A (also known as “LALA”), the L235A/G237A modification (also known as “LAGA” and described in Liu et al., Antibodies. 2020;9(4):64; Szapacs et al., Bioanalysis. 2023;15(16):955-6), and the L234A/L235A/P329G modification (also known as LALAPG) and which more completely abrogates CD16 binding compared to the LALA modification. In embodiments wherein an antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an antigen-binding region that specifically binds an epitope of a y6 TCR, it is preferred that the antigen binding protein comprises an Fc region that is modified to reduce or eliminate binding to CD16A.
Modification of the Fc region that reduce or eliminate its binding to CD16A can be useful to prevent or at least reduce the “sink effect”, wherein at least a fraction of the administered amount of multispecific antigen binding protein is lost by binding to NK cells or monocytes. Modification of the Fc region that reduce or eliminate its binding to CD16A can also be useful in an antigen binding protein to further attenuate apparent affinity to NK cells reducing activity in absence of target cells. Modification of the Fc region that reduce or eliminate its binding to CD16A can also be useful in an antigen binding protein to reduce or avoid NK cell fratricide. The lack of NK cell fratricide can be an advantageous feature for the antigen binding protein described herein. NK cell cross-linking with NK cells or other immune cells is expected to reduce therapeutic efficacy of NK cell-engagement. Most importantly, cross-linking of a NK cell with one or more NK cells or other immune cells through bivalent or multivalent interactions with FcRy or in combination with a second immune cell antigen (e.g. NKp46, NKG2D, NKp30, SLAMF7 or CD38) can cause immune cell activation. This might lead to induction of target cell-driven fratricide or immune cell killing (e.g. NK-NK cell lysis), ultimately resulting in efficient NK cell depletion in vivo, as previously described for a CD16-directed murine IgG antibody (3G8), the CD38-directed antibody daratumumab and other approaches (Choi et al 2008 Immunology 124 (2) 215-22; DOI: 10.111 l/j.1365-2567.2007.02757.x; Yoshida 2010 Front. Microbiol 1 : 128 DOI: 10.3389/fmicb.2010.00128; Wang et al 2018 Clin Cancer Res, 24(16): 4006- 4017; DOI: 10.1158/1078-0432. CCR-17-3117; His et al 2008; Nakamura 2013 PNAS; 110(23) 9421-9426; DOI: 10.1073/pnas.1300140110; Breman et al 2018 Front Immunol, 12(9)2940; DOI: 10.3389/fimmu.2018.02940).
The person of skill in the art will appreciate that other configurations for modification of Fc regions can be implemented. For example, substitutions into human lgG1 or lgG2 residues at positions 233-236 and lgG4 residues at positions 327, 330 and 331 were shown to greatly reduce binding to Fey receptors and thus ADCC and CDC. Furthermore, Idusogie et al. (2000) J. Immunol. 164(8):4178-84 demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation.
In one embodiment, the antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an Fc region or portion thereof that binds CD16A that is modified to have reduced binding affinity for CD16A, has a binding affinity for human CD16A that is at least 1 , 2 or 3 log less than that of a conventional or wild-type human IgG 1 antibody, e.g., as assessed by surface plasmon resonance.
In one embodiment, the antigen binding protein in a conjugate with an IL-21 mutein as described herein comprises an Fc region that has an amino acid sequence having at least 85, 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 100% amino acid identity with an Fc region in at least one of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 - 19 and 23, and preferably having one or more of the above structural and/or functional features.
In one embodiment, in an antigen binding protein in a conjugate with an IL-21 mutein as described herein, third antigen-binding region can be an antigen-binding region that specifically binds an NK cell activating receptor, such as described above for the second antigen-binding region. In one embodiment, at least one of the second or third antigen-binding region activates the NK cell activating receptor.
Additional agonists for activation of NK cells, a y<5 T cells and/or co-stimulation of y<5 T cells
In one embodiment, an antigen binding protein in a conjugate with an IL-21 mutein as described herein, comprises at least one further agonist, which further agonist can be at least one of: i) a further NK cell-activating cytokine, in addition to the IL-21 mutein, ii) a further y6 T cellactivating agonist, in addition to the IL-21 mutein; and iii) a y6 T cell co-stimulatory agonist.
In one embodiment of the conjugate, the further agonist is the heterologous moiety. In another embodiment of the conjugate, the further agonist is directly attached to the heterologous moiety, or the further agonist is attached to the heterologous moiety via a linker, preferably a (flexible) peptidyl linker as described above, e.g. (GGGGS)n, wherein the copy number “n” can be 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10.
In one embodiment of the conjugate, the further NK cell-activating cytokines can be selected from the group consisting of a 4-1 BB agonist, an IL-15 receptor agonist, an IL-2 receptor agonist, a type I interferon (IFN-1) receptor agonist, an IL-12 receptor agonist and an IL-18 receptor agonist, as further detailed below.
In one embodiment of the conjugate, the further y6 T cell-activating agonist can be selected from the group consisting of: an IL-15 receptor agonist, an IL-2 receptor agonist, a type I interferon (IFN-1) receptor agonist, an IL-12 receptor agonist and an IL-18 receptor agonist, as further detailed below.
In one embodiment of the conjugate, the y6 T cell co-stimulatory agonist is selected from the group consisting of: a 4-1 BB agonist, a CD27 agonist and a GITR agonist. In one embodiment, the conjugate with an IL-21 mutein as described herein thus at least comprises a 4-1 BB agonist.
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, the disclosure of which is incorporated herein by reference. 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 ofT, 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.
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 conjugate as described herein comprising a 4-1 BB agonist.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises or consist of at least one 4-1 BBL ECD or at least one agonistic antigen-binding region that specifically binds 4-1 BB.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that has reduced 4-1 BB agonist activity as compared to human wild type 4-1 BBL or the anti-4-1 BB agonist antibody 2A. In one embodiment, the 4-1 BB agonist has a 4-1 BB agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type 4-1 BBL or the anti-4-1 BB agonist antibody 2A.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that has enhanced 4-1 BB agonist activity as compared to human wild type 4-1 BBL. In one embodiment, the 4-1 BB agonist has a 4-1 BB agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 higher than that of the ECD of human wild type 4-1 BBL or the anti-4-1 BB agonist antibody 2A.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist comprising at least one 4-1 BBL ECD comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 37, and preferably having an 4-1 BB agonist activity as defined above, and/or preferably having an affinity for 4-1 BB as defined below.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist of which the affinity for the 4-1 BB is reduced or enhanced as compared to the ECD of human wild type 4- 1 BBL. The affinity of a 4-1 BB agonist of the affinity for the 4-1 BB can be assayed using methods generally known in the art, such as surface plasmon resonance.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that has reduced affinity for 4-1 BB as compared to human wild type 4-1 BBL. In one embodiment, the affinity of the 4-1 BB agonist for 4-1 BB is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type 4-1 BBL or the anti-4-1 BB agonist antibody 2A.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that has enhanced affinity for 4-1 BB as compared to human wild type 4-1 BBL. In one embodiment, the affinity of the 4-1 BB agonist for 4-1 BB is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 higher than that of the ECD of human wild type 4-1 BBL or the anti-4-1 BB agonist antibody 2A.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises or consists of the ECD of 4-1 BBL or a fragment thereof that has 4-1 BB agonist activity. In one embodiment, the 4-1 BB agonist in the conjugate is a mutein 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 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
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 1 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.
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: 100, 101 , 110, 114, 1 15, 116, 153, 154, 171 , 227 and 230.
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.
In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution selected from the group consisting of: V100T, V100Q, V100S, V100A, V100G, V100N, V100D, V100E, V100K, V100R, L101 N, L101 E, L101 Q, L101 D L101 R, L101 K, Y110Q, Y110E, Y110N, Y1 10D, Y110R, Y110K, Y110S, Y110A, Y1 10G, Y110T, G1 14K, G114R, G114Q, G114D, G114E, G114N, G114S, G114A, G114G, G114T, L1 15R, L115K, L115Q, L115N, L1 15D, L115E, L115S, L115A, L115G, L115T, A116D, A116E, A116R, A116K, A116Q, A116N, A116Y, A116H, V153Q, V153N, V153R, V153K, V153D, V153E, V153S, V153G, A154D, A154E, A154R, A154K, A154Q, A154N, A154Y, A154H, 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.
In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution selected from the group consisting of: A154D, Y110Q, V153Q, V100T, V100Q, L101 N, G114K, L115R, A116D, R171 D, Q227E, Q227R, Q230S and Q230K, of which A154D, Y110Q, and V153Q are preferred, of which A154D and Y110Q are more preferred, and of which A154D is most preferred.
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: V100T, V100Q, V100S, V100A, V100G, V100N, V100D, V100E, V100K, V100R, L101 N, L101 E, L101Q, L101 D L101 R, L101 K, Y110Q, Y110E, Y110N, Y1 10D, Y110R, Y110K, Y110S, Y110A, Y110G, Y1 10T, 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, V153Q, V153N, V153R, V153K, V153D, V153E, V153S, V153G, A154D, A154E, A154R, A154K, A154Q, A154N, A154Y, A154H, 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.
In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least two, three, four or five amino acid substitution selected from the group consisting of: A154D, Y110Q, V153Q, V100T, V100Q, L101 N, G1 14K, L115R, A116D, G155Q, R171 D, Q227E, Q227R, Q230S and Q230K, of which A154D, Y110Q, and V153Q are preferred, of which A154D and Y110Q are more preferred, and of which A154D is most preferred.
In one embodiment, there is provided a 4-1 BBL ECD mutein comprising at least one amino acid substitution a position in SEQ ID NO: 37 selected from the substitutions listed in Table C.
Table C. Additional substitutions, deletions and insertions for 4-1 BBL ECD muteins
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 an A154D or an A154E substitution, of which the A154D substitution is preferred. In one embodiment, there is provided a 4-1 BBL ECD mutein comprising an A154D substitution in combination with at least one, two, three, four or five amino acid substitutions selected from the group consisting of: V100T, V100Q, L101 N, Y110Q, G1 14K, L115R, A116D, V153Q, 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: A154D and V100T; A154D and V100Q; A154D and L101 N; A154D and Y110Q; A154D and G114K; A154D and L1 15R; A154D and A116D; A154D and V153Q; A154D and G155Q; A154D and R171 D; A154D and Q227E; A154D and Q227R; A154D and Q230S; and A154D and Q230K.
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 153, and wherein preferably the 4-1 BBL ECD mutein comprises a V153Q 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, V100Q, L101 N, Y110Q, G1 14K, L115R, A116D, A154D, 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 V100Q; V153Q and L101 N; V153Q and Y110Q; V153Q and G1 14K; V153Q and L115R; V153Q and A116D; V153Q and A154D; V153Q and R171 D; V153Q and Q227E; V153Q and Q227R; V153Q and Q230S; and V153Q 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, V153Q and Q227E; L101 N, Y110Q and V153Q; V100Q, Y110Q and V153Q; L101 N, V153Q and Q227E; Y110Q, A154D and Q227E; Y110Q and Q227E; and, L101 N and Q227E.
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; L1 15D and A116X; A116D and G117X; 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.
In one embodiment, there is provided an antigen binding protein in a conjugate as described herein comprising a combination of an IL-21 mutein as described herein and a 4-1 BBL mutein as described herein, as shown in Table D.
Table D. Combination of 4-1 BBL and IL-21 muteins conjugated to an antigen binding protein.
In specific embodiments 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- I66-); 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- I66-); 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- 166-); 4-1 BBL mutein Q227E and IL-21 mutein L74F; 4-1 BBL mutein Q227R and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein Q227R and IL-21 mutein L20W; 4-1 BBL mutein Q227R and IL-21 mutein L74D; 4-1 BBL mutein Q227R and IL-21 mutein L20N; 4-1 BBL mutein Q227R and IL-21 mutein I67N; 4-1 BBL mutein Q227R and IL-21 mutein L20S; 4-1 BBL mutein Q227R and IL-21 mutein L13E; 4-1 BBL mutein Q227R and IL-21 mutein I8H; 4-1 BBL mutein Q227R and IL-21 mutein (N63- E64- R65- I66-); 4-1 BBL mutein Q227R and IL-21 mutein L74F; 4- 1 BBL mutein L101 N and IL-21 mutein (N82- A83- G84- R85- R86- Q87- K88-); 4-1 BBL mutein
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 V1 OOQ and IL-21 mutein L20S; 4-1 BBL mutein V1 OOQ 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.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises or consists of 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 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)s, (GGGGS)4, (GGGGS)5, GGGSGGG, GGSGGGGSGG and G, of which (GGGGS)4 is 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. In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises or consists of a fusion protein comprising three 4-1 BBL ECD monomers fused together in a single polypeptide chain, e.g. comprising the amino acid sequence of SEQ ID NO: 36, except for the mutein-specific mutations.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises three 4-1 BBL ECD monomers that are present in more than one polypeptide chain of the conjugate. For example, two 4-1 BBL ECD monomers can be fused together in a single polypeptide chain, optionally connected through polypeptide linker as described above, that is part of a first polypeptide chain of the conjugate and the third 4-1 BBL ECD monomer is part of a second polypeptide chain of the conjugate, as e.g. described in WO2016/075278. The first and second polypeptide chains of the conjugate can be the chains comprising the heavy and light chains of an antigen binding protein in the conjugate, respectively, or vice versa, whereby preferably the 4-1 BBL ECDs are fused to the N-termini of the variable domains. Alternatively, the first and second polypeptide chains of (an antigen binding protein in) the conjugate can be the two chains comprising the two heavy chains, whereby preferably the 4-1 BBL ECDs are fused to the C-termini of the constant domains.
In one embodiment, a conjugate as described herein comprises a 4-1 BB agonist that comprises an antigen-binding region that specifically binds 4-1 BB and that has 4-1 BB agonistic activity. Antibodies against 4-1 BB are e.g. described in W02005035584, W02006088464, US2006188439.
In one embodiment, a conjugate as described herein comprises more than one 4-1 BB agonist as described above. Thus, in one embodiment, the conjugate has a 4-1 BB agonist-valency that is higher than one. The 4-1 BB agonist-valency of a conjugate can for example be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more.
In further embodiments, the conjugate as described herein can comprise, in addition to the IL-21 mutein, and optionally a 4-1 BB agonist, a further NK cell-activating cytokine and/or further y6 T cell-activating agonist selected from the group consisting of: an IL-15 receptor agonist, type I interferon (IFN-1) receptor agonist, an IL-2 receptor agonist, an IL-12 receptor agonist and an IL- 18 receptor agonist. In one embodiment, the IL-15 receptor agonist is an IL-15 polypeptide or an agonistic antigen-binding region that specifically binds the IL-15 receptor. In one embodiment, 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. In one embodiment, the IFN-1 polypeptide is an IFN-1 mutein that is modified to reduce (or enhance) affinity for the IFN-a receptor, relative to a corresponding wild type IFN-1 polypeptide. In one embodiment, the IL-2 receptor agonist is an IL-2 polypeptide or an agonistic antigen-binding region that specifically binds the IL-2 receptor. In one embodiment, the IL-12 receptor agonist is an IL-12 polypeptide or an agonistic antigen-binding region that specifically binds the IL-12 receptor. In one embodiment, the IL-18 receptor agonist is an IL-18 polypeptide or an agonistic antigen-binding region that specifically binds the IL-18 receptor.
In one embodiment, a conjugate as described herein comprises an IL-15 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 169 (i.e. the functional domain of IL-15, corresponding to amino acids 49-162 of Uniprot acc. No. P40933). The IL-15 polypeptide preferably has IL-15 receptor agonist activity as can be assayed as described in the art (see e.g. US 20230158164), e.g. by assaying STAT5 phosphorylation in a bio-assay, using reporter cells (e.g. https://nld.promeqa.com/products/reporter-bioassays/cytokine-and-qrowth-factor-bioassays/il-15- The IL-15 polypeptide preferably has the ability to specifically bind to an IL-15 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance.
In one embodiment, the IL-15 polypeptide is an IL-15 mutein that is modified to reduce (or enhance) affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide. In one embodiment, the IL-15 mutein has reduced affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide, and wherein the IL-15 mutein has a mutation (i.e. amino acid substitution, deletion or insertion) of one or more amino acids selected from the group consisting of: N1 , D8, D30, H32 L45, E46, I50, V49, S51 , L42, V63, E64, N65, I68, L69, Q108, M109, N1 12 (amino acid positions referring to position in SEQ ID NO: 168 or a corresponding position in an IL-15 allelic variant). Preferably, the IL-15 mutein comprises one or more amino acid substitutions selected from the group consisting of: N1 G, D8S, D30N, D30T, H32D, H32E, H32N, H32Q, E46G, E46K, V49R, V49D, I50D, V63F, V63A, V63K, V63R, E64Q, E64K, E64R, N65D, N65E, N65K, N65R, I68F, I68H, I68D, I68K, I68R, I68E, I68Q, I68G, L69D, L69E, L69K, L69R, Q108A, Q108D, Q108E, Q108F, Q108H, Q108K, Q108L, Q108M, Q108N, Q108S, Q108T, Q108Y, M109A, M109H, M109R, N112D, N112G, N1 12P and N112R.
In one embodiment, the IL-15 mutein has reduced affinity for at least one of IL-15Rp and IL- 15Ry, relative to a corresponding wild type IL-15 polypeptide. IL-15 muteins with an amino acid substitution in D8 or Q108 (e.g. D8S or Q108S) have a reduced affinity for IL-15R0 and IL-15Ry, respectively.
In a preferred embodiment, the IL-15 mutein has reduced affinity for IL-15R0, relative to a corresponding wild type IL-15 polypeptide. Thus, a preferred IL-15 mutein has an amino acid substitution in at least one of D8 (e.g. D8S), E64, N65, I68, and L69.
In one embodiment, a conjugate as described herein comprises an IL-2 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 170 (i.e. the functional domain of IL-2, corresponding to amino acids 21-153 of Uniprot acc. No. P60568). The IL-2 polypeptide preferably has IL-2 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT5 phosphorylation in a bio-assay, using reporter cells (e.g. https://nld.promeqa.com/products/reporter-bioassavs/cytokine-and-arowth-factor-bioassays/il2- The IL-2 polypeptide preferably has the ability to specifically bind to an IL-2 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance. In one embodiment, the IL-2 polypeptide is an IL-2 mutein that is modified to reduce (or enhance) affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide.
In one embodiment, a conjugate as described herein comprises an IL-12 polypeptide comprising i) an IL-12a polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 172 (i.e. the functional domain of IL-12 a, corresponding to amino acids 23-219 of Uniprot acc. No. P29459), and, ii) an IL-120 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 172 (i.e. the functional domain of IL-120, corresponding to amino acids 23-328 of Uniprot acc. No. P29460). The IL-12 polypeptide preferably comprises, in an N- to C-terminal direction, an aforementioned IL-120 polypeptide linked to an aforementioned IL-12a polypeptide, whereby the IL-120 polypeptide is linked to the IL-12a polypeptide through a peptidyl linker as herein described, such as (GGGGS)n , whereby n = 3, 4, 5, 6 or 7. The IL-12 polypeptide preferably has IL-12 receptor agonist activity as can be assayed as described in the art, e.g. by assaying STAT4 phosphorylation in a bio-assay, using reporter cells (e.g. https://www.invivogen.com/hek-blue-il12). The IL-12 polypeptide preferably has the ability to specifically bind to an IL-12 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance. In one embodiment, the IL-12 polypeptide is an IL-12 mutein that is modified to reduce (or enhance) affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide.
In one embodiment, a conjugate as described herein comprises an IL-18 polypeptide comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 173 (i.e. the functional domain of IL-18, corresponding to amino acids 37-193 of Uniprot acc. No. Q14116). The IL-18 polypeptide preferably has IL-18 receptor agonist activity as can be assayed as described in the art, e.g. by assaying NF- kB activation in a bio-assay, using reporter cells (e.g. https://www.invivoqen.com/hek-blue-il18). The IL-18 polypeptide preferably has the ability to specifically bind to an IL-18 receptor, as can be determined using methods generally known in the art, such as surface plasmon resonance. In one embodiment, the IL-18 polypeptide is an IL-18 mutein that is modified to reduce (or enhance) affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide. In one embodiment, the conjugate with an IL-21 mutein as described herein thus at least comprises a CD27 agonist, e.g. as a y6 T cell co-stimulatory agonist. CD27 is a member of the tumor necrosis factor receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 7 (TNFRSF7) or S 152. CD274-1 BB is encoded by the TNFRSF7 gene (Entrez Gene ID: 939). An amino acid sequence for human CD27 is described in GenBank Number NP_001233 or Uniprot accession numbers P26842. CD27 known as a co-stimulatory immune checkpoint molecule. CD27 is expressed on both naive and activated effector T cells as well as NK cells and activated B cells. It is a type I transmembrane protein with cysteine-rich domains. The ligand for CD27 is CD70, also known as TNFSF7 or CD27L.
CD70 is a protein that in humans is encoded by the TNFSF7 gene (Entrez Gene ID: 970). An amino acid sequence for human CD70 is described in NCBI accession number NP_001243 (SEQ ID NO: 535).
As used herein, a “CD27 agonist” is an agent that has “agonist” activity at the CD27, which means that the agent that can cause or increase "CD27 signaling". “CD27 signaling” refers to an ability of CD27, e.g. when expressed on the surface of T, B and NK cells and triggered by its natural ligand CD70, to activate or transduce an intracellular signaling pathway. The “natural CD27 ligand” is herein understood as the extracellular domain (ECD) of a human wild type CD70 comprising or consisting of an amino acid sequence from position 54 to 193 of the amino acid sequence of human CD70 (i.e. SEQ ID NO: 535). A CD70 extracellular domain (ECD) is herein thus understood as a polypeptide comprising or consisting of an amino acid sequence from positions 54 to 193 of human CD70, which amino acid sequence is depicted in SEQ ID NO: 536, or a fragment thereof having CD27 agonist activity.
The interaction between CD27 and CD70 leads to the recruitment of intracellular adaptor proteins, such as TRAF2 and TRAF5, which then activate signaling pathways, including the NF-KB and JNK pathway. CD27 agonist activity, i.e. changes in CD27 signaling activity, can be measured, for example, by assays designed to measure changes in the CD27 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 CD27 (e.g. production of specific cytokines). A suitable cell-based assay for in vitro biological activity of a CD27 agonist, is e.g. described in Wyzgol et al. 2009 (J. Immunol. 183(3): 1851 -61 ). Natural CD70 or a CD70 ECD trimer, e.g. a CD70 ECD trimer comprising the amino acid sequence of SEQ ID NO: 537 can serve as a positive control in an assay for CD27 agonist activity and can also be used as a reference for the amount of CD27 agonist activity of a given non-natural CD27 agonist, such as a conjugate with an IL-21 mutein as described herein comprising a CD27 agonist.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that has reduced CD27 agonist activity as compared to human wild type CD70. In one embodiment, the CD27 agonist has a CD27 agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type CD70. In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that has enhanced CD27 agonist activity as compared to human wild type CD70. In one embodiment, the CD27 agonist has a CD27 agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 1 ,000 or 10,000 higher than that of the ECD of human wild type CD70.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist comprising at least one CD70 ECD comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 536 359, and preferably having an CD27 agonist activity as defined above, and/or preferably having an affinity for CD27 as defined below.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist of which the affinity for the CD27 is reduced or enhanced as compared to the ECD of human wild type CD70. The affinity of a CD27 agonist of the affinity for the CD27 can be assayed using methods generally known in the art, such as surface plasmon resonance.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that has enhanced affinity for CD27 as compared to human wild type CD70 or wild type CD70 ECD. In one embodiment, the affinity of the CD27 agonist for CD27 is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 higher than that of the ECD of human wild type CD70 or wild type CD70 ECD.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that has reduced affinity for CD27 as compared to human wild type CD70 or wild type CD70 ECD. In one embodiment, the affinity of the CD27 agonist for CD27 is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type CD70 or wild type CD70 ECD.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that is a CD70 ECD mutein as described herein below and that has reduced affinity for CD27 as compared to human wild type CD70.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that comprises or consists of the ECD of CD70 or a fragment thereof that has CD27 agonist activity. In one embodiment, the CD27 agonist in the conjugate is a mutein of the CD70 ECD comprising at least one substitution, deletion and/or insertion. Amino acid substitutions, deletions and insertions in a CD70 ECD mutein provided herein are indicated relative to the wild-type human CD70 ECD amino acid sequence, which is provided herein as SEQ ID NO: 536. Hence, to allow for allelic variation, a wild-type human CD70 ECD preferably comprises an amino acid sequence having, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 536.
Liu et al. (2021 , J. Biol. Chem. 297(4) 101102) disclose amino acid residues of CD70 that reduce its affinity for CD27 as compared to wild type CD70, which residues include Q61 , A80, S137, S146 and H148.
Thus, in one embodiment, there is provided a CD70 ECD mutein comprising at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 536 selected from the group consisting of: Q61 , A80, S137, S146 and H148. In one embodiment, there is provided a CD70 ECD mutein comprising at least one amino acid substitution selected from the group consisting of: Q61A, A80F, A80R, S137A, S137E, S137K, S146A, H148A, H148E and H148D.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that comprises or consists of a fusion protein comprising three CD70 ECD monomers fused together in a single polypeptide chain. It is understood herein that three CD70 ECD monomers fused together in a single polypeptide chain can be three monomers of any of the CD70 ECD muteins described herein. In one embodiment, the three CD70 ECD monomers are connected by polypeptide linkers. In one embodiment, the three CD70 ECD monomers are connected by polypeptide linkers selected from the group consisting of: GGGSGGG, GGSGGGGSGG, (GGGGS)n, (GGGSS)n, (GGSSS)n and (GSSSS)n, whereby n = 3, 4, 5, 6 or 7, of which 4 is preferred and of which (GGGGS)4 is more preferred. Other suitable flexible polypeptide linker(s) are described herein below. In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that comprises or consists of a fusion protein comprising three CD70 ECD monomers fused together in a single polypeptide chain, e.g. comprising the amino acid sequence of SEQ ID NO: 537.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that comprises three CD70 ECD monomers that are present in more than one polypeptide chain of the conjugate. For example, two CD70 ECD monomers can be fused together in a single polypeptide chain, optionally connected through polypeptide linker as described above, that is part of a first polypeptide chain of the multispecific antigen binding protein and the third CD70 ECD monomer is part of a second polypeptide chain of the conjugate, as e.g. described in WO2016/075278. The first and second polypeptide chains of the antigen binding protein in the conjugate can be the chains comprising the heavy and light chains of an antigen binding protein in the multispecific antigen binding protein, respectively, or vice versa, whereby preferably the CD70 ECDs are fused to the N-termini of the variable domains. Alternatively, the first and second polypeptide chains of (an antigen binding protein in) the conjugate can be the two chains comprising the two heavy chains, whereby preferably the CD70 ECDs are fused to the C-termini of the constant domains.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a CD27 agonist that comprises an antigen-binding region that specifically binds CD27 and that has CD27 agonistic activity.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises more than one CD27 agonist as described above. Thus, in one embodiment, the conjugate has a CD27 agonist-valency that is higher than one. The CD27 agonist-valency of a conjugate can for example be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 or more.
In one embodiment, the conjugate with an IL-21 mutein as described herein thus at least comprises a GITR agonist, e.g. as a y6 T cell co-stimulatory agonist. GITR (glucocorticoid-induced TNFR-related protein) is a member of the tumor necrosis factor receptor family. Its alternative names are tumor necrosis factor receptor superfamily member 18 (TNFRSF18) and CD357. GITR is encoded by the human TNFRSF18 gene (Entrez Gene ID: 8784). An amino acid sequence for human GITR is described in NCBI accession numbers NP_004186, NP_683699 and NP_683700. GITR is constitutively expressed on CD25+CD4+ regulatory T cells and its expression is upregulated on all T cell subsets after activation. GITR is also expressed on neutrophils and NK cells. GITR is co-stimulatory surface receptorforT cells and after interaction with GITRL maintain T cell activation, proliferation, cytokine production, and rescue T cells from anti-CD3-induced apoptosis. GITR can be used as Treg marker and its signaling abrogates the suppressive function of regulatory T cells.
GITR ligand (GITRL), also known as TNFSF18, is a protein that in humans is encoded by the TNFSF18 gene (Entrez Gene ID: 8995). An amino acid sequence for human GITRL is described in NCBI accession number NP_005083 (SEQ ID NO: 538). The human GITR/GITRL complex consists of three monomeric GITRs bound to a trimeric GITRL (Wang et al. 2021 , Nature Comm. 12: 1378).
As used herein, an “GITR agonist” is an agent that has “agonist” activity at GITR, which means that the agent that can cause or increase "GITR signaling". “GITR signaling” refers to an ability of GITR, e.g. when expressed on the surface of T, B and NK cells and triggered by its natural ligand GITRL, to activate or transduce an intracellular signaling pathway, which signaling is propagated via recruiting TRAF-family members, specifically TRAF1 , TRAF2 and TRAF5, to the GITR-signaling complex. The signaling is then mediated through NF-kB and MAPK pathways. The “natural GITR ligand” is herein understood as the extracellular domain (ECD) of a human wild type GITRL comprising or consisting of an amino acid sequence from position 50 to 177 of the amino acid sequence of human GITRL (i.e. SEQ ID NO: 539). A GITRL extracellular domain (ECD) is herein thus understood as a polypeptide comprising or consisting of an amino acid sequence from positions 50 to 177 of human GITRL which amino acid sequence is depicted in SEQ ID NO: 539, or a fragment thereof having GITR agonist activity.
GITR agonist activity, i.e. changes in GITR signaling activity, can be measured, for example, by assays designed to measure changes in the GITR 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 GITR (e.g. production of specific cytokines). A suitable cell-based assay for in vitro biological activity of a GITR agonist, is e.g. described in Wang et al. (2021 , supra), using measurement of IL-2 production from 3A9 cell lines expressing wild-type hGITRs in microtiter plates precoated with an anti-mCD3 monoclonal antibody (BD Biosciences #553058 or BioLegend #100340). Alternatively, GITR agonist activity can be assayed using NFKB-luciferase reporter cells stably expressing GITR and measuring luminescence, driven by agonism of GITR and subsequent activation of the NFkB pathway, as described in WQ2017/025610.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that has reduced GITR agonist activity as compared to human wild type GITR. In one embodiment, the GITR agonist has a GITR agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type GITRL. In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that has enhanced GITR agonist activity as compared to human wild type GITRL. In one embodiment, the GITR agonist has a GITR agonist activity that is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 higher than that of the ECD of human wild type GITRL.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist comprising at least one GITRL ECD comprising an amino acid sequence with at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 96, at least 97, at least 98, at least 99, or 100% sequence identity to SEQ ID NO: 539, and preferably having an GITR agonist activity as defined above, and/or preferably having an affinity for GITR as defined below.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist of which the affinity for the GITR is reduced or enhanced as compared to the ECD of human wild type GITRL. The affinity of a GITR agonist of the affinity for the GITR can be assayed using methods generally known in the art, such as surface plasmon resonance.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that has reduced affinity for GITR as compared to human wild type GITRL. In one embodiment, the affinity of the GITR agonist for GITR is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 less than that of the ECD of human wild type GITRL.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that has enhanced affinity for GITR as compared to human wild type GITRL. In one embodiment, the affinity of the GITR agonist for GITR is a factor 2, 5, 10, 20, 50, 100, 200, 500 or 1000 higher than that of the ECD of human wild type GITRL.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that comprises or consists of the ECD of GITRL or a fragment thereof that has GITR agonist activity. In one embodiment, the GITR agonist in the conjugate is a mutein of the GITRL ECD comprising at least one substitution, deletion and/or insertion. Amino acid substitutions, deletions and insertions in a GITRL ECD mutein provided herein are indicated relative to the wild-type human GITRL ECD amino acid sequence, which is provided herein as SEQ ID NO: 539. Hence, to allow for allelic variation, a wild-type human GITRL ECD preferably comprises an amino acid sequence having, with increasing preference, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 539.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that comprises or consists of a fusion protein comprising three GITRL ECD monomers fused together in a single polypeptide chain. It is understood herein that three GITRL ECD monomers fused together in a single polypeptide chain can be three monomers of any of the GITRL ECD muteins described herein. In one embodiment, the three GITRL ECD monomers are connected by polypeptide linkers. In one embodiment, the three GITRL ECD monomers are connected by polypeptide linkers selected from the group consisting of: GGGSGGG, GGSGGGGSGG, (GGGGS)n, (GGGSS)n, (GGSSS)n and (GSSSS)n, whereby n = 3, 4, 5, 6 or 7, of which 4 is preferred and of which (GGGGS)4 is more preferred. Other suitable flexible polypeptide linker(s) are described herein below.
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises a GITR agonist that comprises an antigen-binding region that specifically binds GITR and that has GITR agonistic activity. Agonistic antibodies against GITR are e.g. described by Liu et al. (2022, Cell Reports Medicine 3, 100660).
In one embodiment, a conjugate with an IL-21 mutein as described herein comprises more than one GITR agonist as described above. Thus, in one embodiment, the conjugate has a GITR agonist-valency that is higher than one. The GITR agonist-valency of a conjugate can for example be 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or more.
Structure of a conjugate comprising an IL-21 mutein and an antigen binding protein
In one embodiment, in a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, the at least one of the first and second antigen-binding regions (that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR) is conjugated to the third antigen-binding region that has or can have affinity for a surface antigen expressed on NK cells. Preferably, the at least one of the first and second antigen-binding regions that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR is conjugated to at least one polypeptide chain of the third antigen-binding region. The conjugation of the two domains/regions is understood to mean that they are covalently linked to each other. The two domains/regions can be chemically cross-linked to each other, using a cross-linker for linking two proteinaceous molecules, as are well-known in the art. There are a number of commercially available crosslinking reagents for preparing protein or peptide bioconjugates. Many of these crosslinkers allow dimeric homo- or heteroconjugation of biological molecules through free amine or sulfhydryl groups in protein side chains. Other crosslinking methods involve coupling through carbohydrate groups with hydrazide moieties. For cross-linking the first and/or second binding regions to the third binding region it is preferred that cross-linkers with heterofunctional specificity are used. In one embodiment, the cross-linker comprises a flexible spacer, to provide flexibility or freedom of motion of the two regions with respect to each other.
In one embodiment, however, the at least one of the first and second antigen-binding regions is conjugated to the third antigen-binding region by being comprised in a single polypeptide chain. As an antigen-binding region can comprise two polypeptide chains, such as a VH and a VL chain, in one embodiment, at least one polypeptide chain in an antigen-binding region that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR, forms a single polypeptide chain with at least one polypeptide chain of the second antigen-binding region. Likewise, as also the third antigen-binding region that has or can have affinity for a surface antigen expressed on NK cells can comprise two polypeptide chains, such as a dimeric Fc region of an antibody, in one embodiment, at least one polypeptide chain of the third antigen-binding region forms a single polypeptide chain with the at least one polypeptide chain in at least one of the first and second antigen-binding region. Thus, in one embodiment, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein comprises a single polypeptide chain that comprises in an N- to C- terminal order: i) at least one polypeptide chain in the at least one of the first and second antigenbinding region; ii) optionally a flexible linker; and iii) (at least one polypeptide chain of) the third antigen-binding region. The flexible linker can be an immunoglobulin hinge region or can be linker as described herein below.
In one embodiment, the third antigen-binding region, i.e. the domain that has or can have affinity for a surface antigen expressed on NK cells, is a dimeric immunoglobulin Fc region, wherein each of the two polypeptide chains of the Fc region is linked to a CH1 domain, each of which CH1 domains is linked to an immunoglobulin variable region of the at least one of the first and second antigen-binding region (that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR). The dimeric immunoglobulin Fc region preferably is a dimer of an Fc region as herein described above. The immunoglobulin variable region can be an 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. In one embodiment, the immunoglobulin variable region that is linked to the CH1 domain is a VH domain that is paired with a VL domain linked to a CK or CA domain. In this embodiment, preferably, the VH and VL domains together specifically bind the TAA or the NK cell activating receptor.
In one embodiment, the two immunoglobulin variable regions bind the same TAA. In another embodiment, the two immunoglobulin variable regions each bind a different TAA. In one embodiment, the first immunoglobulin variable region binds a TAA and the second immunoglobulin variable region binds an NK cell activating receptor. In one embodiment, the first and the second immunoglobulin variable regions both bind an NK cell activating receptor. The two immunoglobulin variable regions can each bind a different NK cell activating receptor or they can both bind the same NK cell activating receptor. In one embodiment, the first immunoglobulin variable region binds a TAA and the second immunoglobulin variable region binds a y6 TCR. In one embodiment, the first and the second immunoglobulin variable regions both bind a y6 TCR. The two immunoglobulin variable regions can each bind a different a y6 TCR or they can both bind the same a y6 TCR.
With respect to its specificity, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein can thus be homodimeric, with two identical immunoglobulin variable regions that both bind the same TAA. Alternatively, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein can thus be heterodimeric with respect to the specificity for TAAs NK cell activating receptor and/or y6 TCR, wherein each of the two immunoglobulin variable regions each bind a different TAA, a TAA and an NK cell activating receptor or a TAA and a y6 TCR. In embodiments, wherein the antigen binding protein is bispecific, it is preferred that one of the two immunoglobulin variable regions is an immunoglobulin single variable domain, while the other immunoglobulin variable region is not. Next, the assembly of heterodimeric antibody heavy chains can be accomplished by expressing two different antibody heavy chain sequences in the same cell, which may lead the assembly of homodimers of each antibody heavy chain as well as assembly of heterodimers. Promoting the preferential assembly of heterodimers can be accomplished by incorporating different mutations in the CH3 domain of each antibody heavy chain constant region as shown in US13/494.870, US16/028850, US11/533,709, US12/875.015, US13/289,934, US14/773,418, US12/811 ,207, US13/866,756, US14/647,480, US 14/830,336 and WO2019/195409. For example, mutations can be made in the CH3 domain based on human Ig G 1 and incorporating distinct pairs of amino acid substitutions within a first polypeptide and a second polypeptide that allow these two chains to selectively heterodimerize with each other. For example, CH3 domains which comprise amino acid substitutions, wherein the CH3 domain interface of the antibody Fc region is mutated to create altered charge polarity across the Fc dimer interface such that co-expression of electrostatically matched Fc chains supports favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation.
In one embodiment, a “knob-into-holes” approach is used in which the CH3 domain interface of the antibody Fc region is mutated so that the antibodies preferentially form heterodimers (further including the attached light chains). These mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. For example, one heavy chain comprises a T366W substitution and the second heavy chain comprises a T366S, L368A and Y407V substitution, see, e.g. Ridgway et al (1996) Protein Eng., 9, pp. 617-621 ; Atwell (1997) J. Mol. Biol., 270, pp. 26-35; and W02009/089004, the disclosures of which are incorporated herein by reference. In another approach, one heavy chain comprises a F405L substitution, and the second heavy chain comprises a K409R substitution, see, e.g., Labrijn et al. (2013) Proc. Natl. Acad. Sci. U.S.A., 110, pp. 5145-5150. In another approach, one heavy chain comprises T350V, L351Y, F405A, and Y407V substitutions and the second heavy chain comprises T350V, T366S, K392L, and T394W substitutions, see, e.g. Von Kreudenstein et al., (2013) mAbs 5:646-654. In another approach, one heavy chain comprises both K409D and K392D substitutions and the second heavy chain comprises both D399K and E356K substitutions, see, e.g. Gunasekaran et al., (2010) J. Biol. Chem. 285:19637-19646. In another approach, one heavy chain comprises D221 E, P228E and L368E substitutions and the second heavy chain comprises D221 R, P228R, and K409R substitutions, see, e.g. Strop et al., (2012) J. Mol. Biol. 420: 204-219. In another approach, one heavy chain comprises S364H and F405A substitutions and the second heavy chain comprises Y349Tand, T394F substitutions, see, e.g. Moore et al., (2011) mAbs 3: 546-557. In another approach, one heavy chain comprises a H435R substitution, and the second heavy chain optionally may or may not comprise a substitution, see, e.g. US Patent no. 8,586,713. When such heteromultimeric antibodies have Fc regions derived from a human lgG2 or lgG4, the Fc regions of these antibodies can be engineered to contain amino acid modifications that permit CD16 binding or that avoid CD16 binding. In some embodiments, the antibody may comprise mammalian antibody-type N-linked glycosylation at residue N297 (Kabat EU numbering).
In one preferred embodiment, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein comprises a dimeric immunoglobulin Fc region that is a (homo or hetero) dimer of at least one Fc region as herein described above, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR. Apart from the presence of the IL-21 mutein (and optional further agonist), the antigen binding protein in a conjugate comprising such a dimeric Fc linked to two Fabs can thus form an immunoglobulin structure, such as a conventional IgG immunoglobulin.
In one embodiment, in a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, at least one of the IL-21 mutein and the further agonist , is conjugated to the at least one of the first and second antigen-binding region that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR, or to the third antigen-binding region. As indicated above conjugation of two proteinaceous entities is understood to mean that they are covalently linked to each other, which can be done by chemical cross-linking, using cross-linkers for linking two proteinaceous molecules, as are well-known in the art, which cross-linker can comprise a flexible spacer.
In one embodiment, however, the at least one of the IL-21 mutein and the further agonist forms a single polypeptide chain with at least one of: i) at least one polypeptide chain in the at least one of the first and a second antigen-binding regions that specifically binds a TAA, NK cell-activating receptor or an epitope of a y6 TCR; and, ii) (at least one polypeptide chain in) the second antigenbinding region that has or can have affinity for a surface antigen expressed on NK cells. In one embodiment, a flexible linker (as described below) is present between the agonist and the region defined in i) or ii).
In one embodiment, the at least one of the IL-21 mutein and the further agonist forms a single polypeptide chain with at least one of: i) a light chain in at least one of the two Fabs that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR; and, ii) at least one of the two Fc chains in the dimeric immunoglobulin Fc region. In one embodiment, a flexible linker (as described below) is present between the at least one of the IL-21 mutein and the further agonist and the light chain defined in i) or the Fc chain defined in ii).
In one embodiment, the at least one of the IL-21 mutein and the further agonist is fused to at least one of: i) the N-terminus of the light chain in at least one of the two Fabs that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR, optionally through a flexible linker; ii) the C-terminus of the light chain in at least one of the two Fabs that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR, optionally through a flexible linker; iii) the N- terminus of the heavy chain in at least one of the two Fabs that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR; and, iv) the C-terminus of the heavy chain in at least one of the two Fc chains in the dimeric immunoglobulin Fc region, optionally through a flexible linker, whereby the flexible linker can be as described below.
In one embodiment, wherein the conjugate comprises a (homo- or hetero) dimeric antigen binding protein as described above, the dimer can comprise at least one of the IL-21 mutein and the further agonist on only one of the two monomers in the dimer, or the dimer can comprise at least one of the IL-21 mutein and the further agonist on each (i.e. both) of the two monomers in the dimer. Thus, in one embodiment, wherein the conjugate comprises an immunoglobulin structure, at least one of the IL-21 mutein and the further agonist can be present on at least one or on both sides of the immunoglobulin structure. In embodiments wherein at least one of the IL-21 mutein and the further agonist is present on each of the two monomers in the dimer or immunoglobulin structure, the antigen binding protein in the conjugate can for example comprises an IL-21 mutein on both monomers, a further agonist (e.g. a 4-1 BB agonist) on both monomers or, an IL-21 mutein on a first monomer and a further agonist (e.g. a 4-1 BB agonist) on a second monomer. It is understood that when the antigen binding protein in the conjugate comprises heterodimeric heavy chains, the "knob- into-hole" technology as described above can be applied, wherein the CH3 domain of the first chain is modified to have a "protuberance" ("knob") and the second chain is modified to have a corresponding "cavity" ("hole").
Thus, in one embodiment, a conjugate comprising antigen binding protein as described herein is heterodimeric with respect to at least one of: i) the first and second antigen-binding regions; and ii) at least one of the fused IL-21 mutein and the fused further agonist, and the dimeric Fc region comprises different first and a second polypeptide chains comprising knob-into-hole modifications promoting association of the first and the second polypeptide chains of the Fc region.
Amino acid sequences of suitable peptidyl linkers for linking the various functional domains and regions in a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein are known in the art as described herein above.
In one embodiment, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein is a conjugate as exemplified herein, such as e.g. AVC22 - AVC40 and AVC114 - AVC126 (see Table 1.1.10.1) or a derivative thereof wherein the trastuzumab variable heavy (VH) and variable light (VL) domains are replaced by variable heavy (VH) and variable light (VL) domains from another monoclonal antibody against a TAA or an NK cell activating receptor, for example, from a monoclonal antibody against a TAA known in the art as herein described above.
In one embodiment, a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, and further comprising a 4-1 BBL mutein, is a conjugate as exemplified herein, such as e.g. AVC72 - AVC107, AVC176, AVC187 - AVC189 and AVC267 (see Tables 1.1 .10.1 and 1 .1 .10.2) or a derivative thereof wherein the trastuzumab variable heavy (VH) and variable light (VL) domains are replaced by variable heavy (VH) and variable light (VL) domains from another monoclonal antibody against a TAA (e.g. such as sacituzumab in AVC267) or an NK cell activating receptor, for example, from a monoclonal antibody against a TAA known in the art as herein described above (such as sacituzumab in AVC267).
In one embodiment, the conjugate comprising an IL-21 mutein and an antigen binding protein comprises: a) a first heavy chain comprising an amino acid sequence with at least 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID NO.’s: 11 , 196 - 218 and 585; b) a second heavy chain comprising an amino acid sequence with at least 95, 96, 97, 98, 99 or 100% sequence identity to any one of SEQ ID NO.’s: 12, 177 - 195, 219 - 237 and 674; and c) a light chain comprising an amino acid sequence with at least 95, 96, 97, 98, 99 or 100% sequence identity to SEQ ID NO: 2 or 584. Biological activities of the conjugate comprising an IL-21 mutein and an antigen binding protein
A conjugate comprising an IL-21 mutein or an antigen binding protein as described herein can have a one or more biological activities, including e.g. antigen (TAA) binding, binding to an NK cell or a T cell, the ability to direct an NK cell or a y6 T cell to a target cell expressing the TAA, activating an NK cell or a y6 T cell, including inducing hyper-functionality of the NK cell or y6 T cell, and/or the ability to elicit lysis of target cell by the (activated/hyper-functional) NK cell or y6 T cell.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 , 0.2, 0.5, 1.0, 1.1 , 1.2, 1 .5, 2.0, 5.0, 10, 20 50 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to the increase achieved with the same effector : target cell ratio, with the same NK cells and target cells that are not brought into contact with the conjugate comprising an IL-21 mutein or the antigen binding protein.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein cause an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 , 0.2, 0.5, 1.0, 1.1 , 1.2, 1 .5, 2.0, 5.0, 10, 20, 50, 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to the increase achieved with the same effector : target cell ratio, with the same NK cells and target cells that are brought into contact (under otherwise identical conditions) with a reference antigen binding protein.
In one embodiment, the reference antigen binding protein is a conventional human lgG1 monoclonal antibody that binds to the same TAA, preferably, that binds to the same epitope, more preferably that has same TAA-specific antigen-binding region(s) as the conjugate comprising an IL- 21 mutein or the antigen binding protein. For example, compared to the monoclonal antibody trastuzumab, a conjugate comprising an IL-21 mutein and an antigen binding protein having a HER2-binding region (preferably having the trastuzumab variable domains), is superior in causing an increase in NK cell activities.
In one embodiment, the reference antigen binding protein is a (multispecific) antigen binding protein comprising at least one antigen-binding region that binds to the same TAA, preferably, that binds to the same epitope, more preferably that has same TAA-specific antigen-binding region(s) as the conjugate comprising an IL-21 mutein or the antigen binding protein, and comprising at least one antigen-binding region that specifically binds an NK cell activating receptor such as NKp46, NKp44, NKp30, NKG2D, DNAM1 and CD16A. In one embodiment, the reference antigen binding protein is a (multispecific) antigen binding protein comprising at least one antigen-binding region that binds to the same TAA, preferably, that binds to the same epitope, more preferably that has same TAA-specific antigen-binding region(s) as the conjugate comprising an IL-21 mutein or the antigen binding protein, and comprising at least one NK cell-activating cytokine other than an IL-21 mutein. The NK cell-activating cytokine other than an IL-21 mutein can be an NK cell activating cytokine is selected from the group consisting of a 4-1 BB agonist, an IL-15 receptor agonist, a type I interferon (IFN-1) agonist, an IL-2 receptor agonist, an IL-12 receptor agonist and an IL-18 receptor agonist, as described above. For example an IL-15 receptor agonist, such as IL15, a human modified IL-15 cross-linker as described in US2018282386 and Vallera et al. (2016, Clin Cancer Res.; 22(14): 3440-3450).
In one embodiment, the reference antigen binding protein is an NK cell engager, such as e.g. described in WO2016/207278, WO 2018/148445, WO2018/152518, WO2019195409 US2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021 , supra). One example of a (multispecific) reference antigen binding protein is for example AVC-006 as described in WO2024/056862, comprising one HER2-binding region and one NKG2D-binding region.
Assays which detect the expression of an NK activation marker or which detects NK cell cytotoxicity, or for detecting NK cell activation and cytotoxicity assays (e.g. short and long term cytotoxicity assays) are described in the Examples herein, as well as for example, in Pessino et al, J. Exp. Med, 1998, 188 (5): 953-960; Sivori et al, Eur J Immunol, 1999. 29:1656-1666; Brando et al, (2005) J. Leukoc. Biol. 78:359-371 ; El-Sherbiny et al, (2007) Cancer Research 67(18):8444-9; Nolte-'t Hoen et al, (2007) Blood 109:670-673); WO 2016/207278 and WO 2018/148445.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein has the ability to induce hyper-functionality (or a hyper-functional phenotype) in an NK cell or in a population of NK cells. A hyper-functional NK cell phenotype is herein understood as a phenotype that has one or more of the features of the phenotype that is obtained by expanding NK cells obtained from donors ex vivo by co-culturing them with irradiated K562 feeder cells modified to express membrane bound IL-21 (mblL-21) and 4-1 BB ligand (FC21 feeder cells) as described by Denman et al. (2012, supra). Thus, in one embodiment, ex vivo expansion of donor NK cells by co-culturing (e.g. for 7, 14 or 21 days), with a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein, produces a population of NK cells having one or more (or preferably all) of the features selected from the group: a) the fold expansion of the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.5, 1 .0, 2.0 or 5.0 fold of the percentage telomere length increase of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the expression level on NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded NK cells is at least 0.5, 1.0, 2.0 or 5.0 fold of the secretion of the cytokine by NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; and, e) the cytotoxicity of the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the cytotoxicity of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells. In a preferred embodiment, the ex vivo expansion of donor NK cells further comprises that the NK cells are co-cultured with tumor cells expressing a TAA specifically bound by the conjugate comprising an IL-21 mutein or the antigen binding protein. Protocols for ex vivo expansion of donor NK cells and assays for determining fold expansion, telomere length increase, expression level of NK cell activating receptors, cytokine secretion and cytotoxicity (e.g. short term or long term cytotoxicity assays) are described in Denman et al. (2012, supra) and in the Examples herein.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein cause an increase in at least one y6 T cell characteristic selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 , 0.2, 0.5, 1 .0, 2.0, 5.0, 10, 20 50 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are not brought into contact with the conjugate or antigen binding protein.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein cause an increase in at least one y6 T cell characteristic selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 , 0.2, 0.5, 1 .0, 2.0, 5.0, 10, 20 50 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are brought into contact with a reference antigen binding protein, e.g. a conventional human lgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the conjugate or antigen binding protein.
The increase in cytokine production preferably is an increase in the production and secretion of inflammatory cytokines such as INFy and TNFa, which promote immune licensing. An increase in cytokine production by y6 T cell as induced by a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein can thus be measured by determining the release of at least one of INFy and TNFa by methods known per se in the art.
The increase in y6 T cell differentiation can be an increase in y6 T cell cytotoxicity, an increase in the expression of CD56 and/or a change in the ratio of CD27 to CD45RA, all of which can be determined by methods known per se in the art.
In one embodiment, the reference antigen binding protein is a conventional human lgG1 monoclonal antibody that binds to the same TAA, preferably, that binds to the same epitope, more preferably that has same TAA-specific antigen-binding region(s) as the conjugate or antigen binding protein. For example, compared to the monoclonal antibody trastuzumab, a multispecific antigen binding protein having a HER2-binding region (preferably having the trastuzumab variable domains), is superior in causing an increase in y6 T cell activities.
In one embodiment, the reference antigen binding protein is a bispecific antibody binding a y6 TCR and a TAA such as described in WO2022/034562, wherein preferably, the bispecific antibody binds a V61 chain of a y6 TCR, preferably that has same V61 chain-specific antigenbinding region(s) as the conjugate or antigen binding protein described herein, and the same TAA, preferably, that binds to the same epitope, more preferably that has same TAA-specific antigenbinding region(s) as the conjugate or antigen binding protein described herein. In one embodiment, the reference antigen binding protein is a multispecific antigen binding protein as described herein, which lacks at least one of i) the first and second antigen binding region (binding the target antigen); ii) the third antigen binding region (binding the y6 TCR); iii) the y6 T cell-activating agonist that induces at least one of STAT5 and STAT3 activation; and iv) the costimulatory agonist.
In one embodiment, a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein has the ability to induce hyper-functionality or innate potency in a y6 T cell or in a population of y6 T cells, in vivo and/or in vitro. In one embodiment, y6 T cell innate like potency is determined by measuring the change in the levels of at least one of CD56 and NKp30 present on a V61 + T cell in a model system. These markers are often used as markers of lymphocyte potency (e.g. cytotoxicity against tumor cells or degranulation upon target recognition or T cell receptor engagement with an agonistic agent.) and can be measured following application of a conjugate comprising an IL-21 mutein or an antigen binding protein described herein to a population of y6 T cells, e.g. by flow cytometry. The change in phenotype of a V61 + T cell or population thereof induced by the conjugate or antigen binding protein as described herein can then be compared to the change in phenotype obtained when a reference antigen binding protein as described above, or an alternative comparator antibody is applied (e.g. OKT-3, TS8.2, etc.) to said equivalent y6 T cells. In one embodiment, the expression level of at least one of CD56 and NKp30 present on V61 + T cells contacted with a conjugate comprising an IL-21 mutein or an antigen binding protein described is increased at least a factor 0.1 , 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20 50 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to the increase achieved with a reference antigen binding protein or comparator antibody as described above, preferably as determined by flow cytometry.
In one embodiment, y6 T cell innate like potency is determined by measuring the killing of TAA negative tumor cells V61 + T cells, after the V61 + T cells have been exposed to a conjugate comprising an IL-21 mutein or an antigen binding protein as described herein for a minimum of 7 days, preferably 14 days in an artificial culture system and compared to the killing by V61 + T cells that have not been exposed to the conjugate or antigen binding protein, or that have been exposed to a reference antigen binding protein as described above, or an alternative comparator antibody (e.g. OKT-3, TS8.2, etc.). In one embodiment, the cytotoxicity against TAA negative tumor cells of V61 + T cells contacted with a conjugate comprising an IL-21 mutein or an antigen binding protein described is increased at least a factor 0.1 , 0.2, 0.5, 1.0, 2.0, 5.0, 10, 20 50 100, 110, 120, 150, 200, 210, 220, 250 or 300 higher as compared to the cytotoxicity against TAA negative tumor cells of V61 + T cells contacted with a reference antigen binding protein or comparator antibody as described above.
Protocols for ex vivo proliferation of donor y6 T cells and assays for determining fold expansion, expression level of CD56 and NKp30, cytokine secretion and cytotoxicity (e.g. short term or long term cytotoxicity assays) are described in the Examples herein.
Pharmaceutical compositions In a further aspect, the invention relates to a pharmaceutical composition comprising an IL-
21 mutein as described herein or a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, and a pharmaceutically acceptable carrier (excipient). The pharmaceutically acceptable carrier such as an adjuvant, or vehicle, is for administration of the protein to a subject. Said pharmaceutical composition can be used in the methods of treatment described herein below by administration of an effective amount of the composition to a subject in need thereof. The term "subject", as used herein, refers to all animals classified as mammals and includes, but is not restricted to, primates and humans. The subject is preferably a male or female human of any age or race.
The term "pharmaceutically acceptable carrier", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7th edition, 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) proteins; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter ions such as sodium; metal complexes (e.g. Zn2+ protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).
Supplementary active compounds can also be incorporated into the pharmaceutical composition of the invention. Thus, in a particular embodiment, the pharmaceutical composition of the invention may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a chemotherapeutic agent, a cytokine, an analgesic agent, a thrombolytic or an immunomodulating agent, e.g. an immunosuppressive agent or an immunostimulating agent. The effective amount of such other active agents depends, among other things, on the amount of the protein of the invention present in the pharmaceutical composition, the type of disease or disorder or treatment, etc.
In one embodiment, the protein of the invention is prepared with carriers that will protect said compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems, e.g. liposomes. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. Liposomal suspensions, including targeted liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522,811 or WO2010/095940.
The administration route of the protein of the invention can be parenteral. The term "parenteral" as used herein includes intravenous, intra-arterial, intralymphatic, intraperitoneal, intramuscular or subcutaneous. The intravenous or intramuscular forms of parenteral administration are preferred. By "systemic administration" is meant intravenous, intraperitoneal and intramuscular administration. The amount of the protein required for therapeutic or prophylactic effect will, of course, vary with the protein chosen, the nature and severity of the condition being treated and the patient. In addition, the protein may suitably be administered by pulse infusion, e.g., with declining doses of the protein. Preferably the dosing is given by injections, most preferably intravenous, intramuscular or subcutaneous injections, depending in part on whether the administration is brief or chronic.
Thus, in a particular embodiment, the pharmaceutical composition of the invention may be in a form suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminium monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active compound (e.g a protein of the invention) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
In a particular embodiment, said pharmaceutical composition is administered via intravenous (IV), intramuscular (IM) or subcutaneous (SC) route. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants. The mentioned formulations will be prepared using standard methods for preparing parenterally administrable compositions as are well known in the art and described in more detail in various sources, including, for example, “Remington: The Science and Practice of Pharmacy” (Ed. Allen, L. V. 22nd edition, 2012, www.pharmpress.com).
It is especially advantageous to formulate the pharmaceutical compositions, namely parenteral compositions, in dosage unit form for ease administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound (protein of the invention) calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention is dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
Generally, for the prevention and/or treatment of the diseases and disorders mentioned herein and depending on the specific disease or condition to be treated and its severity, the potency of the specific protein of the invention to be used, the specific route of administration and the specific pharmaceutical formulation or composition used, the protein of the invention will generally be administered in the range of from 0.001 to 1 ,000 mg/kg body weight/day, preferably about 0.01 to about 100 mg/kg body weight/day, most preferably from about 0.05 to 10 mg/kg body weight/day, such as about 1 , 10, 100 or 1000 microgram per kg body weight per day, either continuously (e.g. by infusion), as a single daily dose or as multiple divided doses during the day. The clinician will generally be able to determine a suitable daily dose, depending on the factors mentioned herein. It will also be clear that in specific cases, the clinician may choose to deviate from these amounts, for example on the basis of the factors cited above and his expert judgment. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
Therapeutic use
In another aspect there is provided an IL-21 mutein as described herein or a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein for use as a medicament. In one embodiment, the IL-21 mutein as described herein or the conjugate as described herein is used as an active ingredient, component or substance in a medicament.
In one aspect, the invention pertains to a use of an IL-21 mutein as described herein or a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein for the manufacture of a medicament, e.g. a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient, for the treatment, prevention or diagnosis of a disease in a subject in need thereof.
In one aspect, the invention pertains to an IL-21 mutein as described herein or a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient, for use in the treatment, prevention or diagnosis of a disease in a subject in need thereof.
In one aspect, the invention pertains to a method for the treatment of a disease in a subject in need thereof, wherein the method comprises the step of administering to the subject (an effective amount of) an IL-21 mutein as described herein or a conjugate comprising an IL-21 mutein and an antigen binding protein as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient.
The disease to be treated, prevented or diagnosed using the IL-21 mutein or the conjugate can be a cancer, an infectious disease, an inflammatory disease or an autoimmune disease.
In one embodiment, the disease to be treated, prevented or diagnosed using the IL-21 mutein or the conjugate is a cancer, e.g. a cancer as described below. The cancer preferably is a cancer expressing a TAA as described herein above, more preferably the cancer is expressing a TAA that is bound by the antigen binding protein in the conjugate.
In one embodiment, the treatment can comprise the steps of a) identifying a TAA expressed by (tumor) cells in the cancer; b) selection of conjugate comprising an antigen binding protein as described herein that specifically binds the TAA; c) using the conjugate selected in b) in the treatment of the cancer. The cancer can be a cancer as described below.
In one embodiment, the invention pertains to a method for enhancing anti-tumor activity of at least one of an NK cell and a y6 T cell in a subject, the method comprising the step of administering to the subject an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient. In one embodiment, the subject has cancer, e.g. a cancer as described below. Preferably, the cancer comprises tumor cells expressing a TAA, more preferably the tumor cells express a TAA that is bound by the antigen binding protein in the conjugate.
In one embodiment, the invention pertains to a method for expanding and/or inducing hyperfunctionality NK cells a in a subject, the method comprising the step of administering to the subject an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient. The fold expansion and the hyperfunctionality preferably is as herein described above. In one embodiment, the subject has cancer, preferably a cancer comprising tumor cells expressing the TAA, more preferably the tumor cells express a TAA that is bound by the antigen binding protein in the conjugate.
In one embodiment, there is provided a method for expanding, activating and/or inducing innate potency of y6 T cells in a subject, the method comprising the step of administering to the subject an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient. The fold expansion, activation and the innate potency of y6 T cells preferably is as herein described above. In one embodiment, the subject has cancer, preferably a cancer comprising tumor cells expressing the TAA.
In one embodiment, there is provided a method for redirecting a y6 T cell to a tumor cell expressing the TAA in a subject, the method comprising the step of administering to the subject an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient.
Thus, in one embodiment, there is provided a method of modulating an immune response in a subject in need thereof comprising administering a therapeutically effective amount of an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate multispecific antigen binding protein as an active ingredient. In various embodiments, modulating an immune response in a subject comprises binding or targeting y6 T cells, activating y6 T cells, causing or increasing proliferation of y6 T cells, causing or increasing expansion of y6 T cells, causing or increasing y6 T cell degranulation, causing or increasing y6 T cell mediated killing activity, causing or increasing y6 T cell mediated killing activity while sparing healthy cells, causing or increasing y6 T cytotoxicity, causing or increasing y6 T cytotoxicity while sparing healthy cells, causing or increasing y6 T cell mobilization, increasing survival of y6 T cells, or increasing resistance to exhaustion of y6 T cells.
Subjects having cancer, often present with lower numbers of NK cells and/or y6 T cells and/or with exhausted NK cells and/or y6 T cells. The IL-21 muteins or the conjugates of the invention can thus be advantageously used to expand the numbers of NK cells and/or y6 T cells and/or to induce hyper-functionality of the NK cells and/or y6 T cells in a subject suffering from cancer. A further advantage of the hyper-functionality of NK cells as induced by the IL-21 mutein or the conjugate of the invention, includes their increased secretion of cytokines such as TNF-a, IFN-y and IL-6, which help shape adaptive immune response involving DCs and T cells. Indeed, NK cells have been reported to promote the recruitment to the tumor micro environment of a DC subset specializing in the cross-presentation of tumor antigens to CD8+ T cells, suggesting a crucial role for NK cells in the potentiation of antitumor CD8+ T cell responses (Bottcher et al., Cell, 2018. 172: 1022-1037; and Barry et al., Nat. Med. 2018. 24: 1 178-1191). The contribution of NK cells to the orchestration of antitumor T-cell responses has also been confirmed experimentally in mice, demonstrating that, in addition to their direct effector functions, NK cell can promote T-cell responses and long-lasting immune control of tumors (Bonavita et al., Immunity 2020. 53: 1215-1229).
In various embodiment, there is provided the use of an IL-21 mutein or a conjugate as described herein for the manufacture of a medicament, for example for the administration to a subject, wherein the subject has cancer, an infectious disease or an inflammatory disease.
In one embodiment, the TAA is a TAA as defined herein above and/or an antigen expressed on the surface of a malignant cell of a type of cancer as described below. A subject to be treated in accordance with the methods described herein can have a cancer selected from the group consisting of: acute lymphoblastic, acute myeloid leukemia, adrenocortical carcinoma, appendix cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, brain tumor, brain stem glioma, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma, pineal parenchymal tumors of intermediate differentiation, supratentorial primitive neuroectodermal tumors and pineoblastoma, visual pathway and hypothalamic glioma, brain and spinal cord tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor, gastrointestinal carcinoid tumor, central nervous system atypical teratoid/rhabdoid tumor, central nervous system embryonal tumors, central nervous system lymphoma, cerebellar astrocytoma cerebral astrocytoma/malignant glioma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, esophageal cancer, Ewing family of tumors, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (gist), germ cell tumor, gestational trophoblastic tumor, glioma, glioma brain stem, glioma cerebral astrocytoma, glioma visual pathway and hypothalamic, hairy cell leukemia, head and neck cancer, hepatocellular (liver) cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell tumors, kidney (renal cell) cancer, Langerhans cell histiocytosis, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, non- small cell lung cancer, small cell lung cancer, aids-related lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstrom macroglobulinemia, malignant fibrous histiocytoma of bone and osteosarcoma, medulloblastoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma/plasma cell neoplasm, mycosis, fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, myelogenous leukemia, myeloid leukemia, myeloid leukemia acute, multiple myeloma, myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oral cancer, oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, pancreatic cancer, papillomatosis, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal parenchymal tumors of intermediate differentiation, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma celt neoplasm/multiple myeloma, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, respiratory tract carcinoma involving the nut gene on chromosome 15, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Ewing family of tumors, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, skin cancer (nonmelanoma), skin cancer (melanoma), Merkel cell skin carcinoma, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, stomach (gastric) cancer, supratentorial primitive neuroectodermal tumors, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumor, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. In various embodiments, the cancer that can be treated by the disclosed methods and compositions is treated while healthy cells are spared.
In various embodiments, the inflammatory diseases that can be treated by the disclosed methods and compositions include, but are not limited to Achalasia, Acute disseminated encephalomyelitis (ADEM), Acute motor axonal neuropathy, Acute respiratory distress syndrome (ARDS), Addison’s disease, Adiposis dolorosa, Adult Still's disease, Adult-onset Still's disease, Agammaglobulinemia, Alopecia Areata, Amyloidosis, Amyotrophic lateral sclerosis, Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis, Anti-N- Methyl-D-Aspartate (Anti-NMDA) Receptor Encephalitis, Antiphospholipid syndrome, Antiphospholipid syndrome (APS, APLS), Antisynthetase syndrome, Anti-tubular basement membrane nephritis, Aplastic anemia, Atopic allergy, Atopic dermatitis, Autoimmune angioedema, Autoimmune comorbidities, Autoimmune dysautonomia, Autoimmune encephalomyelitis, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease (AIED), Autoimmune lymphoproliferative syndrome, Autoimmune myocarditis, Autoimmune neutropenia, Autoimmune oophoritis, Autoimmune orchitis, Autoimmune pancreatitis (AIP), Autoimmune peripheral neuropathy, Autoimmune polyendocrine syndrome (APS) type 1 , Autoimmune polyendocrine syndrome (APS) type 2, Autoimmune polyendocrine syndrome (APS) type 3, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura, Autoimmune thyroiditis, Autoimmune urticaria, Autoimmune uveitis, Autoimmune vasculitis, Axonal & neuronal neuropathy (AMAN), Balo concentric sclerosis, Balo disease, Behcet’s disease, Benign mucosal pemphigoid, Bickerstaffs encephalitis, Blau syndrome, Bullous pemphigoid, Castleman disease (CD), Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic obstructive pulmonary disease, Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Complex regional pain syndrome, Congenital heart block, Connective tissue, systemic, and multi-organ, Contact dermatitis, Coxsackie myocarditis, CREST syndrome, Crohn’s disease, Cushing's syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dermatitis herpetiformis, Dermatomyositis, Devic’sDisease (neuromyelitis optica), Diabetes mellitus type 1 , Digestive system, Discoid lupus, Dressier’s syndrome, Drug-induced lupus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic esophagitis (EoE), Eosinophilic fasciitis, Eosinophilic gastroenteritis, Eosinophilic granulomatosis with polyangiitis (EGPA), Eosinophilic pneumonia, Epidermolysis bullosa acquisita, Erythema nodosum, Erythroblastosis fetalis, Esophageal achalasia, Essential mixed cryoglobulinemia, Evans syndrome, Exocrine, Felty syndrome, Fibrodysplasia ossificans progressiva, Fibromyalgia, Fibrosing alveolitis, Gastritis, Gastrointestinal pemphigoid, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture’s syndrome, Granulomatosis with Polyangiitis, Graves' ophthalmopathy, Graves’ disease, Guillain-Barre syndrome, Hashimoto’s thyroiditis, Hashimoto's encephalopathy, Hemolytic anemia, Henoch- Schonlein purpura (HSP), Herpes gestationis or pemphigoid gestationis (PG), Hidradenitis Suppurativa (HS) (Acne Inversa), Hypogammaglobulinemia, Idiopathic giant- cell myocarditis, Idiopathic inflammatory demyelinating diseases, Idiopathic pulmonary fibrosis, IgA Nephropathy, IgA vasculitis (IgAV), lgG4-related disease, lgG4-related sclerosing disease, Immune thrombocytopenic purpura (ITP), Inclusion body myositis (IBM), Inflammatory Bowel Disease, Intermediate uveitis, Interstitial cystitis (IC), Interstitial lung disease, IPEX syndrome, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert- Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus, Lupus nephritis, Lupus vasculitis, Lyme disease chronic, Majeed syndrome, Meniere’s disease, Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD), Mooren’s ulcer, Morphea, Mucha-Habermann disease, MultifocalMotor Neuropathy (MMN) orMMNCB, Multiple sclerosis, Myasthenia gravis, Myocarditis, Myositis, Narcolepsy, Neonatal Lupus, Nervous system, Neuromyelitis optica, Neuromyotonia, Neutropenia, Ocular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Optic neuritis, Ord's thyroiditis, Oshtoran syndrome, Palindromic rheumatism (PR), Paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, ParsPlanitis (peripheral uveitis), Pediatric Autoimmune Neuropsychiatric Disorder Associated with Streptococcus (PANDAS), Pelvic Inflammatory Disease (PID), Pemphigus, Pemphigus vulgaris, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia (PA), Pityriasis lichenoides et varioliformis acuta, POEMS syndrome, Polyarteritis nodosa, Polyglandular syndromes type I, II, III, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Primary biliary cholangitis (PBC), Primary biliary cirrhosis, Primary immunodeficiency, Primary sclerosing cholangitis, Progesterone dermatitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Relapsing polychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Rheumatoid vasculitis, Sarcoidosis, Schizophrenia, Schmidt syndrome, Schnitzler syndrome, Scleritis, Scleroderma, Serum sickness, Sjogren’s Syndrome, Sperm & testicular autoimmunity, Spondyloarthropathy, Stiff person syndrome (SPS), Subacute bacterial endocarditis (SBE), Susac’s Syndrome, Sweet's syndrome, Sydenham's chorea, Sympathetic ophthalmia (SO), Systemic lupus erythematosus (SLE), Takayasu’s arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenia, Thrombocytopenic purpura (TTP), Thyroid gland, Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1 diabetes, Ulcerativecolitis (UC), Undifferentiated connective tissue disease (UCTD), Undifferentiated spondyloarthropathy, Urticarial vasculitis, Uticaria, Uveitis, Vasculitis, Vitiligo, and Vogt-Koyanagi- Harada Disease. In various embodiments, the inflammatory disease that can be treated by the disclosed methods and compositions is treated, while healthy cells are spared. In various embodiments, the infectious disease that can be treated by the disclosed methods and compositions include, but are not limited to Acinetobacter infection, Actinomycosis, Acute Flaccid Myelitis (AFM), African sleeping sickness (African trypanosomiasis), AIDS (acquired immunodeficiency syndrome), Ameba infection, Amebiasis, Anaplasma phagocytophilum infection, Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arboviral diseases, neuroinvasive and non-neuroinvasive, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Avian Influenza, Babesiosis, Bacillus cereus infection, Bacterial infection, Bacterial meningitis, Bacterial pneumonia, Bacterial vaginosis, Bacteroides infection, Balantidiasis, Bartonellosis, Baylisascaris infection, BK virus infection, Black piedra, Blastocystosis, Bolivian hemorrhagic fever, Botulism, Botulism (foodborne), Botulism (infant), Botulism (other), Botulism (wound), Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus), California serogroup virus diseases, Campylobacter, Campylobacteriosis, Candida auris, clinical, Candidiasis (Moniliasis; Thrush), Capillariasis, Carbapenemase Producing Carbapenem-Resistant Enterobacteriaceae (CP-CRE), Carbapenem-resistant Infection (CRE/CRPA), Carrion's disease, Cat- scratch disease, Cellulitis, Chagas disease (trypanosomiasis), Chancroid, Chickenpox, Chikungunya Virus Infection (Chikungunya), Chlamydia, Chlamydia trachomatis, Chlamydophila pneumoniae infection, Cholera, Chromoblastomycosis, Chytridiomycosis, Ciguatera, Clonorchiasis, Clostridium difficile colitis, Clostridium Difficile Infection, Clostridium perfringens, Coccidioidomycosis fungal infection (Valley fever), Colorado tick fever (CTF), Common cold (Acute viral rhinopharyngitis; Acute coryza), Congenital syphilis, Conjunctivitis, COVID-19 (Coronavirus Disease 2019), CP-CRE, Enterobacterspp., CP-CRE, Escherichia coli (E. coli), CP-CRE, Klebsiella spp., Creutzfeldt-Jacob Disease, transmissible spongiform encephalopathy (CJD), Creutzfeldt- Jakob disease (CJD), Crimean-Congo hemorrhagic fever (CCHF), Crusted Scabies, Cryptococcosis, Cryptosporidiosis (Crypto), Cutaneous larva migrans (CLM), Cyclospora, Cyclosporiasis, Cysticercosis, Cytomegalovirus infection, Dengue virus infections, Dengue, 1 ,2, 3, 4 (Dengue Fever), Dengue-like illness, Desmodesmus infection, Diarrheal Illness, Dientamoebiasis, Diphtheria, Diphyllobothriasis, Dracunculiasis, E. coli, E. coli infection, Shiga toxin-producing (STEC), Eastern equine encephalitis virus disease, Ebola Hemorrhagic Fever (Ebola), Echinococcosis, Ehrlichia chaffeensis infection, Ehrlichia ewingii infection, Ehrlichiosis, Anaplasmosis, Encephalitis, Arboviral or parainfectious, Enterobiasis (Pinworm infection), Enterococcus infection,, Enterovirus Infection, D68 (EV- D68), Enterovirus Infection, Non-Polio (Non-Polio Enterovirus), Epidemic typhus, Epstein-Barr virus infectious mononucleosis (Mono), Erythema infectiosum (Fifth disease), Exanthem subitum (Sixth disease), Fasciolasis, Fasciolopsiasis, Fatal familial insomnia (FFI), Fifth Disease, Filariasis, Flu (Seasonal), Food Poisoning, Food poisoning by Clostridium perfringens, Free-living amebic infection, Fungal infection, Fusobacterium infection, Gas gangrene (Clostridial myonecrosis), Genital Herpes, Genital Warts, Geotrichosis, German Measles, Gerstmann-Straussler-Scheinker syndrome (GSS), Giardiasis, Glanders, Gnathostomiasis, Gonorrhea, Granuloma inguinale, Granuloma inguinale (Donovanosis), Group A streptococcal infection, Group A Streptococcus, Group B streptococcal infection, Guanarito virus, Haemophilus Influenza disease, Type B (Hib or H-flu), Haemophilus influenzae infection, Hand, foot and mouth disease (HFMD), Hansen's Disease, Hantavirus infection, Hantavirus Pulmonary Syndrome (HPS), Heartland virus disease, Helicobacter pylori infection, Hemolytic Uremic Syndrome (HUS), Hemorrhagic fever with renal syndrome (HFRS), Hendra virus infection, Hepatitis A (Hep A), Hepatitis B (Hep B), Hepatitis C (Hep C), Hepatitis D (Hep D), Hepatitis E (Hep E), Herpes, Herpes B Virus, Herpes simplex, Herpes Zoster, zoster VZV (Shingles), Hib Disease, Histoplasmosis infection (Histoplasmosis), Hookworm infection, HPV (Human Papillomavirus), Human bocavirus infection, Human ewingii ehrlichiosis, Human granulocytic anaplasmosis (HGA), Human Immunodeficiency Virus/AIDS (HIV/AIDS), Human metapneumovirus infection, Human monocytic ehrlichiosis, Human papillomavirus (HPV) infection, Human parainfluenza virus infection, Hymenolepiasis, Impetigo, Influenza (flu), Influenza (Seasonal), Invasive pneumococcal disease, Isosporiasis, Junin virus,, Kawasaki Syndrome, Keratitis, Kingella kingae infection, Kuru, Lassa fever, Lassa virus, Legionellosis (Legionnaires' disease), Leishmaniasis, Leprosy (Hansens Disease), Leptospirosis, Listeriosis (Listeria), Lujo virus, Lyme disease, Lymphatic filariasis (Elephantiasis), Lymphocytic Choriomeningitis (LCMV), Lymphogranuloma venereum infection (LGV), Machupo virus, Malaria, Marburg virus infection, Measles, Melioidosis (Whitmore's disease), Meningitis, Meningitis - Bacterial, Meningitis - Viral, Meningococcal disease, Metagonimiasis, Microsporidiosis, Middle East respiratory syndrome (MERS), Molluscum contagiosum (MC), Monkeypox, Mononucleosis, Mosquito- borne Illness, MRSA, Mumps,, Murine typhus (Endemic typhus), Mycetoma, Mycoplasma genitalium infection, Mycoplasma pneumonia, Myiasis, Neisseria meningitidis, Neonatal conjunctivitis (Ophthalmia neonatorum), Nipah virus infection, Nocardiosis, Norovirus, Onchocerciasis (River blindness), Opisthorchiasis, Orf Virus (Sore Mouth), Paracoccidioidomycosis (South American blastomycosis), Paragonimiasis, Paralytic Shellfish Poisoning (Paralytic Shellfish Poisoning, Ciguatera), Pasteurellosis, PEP, Parasitic infection, Pertussis (whooping cough), Pink Eye, Pneumococcal Disease, Pneumococcal infection, Pneumocystis pneumonia (PCP), Pneumonia, Pneumonic Plague, Poliomyelitis (Polio), Poliomyelitis, paralytic, Poliovirus infection, Pontiac fever, Powassan virus disease, Prevotella infection, Primary amoebic meningoencephalitis (PAM), Progressive multifocal leukoencephalopathy, Protozoan infection, Psittacosis (Parrot Fever), Pustular Rash diseases (Small pox, monkeypox, cowpox), Rabies, Raccoon Roundworm, Rat Bite Fever, Recreational Water Illnesses, Relapsing fever, Respiratory syncytial virus infection, Reye’s Syndrome, Rhinosporidiosis, Rhinovirus infection, Rickettsial infection, Rickettsiosis (Rocky Mountain Spotted Fever), Rift Valley fever (RVF), Ringworm, Rotavirus infection, Rubella, Sabia virus, Salmonella, Salmonella Paratyphi infection, Salmonella Typhi infection, Salmonellosis, SARS (severe acute respiratory syndrome), Scabies, Scarlet fever, Schistosomiasis, Scombroid, Sepsis, Septic Shock, Septicemic Plague, Severe Acute Respiratory Syndrome (SARS), Shiga toxinproducing Escherichia coli, Shigella, Shigellosis, Shingles, Shingles (Herpes zoster), Smallpox, Sore Mouth (Orf Virus), Sporotrichosis, Spotted fever rickettsiosis, St. Louis encephalitis virus disease, Staphyloccal Infection, Staphyloccal Infection (Methicillin-resistant (MRSA)), Staphylococcal food poisoning, Staphylococcal Infection (Vancomycin Intermediate (VISA)), Strep Throat, Streptococcal Disease, Group A (invasive) (Strep A (invasive)), Streptococcal Disease, Group B (Strep-B), Streptococcal toxic shock syndrome, Strongyloidiasis, Subacute sclerosing panencephalitis, Syphilis, Taeniasis, Tetanus Infection, Tickborne Diseases, Tinea barbae, Tinea capitis, Tinea corporis, Tinea cruris, Tinea manum, Tinea nigra, Tinea pedis, Tinea unguium, Tinea versicolor, Toxic shock syndrome, Toxocariasis (ocular larva migrans (OLM)), Toxocariasis (visceral larva migrans (VLM)), Toxoplasmosis, Trachoma, Trichinellosis, Trichomoniasis, Trichonosis Infection (Trichinosis), Trichuriasis (whipworm infection), Tuberculosis (TB), Tularemia (Rabbit fever), Typhoid fever, Typhoid Fever, Group D, Typhus, Typhus fever, Ureaplasma urealyticum infection, Vaginosis, Valley fever, Variant Creutzfeldt-Jakob disease (vCJD, nvCJD), Varicella (Chickenpox), Venezuelan equine encephalitis, Venezuelan hemorrhagic fever, Vibrio cholerae (Cholera), Vibrio parahaemolyticus enteritis, Vibrio vulnificus infection, Vibriosis, Viral infection, Viral hemorrhagic fever, Viral Hemorrhagic Fever (Ebola, Lassa, Marburg), Viral Hemorrhagic Fevers (VHF), Viral pneumonia, West Nile virus disease, Western equine encephalitis virus disease, White piedra (tinea blanca), Whooping Cough, Yellow Fever, Yersenia (Yersinia), Yersinia pseudotuberculosis infection, Yersiniosis, Zeaspora, Zika fever, Zika Virus, Zika virus disease, congenital, Zika virus disease, non-congenital, Zika Virus Infection (Zika), Zika virus infection, congenital, Zika virus infection, non-congenital, and Zygomycosis. In various embodiments, the infectious disease that can be treated by the disclosed methods and compositions is treated, while healthy cells are spared.
In one embodiment, an IL-21 mutein or a conjugate as described herein can be used as a monotherapy (i.e. without other therapeutic agents). In another embodiment, an IL-21 mutein or a conjugate as described herein can be used in combined treatments.
In one embodiment, an IL-21 mutein or a conjugate as described herein is used in combination with another immunotherapy, e.g. a cellular immunotherapy. The IL-21 mutein or the conjugate can thus be used in combination with the adoptive transfer of immune cells, including the adoptive transfer of T cells, e.g. CAR T cells or y6 T cells, or NK cells. The NK cells or y6 T cells can e.g. be enriched or expanded by methods known in the art or can be ex vivo expanded NK cells or y6 T cell as herein described below.
In one embodiment, an IL-21 mutein or a conjugate as described herein can be used in combined treatments with one or more other therapeutic agents. The additional therapeutic agent or agents normally utilized for the particular therapeutic purpose for which an antibody against a TAA is being administered. The additional therapeutic agent or agents will normally be administered in amounts and treatment regimens typically used for that agent in a monotherapy for the particular disease or condition being treated. Such therapeutic agents when used in the treatment of cancer, include, but are not limited to anti-cancer agents and chemotherapeutic agents. Exemplary therapeutic agents that may be used as part of a combination therapy in treating cancer, include, for example, radiation, mitomycin, tretinoin, ribomustin, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin, nitracrine, zinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasin, sobuzoxane, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxifluridine, etretinate, isotretinoin, streptozocin, nimustine, vindesine flutamide, drogenil, butocin, carmofur, razoxane, sizofilan, carboplatin, mitolactol, tegafur, ifosfamide, prednimustine, picibanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymethoIone, tamoxifen, progesterone, mepitiostane, epitiostanol, formestane, interferon-alpha, interferon-2 alpha, interferon-beta, interferon-gamma, colony stimulating factor-1 , colony stimulating factor-2, denileukin diftitox, interleukin-2, and luteinizing hormone releasing factor.
An additional class of agents that may be used as part of a combination therapy (including the use of an IL-21 mutein or a conjugate as described herein) in treating cancer is immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include agents that inhibit one or more of (i) cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PD-L1 , (iv) LAG3, (v) B7-H3, (vi) B7-H4, and (vii) TIM3. Yet other agents that may be used as part of a combination therapy in treating cancer are monoclonal antibodies against TAA as described herein above.
In some embodiments the administration of an IL-21 mutein or a conjugate as described herein and the other therapeutic agent can elicit an additive or synergistic effect on immunity and/or on therapeutic efficacy.
In one embodiment, an IL-21 mutein or a conjugate as described herein is used as at least one of an neoadjuvant therapy and an adjuvant therapy, in addition to a primary therapy comprising e.g. surgery, chemo and/or radiation therapy. As a neoadjuvant therapy, the IL-21 mutein or the conjugate is administered before the primary treatment, e.g. to help reduce the size of a tumor (such that less extensive surgery and/or radiation therapy is required), kill cancer cells that have spread (e.g. micrometastatic disease) and/or reduce the risk of tumor cells spreading post-surgery. As an adjuvant therapy, the IL-21 mutein or the conjugate is administered after the primary treatment, e.g. to treat minimal residual disease (destroy remaining cancer cells). The IL-21 mutein or the conjugate can also be administered as a maintenance therapy, which is a long-term adjuvant therapy, e.g. administered repeatedly over the course of at least one month or one year. The use of the IL-21 mutein or a the conjugate as an neoadjuvant therapy and/or an adjuvant therapy lowers relapse rates. In the neoadjuvant therapy and/or adjuvant therapy, the IL-21 mutein or the conjugate can be used as monotherapy or in in combined treatments as described above.
Ex vivo methods
In a further aspect, the invention relates to methods wherein an IL-21 mutein or a conjugate as described herein is used for ex vivo (in vitro) treatment of an NK cell or a population of NK cells. The method can be a method for at least one of expanding, pre-activating, activating, enhancing cytotoxicity and/or cytokine production, and inducing a hyper-functional phenotype as defined above. The methods at least comprise the step of contacting an NK cell or a population thereof, with an IL-21 mutein or a conjugate as described herein or with a composition comprising the IL-21 mutein or the conjugate. In a preferred embodiment, the method comprises the further step of coculturing the NK cells with tumor cells expressing a TAA specifically bound by the antigen binding protein in the conjugate. Preferably, the NK cells are co-cultured with the tumor cells expressing the TAA specifically bound by the antigen binding protein in the conjugate, in the presence of the conjugate.
An NK cell or a population of NK cells for ex vivo treatment can be enriched from peripheral blood mononuclear cells (PBMCs). Methods for enrichment and ex vivo treatment of NK cells from PBMCs are e.g. described in Denman et al. (pLoS One. 2012;7(1):e30264) and in US2020/0061115. For example, NK cells enriched from PBMCs can be seeded at 0.1 x 106 NK cells/mL in SCGM (CellGenix, Portsmouth, N.H.), supplemented with 10% FBS, 2 mM Glutamax, 100 U/mL IL-2 (Peprotech, Rocky Hill, N.J.) and 1 , 2, 5, 10, 20, 50, 100, 200, 500, 1000 pg/mL of one or more of the IL-21 muteins or a conjugates as described herein. Media with supplements can be refreshed every 2-3 days.
It is understood that the duration of the contact between the NK cells and an IL-21 mutein or a conjugate as described herein (i.e. the duration of the pre-activation or activation (i.e., the duration of expanding, pre-activating, activating, enhancing cytotoxicity and/or cytokine production and inducing a hyper-functional phenotype) can be for any length of time necessary to achieve the desired phenotype of the NK cells. For example, the contact can be as little as 1 minute or as much as 7 days (for example, culturing the NK cells in the presence of an IL-21 mutein or a conjugate as described herein for 7 days). In one embodiment of the method, the NK cells are contacted with the IL-21 mutein or the conjugate for 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23,
24, 36, or 48 hours. In one embodiment of the method, the NK cells are contacted with the IL-21 mutein or the conjugate for 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,
25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,
51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , or 72 days.
In one embodiment, the ex vivo treated (expanded) NK cells have one or more features selected from: a) the fold expansion of the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.5, 1 .0, 2.0 or 5.0 fold of the percentage telomere length increase of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the expression level on NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded NK cells is at least 0.5, 1.0, 2.0 or 5.0 fold of the secretion of the cytokine by NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; and, e) the cytotoxicity of the expanded NK cells is at least 0.5, 1 .0, 2.0 or 5.0 fold of the cytotoxicity of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells. In yet a further aspect, the invention pertains to a method for the treatment of a disease in a subject in need thereof, wherein the method comprises the step of administering to the subject (an effective amount of) NK cells obtained in the above method for ex vivo treatment of an NK cell or a population of NK cells. Once a sufficient number of NK cells with the desired (hyper-functional) phenotype has been expanded by the ex vivo treatment, the NK cells can be administered to a subject in need thereof.
In one embodiment, the method for the treatment comprises the administration of the ex vivo treated NK cells in combination with an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient.
In one embodiment, the method for the treatment comprises the administration of the ex vivo treated NK cells in combination with another NK cell engager, such as e.g. described in WO2016/207278, WO 2018/148445, WO2018/152518, WO2019195409 US2018282386, Vallera et al. (2016, supra) and Demaria et al. (2021 , supra), or with a multispecific antigen binding protein as described in WO2024/056862 and WO2024/056861 by the same applicant. One example of another NK cell engager is for example AVC-006 as described in WO2024/056862, comprising one HER2-binding region and one NKG2D-binding region. In a further embodiment, the ex vivo treated NK cells can be used in combination with the other engager and with an IL-21 mutein or a conjugate as described herein.
The disease to be treated can be a cancer, an infectious disease, an inflammatory disease or an autoimmune disease, as described above. Preferably, the disease to be treated is a cancer, as described above. The cancer preferably is a cancer expressing a TAA that is specifically bound by the antigen binding protein in the conjugate that is administered in combination with the ex vivo treated NK cells. The administration of the ex vivo treated NK cells in combination with the conjugate will facilitate targeting of the administered ex vivo treated NK cells to tumor cells expressing the TAA that is specifically bound by the antigen binding protein in the conjugate.
In one embodiment, the ex vivo treated NK are autologous to the subject. In another embodiment, the ex vivo treated NK are allogeneic, e.g. derived from donor PBMCs.
In a further aspect, the invention relates to methods wherein an IL-21 mutein or a conjugate as described herein is used for ex vivo (in vitro) treatment of a y6 T cell or a population of y6 T cells. The method can be a method for at least one of expanding, pre-activating, activating, enhancing cytotoxicity and/or cytokine production, and inducing a hyper-functional phenotype as defined above. The methods at least comprise the step of contacting a y6 T cell or a population thereof, with an IL-21 mutein or a conjugate as described herein or with a composition comprising the IL-21 mutein or the conjugate.
A y6 T cell or a population of y6 T cells for ex vivo treatment can be enriched from peripheral blood mononuclear cells (PBMCs) from apheresis by depletion of ap T cells. Methods for enrichment and ex vivo treatment of y6 T cells from PBMCs are e.g. described in Almeida et al. (Clin Cancer Res. 2016; 22(23):5795-5804) and Boucher et al. (J Immunother. 2023; 46(1): 5-13). For example, y6 T cells enriched from PBMCs can be seeded at 0.2 x 106 cells/mL in RPMI, supplemented with 10% FBS, 2 mM Glutamax, 50 U/mL IL-2 (Peprotech, Rocky Hill, N.J.) and 1 , 2, 5, 10, 20, 50, 100, 200, 500, 1000 pg/mL of one or more multispecific antigen binding proteins as described herein. Media with supplements can be refreshed every 2-3 days after day 5.
It is understood that the duration of the contact between the y6 T cells and an IL-21 mutein or a conjugate as described herein (i.e. the duration of the pre-activation or activation (i.e., the duration of expanding, pre-activating, activating, enhancing cytotoxicity and/or cytokine production and inducing a hyper-functional phenotype) can be for any length of time necessary to achieve the desired phenotype of the y6 T cells. For example, the contact can be as little as 1 minute or as much as 7 days (for example, culturing the y6 T cells in the presence of an IL-21 mutein or a conjugate as described herein for 7 days). In one embodiment of the method, the y6 T cells are contacted with the IL-21 mutein or the conjugate for 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,
19, 20, 21 , 22, 23, 24, 36, or 48 hours. In one embodiment of the method, the y6 T cells are contacted with the IL-21 mutein or the conjugate for 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18,
19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44,
45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70,
71 , or 72 days.
In one embodiment, the ex vivo treated (expanded) y6 T cells have one or more features selected from: a) the fold expansion of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the fold expansion of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody (e.g. using the protocol of Almeida et al., 2016, supra) or by co-culturing with irradiated K562 feeder cells modified to express a membrane bound version of the y6 T cell-activating agonist and a membrane bound version of the co-stimulatory agonist; b) the telomere length of the expanded y6 T cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh y6 T cells, preferably, the percentage telomere length increase of the expanded y6 T cells as compared to the telomere length of fresh y6 T cells, is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the percentage telomere length increase of y6 T cells obtained upon ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; c) the expression level of at least one y6 T cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 on the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or5.0 fold of the expression level on expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the secretion of the cytokine by expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co- culturing with the modified irradiated K562 feeder cells; and, e) the cytotoxicity of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the cytotoxicity of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells. It is understood that for appropriate comparison of the fold expansion induced by an IL-21 mutein or a conjugate vs. the feeder cells, the membrane bound versions of the y6 T cell-activating agonist and the co-stimulatory agonist expressed on the in with irradiated K562 feeder cells correspond to the y6 T cell-activating agonist and co-stimulatory agonist as comprised in the particular conjugate that is tested. Hence, if a conjugate comprises IL-21 as y6 T cell-activating agonist and a 4-1 BBL ECD trimer as co-stimulatory agonist, the feeder cells express membrane bound versions of IL-21 and 4-1 BBL.
In yet a further aspect, the invention pertains to a method for the treatment of a disease in a subject in need thereof, wherein the method comprises the step of administering to the subject (an effective amount of) y6 T cells obtained in the above method for ex vivo treatment of a y6 T cell or a population of y6 T cells. Once a sufficient number of y6 T cells with the desired (hyper-functional) phenotype has been expanded by the ex vivo treatment, the y6 T cells can be administered to a subject in need thereof.
In one embodiment, the method for the treatment comprises the administration of the ex vivo treated y6 T cells in combination with an IL-21 mutein or a conjugate as described herein, or a pharmaceutical preparation comprising the IL-21 mutein or the conjugate as an active ingredient.
In one embodiment, the method for the treatment comprises the administration of the ex vivo treated y6 T cells in combination with a multispecific antigen binding protein, e.g. a multispecific antigen binding protein as described in the co-pending application EP 24208721 .1 .
The disease to be treated can be a cancer, an infectious disease, an inflammatory disease or an autoimmune disease, as described above. Preferably, the disease to be treated is a cancer, as described above.
In one embodiment, the ex vivo treated y6 T cells are autologous to the subject. In another embodiment, the ex vivo treated y6 T cells are allogeneic, e.g. derived from donor PBMCs.
Nucleic acids, host cells and methods for producing an IL-21 mutein ora conjugate comprising such mutein
In one aspect, the invention relates to a nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of an IL-21 mutein or a conjugate as described herein. The nucleotide sequence encoding such a polypeptide chain preferably encodes a signal peptide operably linked to the polypeptide chain. A nucleic acid molecule comprising one or more of the nucleotide sequences encoding a polypeptide chain, further preferably comprises regulatory elements for (or conducive to) the expression of the polypeptide chain in an appropriate host cell, which regulatory elements are operably linked to the nucleotide sequence.
In one aspect, the invention relates to a host cell comprising the nucleic acid molecule comprising one or more nucleotide sequences encoding a polypeptide chain of an IL-21 mutein or a conjugate as described herein. In one embodiment, the host cell is an isolated cell or a cultured cell. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, for example Escherichia coll or bacilli. Suitable yeast cells include Saccharomyces cerevisiae and Pichia pastoris. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-1 , COS-7 line of monkey kidney cells (Gluzman et al., 1981 , Cell 23:175), L cells, HEK 293 cells (e.g. Expi293, HEK293-F and HEK293-E cells), C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells (e.g. ExpiCHO cells), HeLa cells, BHK cell lines, e.g. BHK21 , BSC-1 , Hep G2, 653, SP2/0, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI as described by McMahan et al. (1991 , EMBO J. 10: 2821). The host cell may be any suitable species or organism capable of producing N-linked glycosylated polypeptides, e.g. a mammalian host cell capable of producing human or rodent IgG type N-linked glycosylation. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985). Host cells comprising the nucleic acid molecule of the invention can be cultured under conditions that promote expression of the polypeptide.
Thus, another aspect the invention relates to a method for producing an IL-21 mutein or a conjugate as described herein. The method preferably comprises culturing a host cell as described above such that one or more nucleotide sequences are expressed and the IL-21 mutein or the conjugate is produced. The method preferably comprises the step of cultivating a host cell comprising one or more of the nucleotide sequences encoding a polypeptide chain of the IL-21 mutein or the conjugate. The host cell is preferably cultured under conditions conducive to expression of the one or more polypeptide chains. The method can further comprise the step of recovering the IL-21 mutein or the conjugate. The IL-21 mutein or the conjugate can be recovered by conventional protein purification procedures, including e.g. protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, size exclusion chromatograpy or affinity chromatography, using e.g. streptavidin/biotin (see e.g. Low et al., 2007, J. Chromatography B, 848:48-63; Shukla et al., 2007, J. Chromatography B, 848:28-39).
In a further aspect, the invention relates to a method for producing a pharmaceutical composition comprising an IL-21 mutein or a conjugate as described herein, the method comprising the steps of a) producing the IL-21 mutein or the conjugate in a method as defined above; and b) formulating the IL-21 mutein or the conjugate with a pharmaceutically acceptable carrier as defined above, to obtain a pharmaceutical composition.
The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.
Description of the figures
Figure 1. NK boosters comprising IL-21 or 4-1 BBL muteins show impaired capacity to induce proliferation only in the absence of tumor cells. NK booster AVC37 (a conjugate of trastuzumab and the I8H IL-21 mutein and wild type 4-1 BBL ECD) and NK booster AVC52 (a conjugate of trastuzumab and wild type IL-21 and the V153Q 4-1 BBL ECD mutein) show strongly impaired capacity to induce proliferation of NK cells but only in the absence of SKOV3 tumor cells expressing HER2. In the presence of SKOV3 tumor cells the ability of NK boosters AVC37 and AVC52 is comparable to that of control NK booster AVC1 (a conjugate of trastuzumab and wild type IL-21 and wild type 4-1 BBL ECD mutein). NK cells were cultured for 5 days with (dashed lines) or without (solid lines) tumor cells and indicated NK boosters in concentrations as indicated. Data shown is from measurements taken at the end of the co-culture. The darker and lighter grey areas indicate historical averages and the standard deviation obtained with AVC1 , with and without tumor cells present, respectively.
Figure 2. NK boosters comprising various IL-21 muteins as indicated show impaired capacity to induce proliferation as compared to a corresponding control NK booster AVC1 comprising wild type IL-21 . NK cells were cultured for 5 days without tumor cells and indicated NK boosters, data shown is from measurements taken at the end of the co-culture.
Figure 3. NK cell cytotoxicity against SKOV3 tumor cells, as induced by NK boosters comprising IL- 21 or 4-1 BBL muteins, is unaffected by IL-21 or 4-1 BBL mutations. NK cells were co-cultured for 5 days with tumor cells in the presence of NK booster AVC37 (a conjugate of trastuzumab and the I8H IL-21 mutein and wild type 4-1 BBL ECD), NK booster AVC52 (a conjugate of trastuzumab and wild type IL-21 and the V153Q 4-1 BBL ECD mutein) or control NK booster AVC1 (a conjugate of trastuzumab and wild type IL-21 and wild type 4-1 BBL ECD mutein), respectively, in concentrations as indicated. Data shown is from measurements taken at the end of the co-culture.
Figure 4. The ability of NK boosters comprising IL-21 or 4-1 BBL muteins to induce and support long-term NK cell expansion is unaffected. NK cells were expanded for 14 days with SKOV3 tumor cells that were opsonized with indicated NK boosters. Fold expansion of NK cells after 14 days is indicated.
Figure 5. Dose-response curves for induction of proliferation of NK cells by NK booster with IL-21 muteins as indicated, in the presence of SKOV3 tumor cells that express HER2, as compared with that of control NK booster AVC1 with wild type IL-21. A) compares IL-21 mutein NK boosters AVC116, AVC118, AVC119, AVC120 and AVC121 with control NK booster AVC1 . B) compares IL- 21 mutein NK boosters AVC122, AVC123, AVC124, AVC125 and AVC126 with control NK booster AVC1 . NK cells were cultured for 5 days in the presence of SKOV3 tumor cells and NK boosters in concentrations as indicated. Data shown is from measurements taken at the end of the co-culture.
Figure 6. Long-term NK cell cytotoxicity induced by the multispecific antigen binding proteins AVC- 001 and AVC-016, against tumor cell lines expressing the respective tumor-associated antigens: (A) A VC-001 (AVC1) with SKOV-3 tumor cells expressing HER2; (B) AVC-016 (AVC16) with BxPC3 tumor cells expressing TROP2; and (C) AVC-020 (AVC20) with A431 tumor cells expressing EGFR. NK cell cytotoxicity induced by the multispecific antigen binding proteins against tumor cells that express the respective antigens (open squares) is followed over time (hours) and compared to the cytotoxicity of NK cells only (solid circles).
Figure 7. Interferon-gamma levels after co-culture with tumor cells that express the respective antigen. Data is shown as fold change over vehicle (NK cells + tumor cells). (A) AVC-001 (AVC1) with SKOV-3 tumor cells expressing HER2; (B) AVC-016 (AVC16) with BxPC3 tumor cells expressing TROP2; and (C) AVC-020 (AVC20) with A431 tumor cells expressing EGFR.
Figure 8. Long-term cytotoxicity against (TROP2-expressing) BxPC3 tumor cells by NK cells stimulated with NK boosters AVC16 (based of Fab-fragments from sacituzumab and comprising wild type IL-21 and a trimer of wild type 4-1 BBL ECD), AVC267 (based of Fab-fragments from sacituzumab and comprising the IL-21 L20W mutein and a trimer of wild type 4-1 BBL ECD) and control protein AVC 137 (a sacituzumab analogue without IL-21 or 4-1 BBL cytokines) upon repeated co-culture with BxPC3 tumor cells. After each 3-day cycle, NK cells were were harvested and used to set up a new cycle of co-culture with fresh target cells at a 1 :1 E:T ratio. The NK boosters were added at the start of each 3-day cycle to expose NK cells to new booster protein at the beginning of every cycle. Control wells contained only NK cells and BxPC3 target cells (NK + BxPC3), or only BxPC3 target cells (BxPC3 only).
Figure 9. Effect of different mutations in IL-21 and/or 41 BBL as present in V61 TCR-targeting y6 T- cell boosters on V61 T cell expansion and phenotype. Effects of avidity attenuating mutations in IL- 21 and/or 41 BBL on V61 T cell expansion (A), PD1 expression on V61 T cells (B) or CD56 expression on V61 T cells (C) after 14 days of expansion. All values have been normalised to AVC160 which uses IL-21 and 41 BB-ligand wildtype in its design. A summary of the IL-21 and 41 BBL mutations used in these boosters is provide in Table 1 .1.10.4.
Examples
Example 1
1.1 Methods and materials
1.1.1 Reagents NK cell expansion
SK-OV-3 cell line medium
Effector cells (NK) complete media 1 .1 .2 Maintenance of SKOV3 WT cell line SK-OV-3
Split sub-confluent cultures (70-80%)
1 . Remove and discard culture medium.
2. Briefly rinse the cell layer with 0.25% (w/v) Trypsin- 0.53 mM EDTA solution to remove all traces of serum that contains trypsin inhibitor.
3. Add 2 to 3 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal.
4. Add 6 to 8 mL of complete growth medium and aspirate cells by gently pipetting.
5. Add appropriate aliquots of the cell suspension to new culture vessels.
6. Incubate cultures at 37°C.
Sub cultivation Ratio: A sub cultivation ratio of 1 :2 to 1 :3 is recommended i.e., seeding at 3-6x10,000 cells/cm2
Medium Renewal: Every 2 to 3 days
1.1.3 Media
Growth and Test medium
McCoy’s 5a medium modified + 2mM glutamine + 15% FBS + 1 % Penicillin Streptomycin
Freezing medium
FCS + 10% DMSO
1 .1 .4 PBMC sort and NK isolation
Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors using a density gradient centrifugation method. Briefly, the buffy coat fraction was overlaid onto Histopaque in a falcon tube for density centrifugation. After centrifugation at 500g for 30 min at RT the mononuclear cell fraction was harvest from the interface into a new falcon tube. Cells underwent several washes steps and lysis to remove red blood cell contamination. Subsequently, CD56+ cells were obtained by magnetic cells sorting using the system from Miltenyi Biotec according to the manufacturer's instructions (Miltenyi- 130-097-042). Following separation and an aliquot of sorted NK cells was taken for post-sort purity check.
1 .1 .5 Irradiation of SK-OV-3
E:T ratio= 1 :2 (NK: SK-OV-3)
• 8x106 cells per flask x (X conditions) x (Y Donors) 1. Irradiate extra cells to account for possible cell loss following irradiation. Maintain enough cells to expand for Day 7 restimulation.
2. Resuspend required number of cells @ 5 x 106 cells/mL
3. Perform irradiation at 100Gy.
4. Count cells post-irradiation and adjust to 5 x 106 cells/mL.
1 .1 .6 Opsonisation of SK-OV-3 cells
1 . Prepare test compounds.
2. Spike in required stock volume to irradiated SK-OV-3 to the required concentration (25 nM) and leave to opsonise for 30 minutes in a flask in 37°C incubator.
3. Wash cells by spinning at 300 x g for 5 mins.
4. Count cells and adjust density to 3.2 x 106/mL.
1.1.7 Seeding NK cells for 14-day expansion
1 . Adjust NK cells to required density - 1 .6 x 106/mL in NK media containing IL-2 (50 lU/mL)
2. Seed 2.5 mL of NK cells per flask (4 x 106 total cells per flask).
3. Seed 2.5 mL of 3.2 x 106/mL compound or vehicle opsonised SK-OV-3 cells according to appropriate flask maps.
4. To all flasks, add 17.5 mL of 2 x IL-2 complete media and 17.5 mL of complete media without IL-
2. This is to bring the IL-2 activity to 50 ILI/mL.
5. Total final volume per T75 flask will be 40 mL.
1 .1 .8 Cytotoxicity measurements
NK cells and NucLight-Red (NLR) transduced SKOV3 tumor cells were co-cultured in a 2:1 ratio for 5 days with NK boosters present in different concentrations, using a 7-step 4-fold dilution series starting at 25 nM thus covering a range from 25 nM to 0.01 nM. The number of NLR-positive cells was quantified by an Incucyte automated microscope every 4 hours throughout the duration of culture and compared to the control condition where NK cells were co-cultured with NLR SKOV3 cells in the absence of NK boosters. Cytotoxicity was calculated as follows:
% target cell lysis = 100 100
1.1.9 Proliferation measurements
NK cells were labeled with an amine-reactive cell proliferation dye (CPD), for example CellTrace Violet (Thermo Scientific, cat# C34557). Subsequently, labeled NK cells were used either in a coculture with SKOV3 cells or cultured by themselves with NK boosters present in different concentrations. In the case of a co-culture with SKOV3 cells a 7-step 4-fold dilution series starting at 25 nM thus covering a range from 25 nM to 0.01 nM was used. When only NK cells were cultured an 8-step 3-fold dilution series which covers a range from 100 nM to 0.01 nM was used instead. After a 5 day (co-)culture, NK cells were analyzed by flow cytometry for cell proliferation dye content, and the percentage of CPD-low cells was used as a read-out for proliferation.
1.1.10 Production of NK boosters NK cell boosters (also referred to conjugates herein) as listed in Table 1.1.10, having amino acid sequences as shown in the sequence listing, were prepared, purified and characterized essentially as described in application WO2024/056862, except that neutralization of the eluates from the HiScreen Fibro PrismA column was done by mixing-in of 1 M Tris pH 8.0 for neutralisation to pH 7 in a 1 .0 / 0.2 ratio, after which 12.5 ml fractions were collected.
Table 1 .1 .10.1 . Overview of the HER2-targeting NK cell boosters with 4-1 BBL and/or IL-21 muteins as indicated and their corresponding amino acid sequences in the sequence listing. AVC1 is the control NK cell booster with wild type 4-1 BBL and IL-21 .
Table 1 .1 .10.2. Overview of the TROP2-targeting NK cell boosters with 4-1 BBL and/or IL-21 muteins and control protein AVC137 (without 4-1 BBL and IL-21),, as indicated and their corresponding amino acid sequences in the sequence listing.
Table 1.1.10.3. EGFR-targeting NK cell booster with 4-1 BBL and/or IL-21 and their corresponding amino acid sequences in the sequence listing.
Table 1 .10.4. Overview of y6 T-cell-boosters comprising anti-V61 TOR VH and VL and wild type or mutein 4-BBL and IL-21 as indicated.
1.1.11 Affinity of NK boosters for IL21 R
Affinity of the boosters for IL21 R was analyzed using a Biacore T200 instrumentat 25 °C and a flow rate of 50 pl/min. A C1 sensor chip was functionalized for a reversible biotin capture assay in all flow cells (fc1-fc4). The analysis buffer consisted of 10 mM HEPES (pH 7.4), 150 mM NaCI, 0.05% Tween 20, and 3 mM EDTA. Each assay cycle proceeded as follows: the biotin capture reagent was introduced over all surfaces (fc1-fc4), IL-21 R was captured specifically on flow cell 2 (fc2), and the boosters were injected in five serial dilutions (starting at different concentrations) for a 60- second association phase and a 600/1200s dissociation phase (the longer dissociation was applied after the highest concentration). A standard regeneration protocol was then applied to completely remove the ligand-receptor complex from all surfaces, restoring the chip to baseline before the next cycle.
1.1.12 y5 T Cell ex-vivo expansion
Protocols used for ex vivo expansion of donor y6 T-cells are adapted from DOT cell expansion protocols as described in Almeida et al. (2016, supra). Briefly, y6 T-cells are enriched from 2 healthy donor derived apheresis through the depletion of ap T cells. At day 0, ap T cell depleted cells are seeded at a density of 0.2 x 106 cells/mL in 8mL (24-well GRex) of OpTimizer CTSTM culture medium (Life Technologies) supplemented with OpTimizer CTS T-cell Expansion Supplement, CTS Glutamax (1x) and human heat-inactivated plasma (2.5%) (Octapharm). The media is supplemented with y6 T-cell boosters (see Table 1.1.10.4) at 42ng/mL, 30ng/ml of IL-4, 4.5ng/ml IL-1 p and 21 ng/mL IFNy. At day 7, y6 T-cell expansion cultures are fed with multispecific antigen binding proteins at 300ng/mL and 21 ng/mL of IL-15. At day 11 , y6 T-cell cultures are fed with y6 T- cell boosters at 1 pg/mL and 10Ong/mL of IL-15. As a control, ap T cell depleted cells were stimulated using OKT3, as described by Almeida et al. Cultures are harvested, characterized and cryopreserved at day 14.
1.1.13 Phenotypic changes of y5 T cells and differentiation assessment y6 T-cells were expanded for 14 days as described in method 1. y6 T-cells are collected and analyzed via Flow Cytometry for the following markers: pan y6 TCR, V61 TCR, CD56, DNAM-1 and PD-1.
1.2 Results Figure 1 shows that NK boosters comprising IL-21 and 4-1 BBL ECD muteins show impaired capacity to induce proliferation, but only in the absence of tumor cells. Figure 1 shows doseresponse curves for NK booster AVC37 (comprising wild type 4-1 BBL ECD and IL-21 I8H mutein), NK booster AVC52 (comprising 4-1 BBL ECD V153Q mutein and wild type IL-21) and control NK booster AVC1 (comprising wild type 4-1 BBL ECD and wild type IL-21). NK boosters AVC37 and AVC52 show strongly impaired capacity to induce proliferation of NK cells but only in the absence of SKOV3 tumor cells that express HER2. In the presence of SKOV3 tumor cells the ability of NK boosters AVC37 and AVC52 to induce NK cell proliferation is comparable to that of control NK booster A VC 1 .
Figure 2 shows dose-response curves for various further NK boosters comprising different IL-21 muteins as indicated on their ability to induce NK cell proliferation in the absence of tumor cells. As can be seen, the various NK boosters with different IL-21 muteins have reduced abilities to different degrees for inducing NK cell proliferation in the absence of tumor cells, as compared to a corresponding control NK booster AVC1 comprising wild type IL-21.
Figure 3 shows that NK cell cytotoxicity against SKOV3 tumor cells, as induced by NK boosters AVC37 (comprising wild type 4-1 BBL ECD and IL-21 I8H mutein) and AVC54 (comprising 4-1 BBL ECD Q227E mutein and wild type IL-21), is unaffected by the IL-21 or 4-1 BBL ECD mutations in AVC37 and AVC54, respectively. The dose-response curves of AVC37 and AVC54 for inducing NK cell cytotoxicity against SKOV3 tumor cells does not significant differ from that of the corresponding control NK booster AVC1 comprising wild types of IL-21 and 4-1 BBL ECD.
Figure 4 shows that the ability of NK boosters AVC37 or AVC52, comprising IL-21 or 4-1 BBL ECD muteins respectively, to induce and support long-term NK cell expansion is hardly affected.
Table 1.2.1 presents the reduced binding affinities for IL-21 R of NK boosters comprising IL-21 muteins as indicated, as compared to a corresponding control NK booster AVC1 , comprising wild type IL-21 , as well as the EC50 values of the boosters for induction of proliferation of NK cells in the presence of SKOV3 tumor cells (in a normalized 5-day NK cell proliferation assay as described in Example 1 .1 .9 above).
Table 1 .2.1 . Overview of the reduced binding affinities for IL-21 R and of EC50 values for induction of proliferation of NK cells in the presence of SKOV3 tumor cells (ND = not determined).
EC50 in nM for induction of proliferation of NK cells in the presence of SKOV3 tumor cells.
As can be seen in Table 1 .2.1 , the affinity for IL-21 R of most of the NK boosters with IL-21 muteins is reduced up to several orders of magnitude compared the AVC1 booster with wild type IL-21 , except for AVC118 (I67T) and AVC126 (G84GGGGG). A subset of boosters with IL-21 muteins was also tested fortheir ability to induce proliferation of NK cells in the presence of SKOV3 tumor cells that express HER2. While these booster have reduced affinity for IL-21 R, they retain their ability to induce proliferation of NK cells in the presence of SKOV3 tumor cells to a similar degree as WILD TYPE IL-21 , as their the EC50 values for induction of such proliferation differ by no more than a factor 2.5.
These findings are confirmed in Figures 5A and B, which show that the corresponding doseresponse curves for induction of proliferation of NK cells in the presence of SKOV3 tumor cells of NK booster with IL-21 muteins are comparable to the dose-response curve of control NK booster AVC1 comprising wild type IL-21. Figure 5A compares IL-21 mutein NK boosters AVC116, AVC118, AVC119, AVC120 and AVC121 with control NK booster AVC1 and Figure 5B compares IL-21 mutein NK boosters AVC122, AVC123, AVC124, AVC125 and AVC126 with control NK booster AVC1.
In Table 1.2.2, the boosters’ ability to induce maximal proliferation of NK cells in the presence of SKOV3 tumor cells is determined at a saturating concentration of 25 nM booster. The boosters’ ability to induce maximal NK cell proliferation is determined of boosters comprising IL-21 muteins combined with either wild type 4-1 BBL ECD or the 4-1 BBL A154D mutein with reduced affinity for its cognate receptor 4-1 BB and compared with control booster AVC1. Als presented in Table 1.2.2 for each of the boosters are their pECso values for the induction of proliferation of NK cells in the presence of SKOV3 tumor cells. Table 1 .2.2. Overview of the ability of induction of proliferation of NK cells in the presence of SKOV3 tumor cells at saturating concentration (25 nM) of boosters comprising IL-21 muteins combined with wild type 4-1 BBL ECD or the 4-1 BBL A154D mutein as indicated, expressed as percentage of control booster AVC1 (with wild type 4-1 BBL and wild type IL-21). pECso values of the boosters for the induction of proliferation of NK cells in the presence of SKOV3 tumor cells are also shown.
As can be seen in Table 1.2.2, the ability to induce proliferation of NK cells in the presence of SKOV3 tumor cells of NK cell boosters comprising a wild type 4-1 BBL ECD and a number of selected IL-21 muteins with reduced affinity for IL-21 R is hardly affected compared to the corresponding booster with wild type IL-21. The pECso values are not reduced by more than 0.5 compared to the control booster and the maximum induced proliferation at saturating booster concentrations is not reduced by more than 10% compared to the control booster.
However, when the wild type 4-1 BBL ECD in these boosters is substituted for the reduced affinity- 4-1 BBL ECD A154D mutein, the pECso values are still not reduced by more than 0.5 compared to the control booster but the maximum induced proliferation at saturating booster concentration is significantly reduced up to more than 50% compared to control for at least two of the IL-21 muteins. Only the IL-21 L20W mutein retains most of its ability to maximally induce NK cell proliferation in combination with the 4-1 BBL mutein, with no more than about 10% reduction in maximally induced proliferation as compared to the corresponding wild type control booster AVC1.
Similar results as those obtained above with boosters targeting the HER2 as TAA are also obtained with boosters targeting either TROP2 or EGFR as TAA. The ability to induce NK cell cytotoxicity was compared for boosters AVC1 and AVC16, targeting HER2 and TROP2, respectively. Longterm NK cell cytotoxicity induced by the conjugates AVC1 and AVC16 was determined essentially as described in Example 1.1.13, except that only a single round co-culture of NK cells and target cells was performed and followed for 96 hours. Briefly, NK cell cytotoxicity was determined against tumor cell lines expressing a TAA specifically bound by the conjugates AVC1 and AVC16 as indicated in Table 1.2.3. A single round of co-culture of NK cells and target cells, in a E:T ratio of 2:1 (20,000 NK cells : 10,000 tumor cells) was followed for 96 hours, in the absence or presence of the multispecific antigen binding proteins at 25 nM. Control wells thus contained only NK cells and the respective target tumor cells. Assays were performed in triplicate. Interferon-gamma levels were determined in the supernatant using the MACSplex cytotoxic IFN-y kit (cat #130-125-800).
Table 1.2.3 Target tumor cell lines used for determination of NK cell cytotoxicity induced by the multispecific antigen binding proteins AVC1 , AVC16 and AVC20.
The results for AVC1 , AVC16 and AVC20 are shown in Figures 6A, 6B and 6C, respectively. Each of the multispecific antigen binding proteins AVC1 , AVC16 and AVC20 significantly induce increased cytotoxicity against tumor cells that express the respective antigens compared to NK cells only. Similarly, Figures 7 A, 7B and 7C show that the same set of multispecific antigen binding proteins induce increase interferon-gamma production in response to co-culture with tumor cells that express the respective antigens. Hence, similar stimulatory effects of the multispecific antigen binding proteins, in terms of inducing TAA-targeted NK cell cytotoxicity or induction of interferon- gamma production, are achieved when targeting either HER2, TROP2 or EGFR as tumor- associated antigen.
Figure 8 shows a comparison of NK boosters AVC16 (based of Fab-fragments from sacituzumab and comprising wild type IL-21 and a trimer of wild type 4-1 BBL ECD), AVC267 (based of Fab- fragments from sacituzumab and comprising the IL-21 L20W mutein and a trimer of the 4-1 BBL ECD A154D mutein) and control protein AVC 137 (a sacituzumab analogue without IL-21 or 4-1 BBL cytokines) in the long-term repeated NK cell cytotoxicity assay as described in Example 1.1.13. Further controls only NK cells and BxPC3 target cells (i.e. without Booster) or only BxPC3 target cells. As can be seen in Figure 8, the NK booster AVC267 comprising the IL-21 mutein L20W shows a similar long-term in vitro tumor control as the AVC16 booster with wild type IL-21 .
Figure 9 shows the effect of different mutations in IL-21 and/or 41 BB-L on V61 expansion and phenotype. y6 T-cell boosters comprising anti-V61 TCR VH and VL and wild type or mutein 4-BBL and IL-21 as indicated in Table 1 .1 .10.4, were used to induce expansion of of donor y6 T-cells as described in Example 1 .1 .14. After 14 days expansion of y6 T-cells (Figure 16A), PD1 expression on V61 T cells (Figure 16B) and CD56 expression on V61 T cells (Figure 16C) were determined as described in Example 1.1.15. All values have been normalised to AVC160 which uses IL-21 and 41 BB-ligand wildtype in its design. Figure 16A shows that all of the boosters with IL-21 and/or 41 BBL muteins are capable of inducing expansion of y6 T-cells, albeit to a somewhat lower rates than the AVC160 booster with wild types IL-21 and 41 BBL. However, compared to the AVC160, many of the boosters with muteins show a higher induction of CD56 expression on the expanded y6 T-cells, which is a marker for potency y6 T-cell. Induction of PD1 expression by the mutein boosters is higher compared to than the wild type AVC160 booster.
Numbered embodiments
1. An IL-21 mutein, the amino acid sequence of which differs from a wild type human IL-21 amino acid sequence of SEQ ID NO: 38 in that the IL-21 mutein comprises at least one amino acid substitution, deletion or insertion selected from the group consisting of: L20W, L74D, L20N, I67N, L20S, L13E, I8H, (N63-, E64-, R65- and I66-), L74F, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, 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), or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions, deletions or insertions.
2. An IL-21 mutein according to embodiment 1 , wherein the IL-21 mutein comprises at least two, three or four amino acid substitutions, deletions or insertions selected from the group consisting of: L20W, I8H, I8V, I8Q, I8F, I8W, I8Y, I8L, D4H, D4R, D4K, D4Q, D4N, R11 E, R1 1 Q, R11 N, R11Y, Q12K, Q12R, L13E, L13S, L13V, L13T, L13G, Q19S, Q19E, Q19K, Q19R, Q19H, Q19G, Q19T, L20S, L20D, L20E, L20R, L20K, L20Q, L20H, L20G, L20N, K21 H, K21 N, K21 Q, K21 E, K21 D, I67T, I67N, I67D, I67E, I67K, I67R, I67Q, I67S, I67G, L74G, L74D, L74F, 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-), (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) or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions, deletions or insertions.
3. An IL-21 mutein according to any one of the preceding embodiments, wherein the IL-21 mutein binds to a human IL-21 receptor with a reduced affinity, relative to the affinity of wildtype IL-21 for the human IL-21 receptor, wherein preferably the IL-21 mutein binds with reduced affinity to a human IL-21 receptor having an amino acid sequence of SEQ ID NO: 173 or the IL-21 mutein binds with reduced affinity to an IL-21 receptor gamma chain having an amino acid sequence of SEQ ID NO: 174. 4. An IL-21 mutein according to embodiment 3, wherein the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W, R5H, I8V, I8Q, Q12K, K73Y, K75E, L13E, D4H, I8H, (N63- E64- R65- I66-), I67N, L74D, L74F, L20S, and L20N, or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions or deletions.
5. An IL-21 mutein according to any one of the preceding embodiments, wherein the IL-21 mutein exhibits at least a 2-fold, 5-fold, 10-fold, 20-fold or 45-fold reduction in binding affinity for the human IL-21 receptor, relative to the affinity of wild-type IL-21 for the human IL-21 receptor.
6. An IL-21 mutein according to embodiment 4 or 5, wherein the IL-21 mutein exhibits no more than a 1000-fold, 500-fold, 200-fold, 100-fold, or 50-fold reduction in binding affinity for the human IL-21 receptor, relative to the affinity of wild-type IL-21 for the human IL-21 receptor.
7. An IL-21 mutein according to any one of the preceding embodiments, wherein the IL-21 mutein exhibits at least a 10-fold reduction in activity as measured by a STAT3 phosphorylation assay, relative to the activity of wild-type IL-21 under corresponding conditions.
8. An IL-21 mutein according to any one of the preceding embodiments, wherein the IL-21 mutein, when present in a conjugate with trastuzumab, which conjugate further comprises a trimer of the wild type 4-1 BB ligand extracellular domain (4-1 BBL ECD), has an EC50 for induction of proliferation of NK cells in a normalized 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not is more than a factor 2.5 higher than the EC50 of a corresponding control conjugate comprising wild-type IL-21 in the same assay.
9. An IL-21 mutein according to embodiment 8, wherein the IL-21 mutein comprises at least one amino acid substitution or deletion selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; L13E; I8H; (N63- E64- R65- 166-); and L74F, or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions or deletions.
10. An IL-21 mutein according to embodiment 9, wherein the IL-21 mutein exhibits a binding affinity for the human IL-21 receptor, expressed in pKo, that is at least 1 .0 lower than the pKo of wild-type IL-21 for the human IL-21 receptor, and wherein the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; I67N; L20S; and L13E, or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions.
11. An IL-21 mutein according to embodiment 10, wherein the IL-21 mutein, when present in a conjugate with trastuzumab, which conjugate further comprises a trimer of the wild type 4- 1 BBL ECD, has an EC50 for induction of proliferation of NK cells in a normalized 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, that is not is more than a factor 2 higher than the EC50 of a corresponding control conjugate comprising wild type wild-type IL-21 in the same assay, and wherein the IL-21 mutein comprises at least one amino acid substitution selected from the group consisting of: L20W; L74D; L20N; and I67N, or wherein the IL-21 mutein comprises no other modifications than the aforementioned amino acid substitutions.
12. An IL-21 mutein according to embodiment 11 , wherein the IL-21 mutein, when present in a conjugate with trastuzumab, which conjugate further comprises a trimer of an 4-1 BBL ECD mutein comprising a substitution A154D, induces a maximal proliferation of NK cells at a saturating concentration of 25 nM of the conjugate in a normalized 5-day NK cell proliferation assay in the presence of SKOV3 tumor cells, which proliferation is not less than 65%, 70%, 75%, 80%, 85%, or 89% 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, and wherein the IL-21 mutein comprises an L20W amino acid substitution, or wherein the IL-21 mutein comprises no other modification than the L20W amino acid substitution.
13. A conjugate comprising an IL-21 mutein according to any one of the preceding embodiments and a heterologous moiety.
14. A conjugate according to embodiment 13, wherein the IL-21 mutein is directly attached to the heterologous moiety or wherein the IL-21 mutein is attached to the heterologous moiety via a linker.
15. A conjugate according to embodiment 14, wherein the linker comprises a peptide, preferably a flexible linker peptide.
16. A conjugate according to embodiment 13 or 14, wherein the conjugate has a IL-21 muteinvalency that is higher than one.
17. A conjugate according to any one of embodiments 13 - 16, wherein the heterologous moiety comprises a polypeptide.
18. A conjugate according to embodiment 16, wherein the polypeptide is an antigen-binding protein or a polypeptide chain of an antigen-binding protein.
19. A conjugate according to embodiment 18, 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 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 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 third antigen-binding region that has affinity for a surface antigen expressed on natural killer (NK) cells, or that comprises or consists of an immunoglobulin Fc region.
20. A conjugate according to embodiment 19, wherein the first and second antigen-binding region comprise at least one immunoglobulin-derived antigen-binding region.
21. A conjugate according to embodiment 20, wherein the immunoglobulin-derived antigenbinding region comprises or consists of a Fab or an immunoglobulin single variable domain (ISVD). A conjugate according to any one of embodiments 18 - 20, wherein the first and second antigen-binding regions are human or humanized antigen-binding regions. A conjugate according to any one of embodiments 18 - 22, wherein the first and second antigen-binding regions bind the same TAA, at least two different TAAs, or at least two different epitopes of the same TAA. A conjugate according to embodiment 23, wherein the first and second antigen-binding regions are identical. A conjugate according to any one of embodiments 18 - 24, wherein the TAA is selected from the group consisting of: 5T4, ADAM9, ADAM10, ADAM12, AFP, ALK, ALPP, ALPP2, ALPPL2, AXL, Angiopoietin-2, Apelin receptor, B7-H3, B7-H4, B7-H6, B7.1 , B7.2, BCMA, BTLA, CA125, CAIX, CCR4, CCR6, CCR7, CD123, CD133, CD138, CD142, CD147, CD166, CD171 , CD19, CD2, CD20, CD205, CD22, CD228, CD24, CD25, CD27, CD276, CD3, CD30, CD317, CD33, CD38, CD3E, CD4, CD40, CD44v6, CD45, CD46, CD47, CD52, CD56, CD70, CD71 , CD73, CD74, CD79, CD79B, CD80, CD80/CD86, CDCP1 , CDH3, CDK4, CEA, CEACAM5, CLDN18, CLEC14A, CLEC4, CSF1 R, CSPG4, CT-7, CTLA4, Cadherin 17, Cadherin 6, CanAg, Claudin 18.2, Claudin 6, cMet, Connexin 37, Cripto-1 , Crypto, DC3, DLK1 , DLL3, DLL4, DR5, E-cadherin, E-selectin, EBV-encoded nuclear antigen (EBNA)-I, EDA, EDB, EDNRB, EGF, EGFR, EGFRvlll, EPCAM, EPHA4, EphAIO, EphA2, EphA3, EphB2, EphB4, ExtradomainB (EDB) fibronectin, F3, FAP, FGFR2, FGFR2b, FGFR4, FOLH1 , FOLR1 , FRa, FSHR, FcRL5/FcRH5, Fibronectin extra-domain B, Flt3, GFRa4, GM3, GPCR5D, GPRC5D, GRP78, GUCY2C, Glycoprotein NMB, Glypican 1 , Glypican 2, Glypican 3, GnT-V, HAVCR2, HER-2/ERBB2, HER-3/ERBB3, HER-4/ERBB4, HER2, HER3, HER4, HLA-G, HSP70, ICAM-1 , IFNG, IGF-1 R, IL-1 accessory protein, IL-6 receptor, IL-8 receptor, IL13Ra2, IL3RA, Ig-idiotype, Integrin beta 6, KAAG-1 , KDR, KLK2, KLRC1 , Killer Ig-Like Receptor, Killer Ig-Like Receptor 3DL2 (KIR3DL2), L1-CAM, L1 CAM, LAG3, LAGE-1 , LGR5, LIV-1 , Lewis-Y, MART-1 /Melan-A, MET, MIC-A/B, MICB, MISIIR, MMP2, MS4A1 , MSLN, MUC1 , MUC1-C, MUC16, MUM-1 , Melanotransferrin, Mesothelin, Mud 6, NAG, NKG2D, NT5E, NTRKR1 (EC 2.7.10.1), NaPi2b, Nectin-4, OLR1 , 0X40, P-cadherin, P1A, PD-L1 , PD1 , PDGF, PDGF alpha receptor, PDGF beta receptor, PDGFR, PDGFRA, PLAUR, PRAME, PSCA, PSMA, PTK7, PTPRC, PVRL4, Plexin-A1 , RAGE, ROBO1 , ROR1 , ROR2, SCP-1 , SEZ6, SLAMF7, SLC3A2, SSTR2, SSX-1 , SSX-2 (HOM-MEL-40), SSX-4, SSX-5, STEAP1 , STEAP2, T-cell receptor/CD3-zeta chain, TACSTD2, TGF-alpha, TIGIT, Tissue factor/TF, TM4SF1 , TMEFF2, TNFRSF10B, TNFRSF17, TNFRSF4, TNFRSF8, 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, avp1 integrins, avp3 integrins, avp6 integrins, adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (ADAbp), anti-Mullerian hormone Type II receptor, brain glycogen phosphorylase, c-erbB-2,, colorectal associated antigen (CRC)-C017-1A/GA733, gastrin releasing peptide receptor antigen, gp100, gp75, gpA33, hCG, human papillomavirus protein, integrin receptors, mmp9, muc17, p15, prostate specific antigen (PSA), protein tyrosine kinase 7(PTK7), receptor protein tyrosine kinase 3 (TYRO-3), sVE-cadherin, scatter factor receptor kinase, a-catenin, a- fetoprotein, allbp3-integrins, p-catenin, and y-catenin.
26. A conjugate according to embodiment 25, wherein at least one of the first and second antigen-binding regions 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 - VH1A/L1); 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); ft) 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); fh) 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); Iw) 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).
27. A conjugate according to embodiment 26, wherein at least one of the first and second antigen-binding region comprises a combination of variable light (VL) and variable heavy (VH) 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: 110 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: 119 (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 (meebotamab); 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 - 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 - 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 - 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 - 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 - VH2/VL2); th) 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). A conjugate according to any one of embodiments 18 - 27, wherein at least one of the first and second antigen-binding region specifically binds an NK cell activating receptor, and wherein preferably at least one of the first and second antigen-binding region is an agonistic antigen-binding region that activates the NK cell receptor. A conjugate according to any one of embodiments 18 - 28, wherein the antigen binding protein comprises two antigen-binding regions that specifically bind an NK cell activating receptor. A conjugate according to embodiment 29, wherein the two antigen-binding regions bind the same NK cell activating receptor or at least two different NK cell activating receptors. A conjugate according to embodiment 30, wherein the two antigen binding regions are identical. A conjugate according to any one of embodiments 18 - 31 , wherein the NK cell activating receptor 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, CD59, PD-L1 , Tim3, CRACC, 4-BB, 0X40, CRTAM, CD27, PSGL1 , CD96, CD100, CEACAM1 , and NTB-A. A conjugate according to any one of embodiments 18 - 27, wherein at least one of the first and second antigen-binding region specifically binds specifically binds an epitope of a y6 TCR. A conjugate according to embodiment 33, at least one first antigen-binding region specifically binds a TAA and at least one second antigen-binding region specifically binds an epitope of a y6 TCR. A conjugate according to any one of embodiments 18 - 34, wherein the antigen-binding region that specifically binds an epitope of a y6 TCR, 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. A conjugate according to any one of embodiments 18 - 35, wherein the antigen-binding region that specifically binds an epitope of a y6 TCR, binds an epitope in the V region of a V61 chain of a y6 TCR. A conjugate according to any one of embodiments 18 - 36, wherein the antigen-binding region that specifically binds an epitope of a y6 TCR, binds an activating epitope of a y6 T cell. A conjugate according to embodiment 37, wherein binding of the antigen-binding region to the activating epitope: (i) downregulates the y6 TCR; (ii) activates degranulation of the y6 T cell; (iii) induces proliferation in a y6 T cell; and/or (iv) promotes y6 T cell mediated killing. A conjugate according to embodiment 37 or 38, wherein the antigen-binding region specifically binds an epitope that upregulates expression of CD107a, CD25, CD56, CD69 and/or Ki67. A conjugate according to any one of embodiments 18 - 39, wherein the antigen-binding region that specifically binds an epitope of a y6 TCR, binds the epitope of a y6 TCR with a binding affinity (KD) as measured by surface plasmon resonance of less than 1.5 x 10-7 M, preferably less than 1 .0 x 10-7 M or less, such as less than 5.0 x 10-8 M, in particular less than 1.0 x 10-8 M. A conjugate according to any one of embodiments 18 - 40, wherein the antigen-binding region that specifically binds an epitope of a y6 TCR, binds the epitope of a y6 TCR with a KD of more than 1 .5 x 10-7 M, preferably more than 1 .0 x 10-6 M, such as more than 5.0 x 10'6 M, in particular more than 1 .0 x 10-5 M. A conjugate according to any one of embodiments 36 - 41 , wherein the antigen-binding region that specifically binds an epitope of a y6 TCR the third antigen-binding region comprises a combination of complementarity-determining regions (CDRs) CDR-H1 , CDR- H2, CDR-H3, CDR-L1 , CDR-L2 and CDR-L3 selected from the group consisting of: a) the CDR-H1 sequence of SEQ ID NO: 511 , the CDR-H2 sequence of SEQ ID NO: 512, CDR-H3 sequence of SEQ ID NO: 513, the CDR-L1 sequence of SEQ ID NO: 514, the CDR-L2 sequence of SEQ ID NO: 515 and the CDR-L3 sequence of SEQ ID NO: 516; b) the CDR-H1 sequence of SEQ ID NO: 517, the CDR-H2 sequence of SEQ ID NO: 518, CDR-H3 sequence of SEQ ID NO: 519, the CDR-L1 sequence of SEQ ID NO: 520, the CDR-L2 sequence of SEQ ID NO: 521 and the CDR-L3 sequence of SEQ ID NO: 522; c) the CDR-H1 sequence of SEQ ID NO: 523, the CDR-H2 sequence of SEQ ID NO: 524, CDR-H3 sequence of SEQ ID NO: 525, the CDR-L1 sequence of SEQ ID NO: 526, the CDR-L2 sequence of SEQ ID NO: 527 and the CDR-L3 sequence of SEQ ID NO: 528, or wherein the third antigen-binding region comprises a combination of variable light (VL) and variable heavy (VH) domains selected from the group consisting of: d) the VH sequence as comprised in SEQ ID NO: 529, and the VL sequence as comprised in SEQ ID NO: 530; e) the VH sequence as comprised in SEQ ID NO: 531 , and the VL sequence as comprised in SEQ ID NO: 532; f) the VH sequence as comprised in SEQ ID NO: 533, and the VL sequence as comprised in SEQ ID NO: 534. A conjugate according to any one of embodiments 18 - 42, wherein the third antigen-binding region comprises or consists of: i) an immunoglobulin Fc region or ii) an antigen-binding region that specifically binds a surface antigen expressed on NK cells. A conjugate according to embodiment 43, wherein the surface antigen expressed on NK cells is an NK cell activating receptor. A conjugate according to embodiment 44, wherein the Fc region is a dimeric Fc region. A conjugate according to any one of embodiments 43 - 45, wherein the Fc region binds to CD16A. A conjugate according to embodiment 46, wherein the Fc region is modified to reduce or enhance affinity for CD16A, relative to a corresponding wild-type Fc region. A conjugate according to embodiment 47, wherein the Fc region is modified to reduce or enhance NK cell activation through CD16A binding, relative to a corresponding wild-type Fc region. A conjugate according to any one of embodiments 42 - 48, wherein the third antigen-binding region activates the NK cell activating receptor. A conjugate according to any one of embodiments 33 - 42, wherein the third antigen-binding region comprises or consists of an immunoglobulin Fc region, preferably is dimeric Fc region, that is modified to reduce or abolish affinity for CD16A, relative to a corresponding wild-type Fc region. A conjugate according to any one of embodiments 13 - 50, comprising, in addition to the IL- 21 mutein, at least one further agonist, wherein the further agonist is at least one of a further NK cell activating cytokine, a further y6 T cell-activating agonist and a y6 T cell co-stimulatory agonist. 52. A conjugate according to embodiment 51 , wherein the further agonist is the heterologous moiety.
53. A conjugate according to embodiment 51 , wherein the further agonist is directly attached to the heterologous moiety or wherein the further agonist is attached to the heterologous moiety via a linker.
54. A conjugate according to any one of embodiments 51 - 53, wherein the further agonist is at least one of: i) a further NK cell activating cytokine selected from the group consisting of: a 4-1 BB 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 further y6 T cell-activating agonist selected from the group consisting of: 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 y6 T cell co-stimulatory agonist selected from the group consisting of: a 4-1 BB agonist, a CD27 agonist and a GITR agonist.
55. A conjugate according to embodiment 54, wherein: i) the 4-1 BB agonist comprises or consist of at least one 4-1 BB ligand (4-1 BBL) extracellular domain (ECD) or at least one agonistic antigen-binding region that specifically binds 4-1 BB; ii) the IL-15 receptor agonist comprises or consists of an IL-15 polypeptide or an agonistic antigen-binding 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.
56. A conjugate according to embodiment 55, wherein: i) the 4-1 BBL ECD comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 37, and preferably has at least one of 4-1 BB agonist activity and affinity for 4-1 BB; ii) the IL-15 polypeptide comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 168, and preferably has at least one of IL-15R agonist activity and affinity for the IL-15R; iiii) the IL-2 polypeptide comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 351 , and preferably has at least one of IL-2R agonist activity and affinity for the IL-2R; iv) the IL-12 polypeptide comprises an IL-12a polypeptide comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 352, and an IL-120 polypeptide comprising an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 353, wherein preferably, in an N- to C-terminal direction, the IL-120 polypeptide is linked, optionally through a peptidyl linker, to the IL-12a polypeptide, and wherein the IL-12 polypeptide preferably has at least one of IL-12R agonist activity and affinity for the IL-12R; v) the IL-18 polypeptide comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 354, and preferably has at least one of IL-18R agonist activity and affinity for the IL-18R; vi) the CD70 ECD comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 536, and preferably has at least one of CD27 agonist activity and affinity for CD27; and, vii) the GITRL ECD comprises an amino acid sequence with at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to SEQ ID NO: 539, and preferably has at least one of GITR agonist activity and affinity for GITR. onjugate according to embodiment 55 or 56, wherein: i) the 4-1 BBL ECD is a mutein that is modified to reduce or enhance affinity, improve stability or improve expression for 4-1 BB, relative to a corresponding wild type 4-1 BBL ECD; ii) the IL-15 polypeptide is an IL-15 mutein that is modified to reduce (or enhance) affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide; iii) the IFN-1 polypeptide is an IFN-1 mutein that is modified to reduce (or enhance) affinity for the IFN-a receptor, relative to a corresponding wild type IFN-1 polypeptide; iv) the IL-2 polypeptide is an IL-2 mutein that is modified to reduce (or enhance) affinity for IL-2R, relative to a corresponding wild type IL-2 polypeptide; v) the IL-12 polypeptide is an IL-12 mutein that is modified to reduce (or enhance) affinity for IL-12R, relative to a corresponding wild type IL-12 polypeptide; vi) the IL-18 polypeptide is an IL-18 mutein that is modified to reduce (or enhance) affinity for IL-18R, relative to a corresponding wild type IL-18 polypeptide; vii) the CD70 ECD is a mutein that is modified to reduce or enhance affinity for CD27, improve stability or improve expression, relative to a corresponding wild type CD70 ECD; and, viii) the GITRL ECD is a mutein that is modified to reduce or enhance affinity for GITR, improve stability or improve expression, relative to a corresponding wild type GITRL ECD.
58. A conjugate according to any one of embodiments 55 - 57, wherein: i) the 4-1 BBL ECD is a mutein with reduced affinity for 4-1 BB, relative to a corresponding wild type 4-1 BBL ECD, and wherein 4-1 BBL ECD mutein provided herein comprises the amino acid sequence:
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 1 amino acid; ii) the IL-15 mutein has reduced affinity for IL-15R, relative to a corresponding wild type IL-15 polypeptide, and wherein the IL-15 mutein has a mutation in at least one amino acid position selected from the group consisting of: N1 , D8, D30, H32 L45, E46, I50, V49, S51 , L42, V63, E64, N65, I68, L69, Q108, M109 and N112; and, iii) the CD70 ECD is a mutein with reduced affinity for CD27 relative to a corresponding wild type CD70 ECD, and wherein CD70 ECD mutein comprises at least one amino acid substitution, deletion or insertion at a position in SEQ ID NO: 536 selected from the group consisting of: Q61 , A80, S137, S146 and H148, wherein preferably, the CD70 ECD mutein comprises at least one amino acid substitution selected from the group consisting of: Q61A, A80F, A80R, S137A, S137E, S137K, S146A, H148A, H148E and H148D.
59. A conjugate according to embodiment 58, wherein: i) the 4-1 BBL ECD comprises one or more amino acid substitutions selected from the group consisting of: A154D, Y110Q, V153Q, V100T, V100Q, L101 N, G1 14K, L115R, A116D, R171 D, Q227E, Q227R, Q230S, Q230K, V100T, Q227R, Q230S and Q230K, of which A154D, Y110Q, and V153Q are preferred, of which A154D and Y110Q are more preferred, and of which A154D is most preferred; ii) the IL-15 mutein comprises a mutation in at least one amino acid position selected from the group consisting of: D8, E64, N65, I68, and L69; and, iii) the CD70 ECD mutein comprises at least one amino acid substitution selected from the group consisting of: Q61A, A80F, A80R, S137A, S137E, S137K, S146A, H148A, H148E and H148D.
60. A conjugate according to any one of embodiments 54 -59, wherein: i) the 4-1 BB agonist comprises or consist of a fusion protein comprising three 4-1 BBL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three 4-1 BBL ECD monomers are connected by polypeptide linkers; ii) the CD27 agonist comprises or consists of a fusion protein comprising three CD70 ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three CD70 ECD monomers are connected by polypeptide linkers; and, iii) the GITR agonist comprises or consists of a fusion protein comprising three GITRL ECD monomers fused together in a single polypeptide chain, and wherein, optionally, the three GITRL ECD monomers are connected by polypeptide linkers.
61 . A conjugate according to any one of embodiments 54 - 60, wherein the conjugate has: i) a 4-1 BB agonist-valency that is higher than one; ii) an IL-15 receptor agonist-valency that is higher than one; iii) a type I interferon (IFN-1) receptor agonist-valency that is higher than one; iv) an IL-2 receptor agonist-valency that is higher than one; v) an IL-12 receptor agonist-valency that is higher than one; vi) an IL-18 receptor agonist-valency that is higher than one; vii) a CD27 agonist-valency that is higher than one; and, viii) a GITR agonist-valency that is higher than one.
62. A conjugate according to any one of the embodiments 19 - 61 , wherein at least one of the first and second antigen-binding regions is conjugated to the third antigen-binding region.
63. A conjugate according to embodiment 62, wherein at least one polypeptide chain in the at least one of the first and second antigen-binding regions forms a single polypeptide chain with at least one polypeptide chain of the third antigen-binding region.
64. A conjugate according to embodiment 63, wherein the single polypeptide chain comprises in an N- to C-terminal order: i) at least one polypeptide chain in the at least one of the first and second antigen-binding region; ii) optionally a flexible linker; and iii) the third antigen-binding region.
65. A conjugate according to embodiment 63 or 64, wherein the third antigen-binding region is a dimeric Fc region, wherein each of the two polypeptide chains of the dimeric Fc region is linked to a CH1 domain, each of which CH1 domains is linked to an immunoglobulin-derived antigen-binding region that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR.
66. A conjugate according to embodiment 65, wherein the two immunoglobulin-derived antigenbinding regions bind the same TAA, or wherein the two immunoglobulin-derived antigenbinding region each bind a different TAA.
67. A conjugate according to embodiment 65 or 66, wherein the protein comprises a dimeric Fc region, wherein each of the two Fc polypeptide chains is operably linked to a Fab that specifically binds a TAA, an NK cell activating receptor or an epitope of a y6 TCR.
68. A conjugate according to any one of embodiments 62 - 67, wherein at least one of the IL-21 mutein and the further agonist, is conjugated to the at least one antigen-binding region that specifically binds a TAA or an NK cell activating receptor or an epitope of a y6 TCR, or to the third antigen-binding region. 69. A conjugate according to embodiment 68, wherein at least one of the IL-21 mutein and the further agonist forms a single polypeptide chain with at least one of: i) at least one polypeptide chain in at least one of the first and second antigen-binding regions; and, ii) at least one polypeptide chain in the third antigen-binding region; wherein optionally, a flexible linker is present between the at least one of the IL-21 mutein and the further agonist and the at least one polypeptide chain in the region defined in i) or ii).
70. A conjugate according to any one of embodiments 67 - 69, wherein at least one of the IL-21 mutein and the further agonist forms a single polypeptide chain with at least one of: i) a light chain in at least one of the two Fabs that specifically bind a TAA an NK cell activating receptor or an epitope of a y6 TCR; and, ii) at least one of the two Fc chains in the dimeric Fc region; wherein optionally, a flexible linker is present between the at least one of the IL-21 mutein and the further agonist and the light chain defined in i) or the Fc chain defined in ii).
71. A conjugate according to embodiment 70, wherein at least one of the IL-21 mutein and the further agonist is fused to at least one of: i) the N-terminus of the light chain in at least one of the two Fab’s that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR, optionally through a flexible linker; ii) the C-terminus of the light chain in at least one of the two Fab’s that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR, optionally through a flexible linker; iii) the N-terminus of the heavy chain in at least one of the two Fab’s that specifically bind a TAA, an NK cell activating receptor or an epitope of a y6 TCR; and, iv) the C-terminus of the heavy chain in at least one of the two Fc chains in the dimeric immunoglobulin Fc domain, optionally through a flexible linker.
72. A conjugate according to embodiment 70 or 71 , wherein at least one of the IL-21 mutein and the further agonist is present on at least one or on both sides of the immunoglobulin structure.
73. A conjugate according to any one of embodiments 65 - 72, wherein the conjugate is heterodimeric with respect to at least one of: i) the first and second antigen-binding regions; and ii) at least one of the fused IL-21 mutein and the fused further agonist, and wherein the dimeric Fc region comprises different first and a second polypeptide chains comprising knob- into-hole modifications promoting association of the first and the second polypeptide chains of the Fc region.
74. A conjugate according to any one of embodiments 13 - 73, wherein the conjugate has at least one biological activity selected from: a) the conjugate causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same NK cells and target cells that are not brought into contact with the conjugate; b) the conjugate causes an increase in at least one NK cell activity selected from CD107a degranulation, CD107 or CD69 expression, IFNy production, NK cell proliferation and NK cell cytotoxicity, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same NK cells and target cells that are brought into contact with a conventional human lgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the conjugate; c) the conjugate causes an increase in at least one y6 T cell activity selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are not brought into contact with the conjugate; and, b) the conjugate causes an increase in at least one y6 T cell activity selected from y6 T cell proliferation, cytokine production, y6 T cell cytotoxicity, y6 T cell differentiation, whereby preferably, the increase is at least a factor 0.1 higher as compared to the increase achieved with the same effector : target cell ratio, with the same y6 T cells and target cells that are brought into contact with a conventional human lgG1 monoclonal antibody that has the same TAA-specific antigen-binding regions as the conjugate binding protein. A conjugate according to any one of embodiments 13 - 74, wherein ex vivo expansion of donor NK cells by co-culturing with the conjugate, produces a population of expanded NK cells having one or more features selected from: a) the fold expansion of the expanded NK cells is at least 0.5, 1 .0, 2.0, or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated K562 feeder cells modified to express membrane bound IL-21 (mblL-21) and 4- 1 BB ligand (FC21 feeder cells); b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.5, 1 .0, 2.0, or 5.0 fold of the percentage telomere length increase of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded NK cells is at least 0.5, 1.0, 2.0, or 5.0 fold of the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; and, e) the cytotoxicity of the expanded NK cells is at least 0.5, 1 .0, 2.0, or 5.0 fold of the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells, wherein, preferably the NK cells are co-cultured with tumor cells expressing a TAA specifically bound by the conjugate, or wherein ex vivo expansion of donor y6 T cells by co-culturing with the conjugate, produces a population of expanded NK cells having one or more features selected from: a) the fold expansion of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the fold expansion of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with irradiated K562 feeder cells modified to express a membrane bound version of the IL-21 mutein and a membrane bound version of the co-stimulatory agonist; b) the telomere length of the expanded y6 T cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh y6 T cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh y6 T cells, is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the percentage telomere length increase of y6 T cells obtained upon ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; c) the expression level of at least one y6 T cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the secretion of the cytokine by expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; and, e) the cytotoxicity of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the cytotoxicity of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co- culturing with the modified irradiated K562 feeder cells wherein, preferably the y6 T cells are co-cultured with tumor cells expressing a TAA specifically bound by the conjugate. A pharmaceutical composition comprising an IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75, and a pharmaceutically acceptable carrier. An ex vivo method for expansion of NK cells, the method comprising the step of contacting an NK cell with a conjugate according to any one of embodiments 13 - 75, or with a composition according to embodiment 76, and wherein preferably, the expanded NK cells have one or more features selected from: a) the fold expansion of the expanded NK cells is at least 0.5, 1 .0, 2.0, or 5.0 fold of the fold expansion of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated K562 feeder cells modified to express membrane bound IL-21 (mblL-21) and 4- 1 BB ligand (FC21 feeder cells); b) the telomere length of the expanded NK cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh NK cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh NK cells, is at least 0.5, 1 .0, 2.0, or 5.0 fold of the percentage telomere length increase of NK cells obtained upon ex vivo expansion in the presence of FC21 feeder cells; c) the expression level of at least one NK cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded NK cells is at least 0.5, 1.0, 2.0, or 5.0 fold of the secretion of the cytokine by expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells; and, e) the cytotoxicity of the expanded NK cells is at least 0.5, 1 .0, 2.0, or 5.0 fold of the cytotoxicity of expanded NK cells obtained by ex vivo expansion by co-culturing with irradiated FC21 feeder cells, wherein, preferably the method comprising the further step of co-culturing the NK cells with tumor cells expressing a TAA specifically bound by the conjugate. An ex vivo method for expansion of y6 T cells, the method comprising the step of contacting a y6 T cell with conjugate according to any one of embodiments 13 - 75, or with a composition according to embodiment 76, and wherein preferably, the expanded y6 T cells have one or more features selected from: a) the fold expansion of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the fold expansion of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with irradiated K562 feeder cells modified to express a membrane bound version of the IL-21 mutein and a membrane bound version of the co-stimulatory agonist; b) the telomere length of the expanded y6 T cells is increased by at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55% as compared to the telomere length of fresh y6 T cells, preferably, the percentage telomere length increase of the expanded NK cells as compared to the telomere length of fresh y6 T cells, is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the percentage telomere length increase of y6 T cells obtained upon ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; c) the expression level of at least one y6 T cell activating receptor selected from NKG2D, NKp30, NKp44, NKp46 and CD16 on the expanded NK cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1.0, 2.0, or 5.0 fold of the expression level on expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; d) the secretion of at least one cytokine of TNF-a, IFN-y and IL-6 by the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the secretion of the cytokine by expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co-culturing with the modified irradiated K562 feeder cells; and, e) the cytotoxicity of the expanded y6 T cells is at least 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.2, 0.5, 1 .0, 2.0, or 5.0 fold of the cytotoxicity of expanded y6 T cells obtained by ex vivo expansion by ap T cell depletion and stimulation with OKT3 antibody or by co- culturing with the modified irradiated K562 feeder cells. An IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75, a composition according to embodiment 76, an ex vivo expanded NK cell obtained in a method according to embodiment 77, optionally in combination with the IL-21 mutein or the conjugate, or an ex vivo expanded y6 T cell obtained in a method according to embodiment 78, optionally in combination with the IL-21 mutein or the conjugate, for use as a medicament. An IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75, a composition according to embodiment 76, an ex vivo expanded NK cell obtained in a method according to embodiment 77, optionally in combination with the IL-21 mutein or the conjugate, or an ex vivo expanded y6 T cell obtained in a method according to embodiment 78, optionally in combination with the IL-21 mutein or the conjugate, for use in the treatment of a cancer, an infectious disease or an inflammatory disease, wherein preferably the cancer is a cancer comprising tumor cells expressing the TAA. An IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75, a composition according to embodiment 76, for use in the treatment of a cancer, preferably a cancer comprising tumor cells expressing the TAA, wherein the IL-21 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, wherein the T cells are preferably y6 T cells.
82. An IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75 or a composition according to embodiment 76, for a use according to embodiment 80 or 81 , wherein at least one of: a) the IL-21 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 IL-21 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.
83. A method for enhancing anti-tumor activity of at least one of an NK cell and a y6 T cell in a subject, the method comprising the step of administering to the subject an IL-21 mutein according any one of embodiments 1 - 12, a conjugate according to any one of embodiments 13 - 75, a composition according to embodiment 76, or an ex vivo expanded NK cell or y6 T cell obtained in a method according to embodiment 77 or 78, optionally, in combination with the IL-21 mutein or the conjugate, or a combination of the IL-21 mutein or the conjugate and an immune cell selected from T cells and NK cells, wherein the T cells are preferably y6 T cells.
84. The method of embodiment 83, wherein the subject has cancer, preferably a cancer comprising tumor cells expressing the TAA.
85. The method of embodiment 84, wherein at least one of: a) the IL-21 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 IL-21 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.
86. A nucleic acid molecule comprising one or more nucleotide sequences encoding an IL-21 mutein according any one of embodiments 1 - 12 or a polypeptide chain of a conjugate according to any one of embodiments 13 - 75.
87. A nucleic acid molecule according to embodiment 86, wherein the one or more nucleotide sequences are operably linked to regulatory sequences for expression of the one or more polypeptide chains in a host cell.
88. A host cell comprising a nucleic acid molecule according to embodiment 86 or 87.
89. A method for producing an IL-21 mutein according any one of embodiments 1 - 12 or a conjugate according to any one of embodiments 13 - 75, the method comprising culturing a host cell according to embodiment 88 such that the one or more nucleotide sequences are expressed, and the IL-21 mutein or the conjugate is produced. A method according to embodiment 89, further comprising the steps of: recovery of the IL- 21 mutein or the conjugate, and, optionally, formulation of the IL-21 mutein or the conjugate with a pharmaceutically acceptable carrier.