Multispecific binding agents targeting dopamine receptor D2 and methods of use thereof

Abstract

The present disclosure generally relates to binding agents capable of targeting tumor cells and immune cells. The binding agents of the present disclosure are multispecific and comprise antigen-binding domains capable of binding to dopamine receptor D2 (DR2), programmed cell death protein 1 (PD-1), and / or cluster of differentiation 47 (CD47). The multispecific binding agents of the present disclosure may be used to treat subjects in need thereof.

Description

[Technical Field]

[0001] The present disclosure generally relates to binding agents capable of targeting tumor cells and immune cells. The binding agents of the present disclosure are multispecific and comprise antigen-binding domains capable of binding to dopamine receptor D2 (DR2), programmed cell death protein 1 (PD-1), and / or cluster of differentiation 47 (CD47). The multispecific binding agents of the present disclosure may be used to treat subjects in need thereof. [Background technology]

[0002] Camelids and cartilaginous fish naturally produce antibodies composed of functional homodimeric heavy chain-only antibodies (HCAbs) (Hamers-Casterman et al., 1993; Muyldermans and Smider, 2016). The heavy chains of HCAbs lack the first constant domain (CH1) and differ from classical antibodies by substitutions of only a few amino acids normally involved in light chain pairing (Muyldermans et al., 1994; Vu et al., 1997). These substitutions (Val37Phe / Tyr, Gly44Glu, Leu45Arg, and Trp47Gly) are present in framework region 2 (FR2). Antigen-binding fragments of HCAbs are called VHHs or nanobodies®. VHHs have a molecular weight of approximately 15 kDa, making them suitable for applications requiring enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic applications, the half-life of VHHs usually needs to be increased to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al., Antibodies 8(1), pp. 1-22, 2019). Methods to increase the half-life of VHHs, such as PEGylation, N-glycosylation, and fusion with HSA or other carrier proteins, have been utilized, but such constructs may introduce immunogenicity or may have limited success.

[0003] VHHs have been utilized as building blocks for generating bispecific and multispecific antibodies, and some studies have shown that bivalent constructs have increased avidity or affinity compared to monovalent forms (Conrath et al., 2001; Coppieters et al., 2006; Hmila et al., 2008; Simmons et al., 2006 and Hultberg et al., 2011; Jahnichen et al. (2010); Fridy et al., 2014).

[0004] Numerous VHH-based therapeutics are currently in late-stage clinical trials or have been approved by the FDA. These include caplacizumab, a bivalent monospecific antibody against the antigen vWF approved for thrombotic thrombocytopenic purpura (Duggan, 2018). ALX-0171, a trivalent nanobody conjugate against RSV, is in late-stage development for respiratory syncytial virus infection (Detallea et al., 2015). ALX-0061, monovalent against the antigen IL-6R but attached to an HSA nanobody to extend its half-life, is in clinical development for RA and SLE indications (Van Roy et al., 2015). The investigational drug ALX-0761 contains three nanobodies against the antigens IL-17A, IL-17F, and HAS and is being developed for psoriasis (Svecova et al., 2019). Anti-RANKL, ALX-0141, is bivalent against the antigen RANKL and is attached to HSA to extend its half-life (Schoen et al., 2013). Ozoralizumab is a bivalent nanobody against the antigen TNFα and is attached to HSA to extend its half-life (Fleischmann et al., 2012).

[0005] The dopamine receptor family includes five G protein-coupled receptors (GPCRs): dopamine receptor D1 (DRD1), dopamine receptor D2 (DRD2), dopamine receptor D3 (DRD3), dopamine receptor D4 (DRD4), and dopamine receptor D5 (DRD5). Based on the G protein that mediates intracellular signaling, it can be divided into the D1-like family of receptors, including DRD1 and DRD5, and the D2-like family of receptors, including DRD2, DRD3, and DRD5. Each dopamine receptor appears to exhibit specific expression patterns across different tumor types. For example, DRD2 shows increased expression in glioblastoma tumor samples (Dolma S. et al., 2016; Prabhu VV et al., 2019). DRD2 is also upregulated in breast cancer, lung cancer, gastric cancer, acute myeloid leukemia (AML), and pancreatic cancer (Gholipour N. et al., 2018, Wu X.-Y. et al., 2018, Carl M. et al., 2021, Jandaghi P. et al., 2016).

[0006] The applicant has developed novel binding agents that target DR2-expressing tumor cells and immune cells for use in methods of treating subjects in need thereof. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] WO / 2021 / 119832 [Patent Document 2] PCT / CA2020 / 051753 [Patent Document 3] WO2022 / 011457 [Patent Document 4] International application PCT / CA2021 / 050951 [Patent Document 5] U.S. Patent No. 8,326,547 [Patent Document 6] US Patent Application Publication No. 2019 / 0010242 [Patent Document 7] U.S. Patent Application Publication No. 20110076275

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[0009] The present disclosure relates generally to binding agents that have the ability to target, inter alia, tumor cells and immune cells.

[0010] Advantageously, the binding agents of the present disclosure reduce tumor growth and / or induce tumor regression in animal models.

[0011] Thus, the binding agents of the present disclosure may be used to treat a subject in need thereof.

[0012] In some embodiments, the binding agents of the present disclosure may be used to induce tumor regression in a subject in need thereof.

[0013] In some embodiments, the binding agents of the present disclosure may be used to slow the progression of cancer in a subject in need thereof.

[0014] In some embodiments, the methods of the present disclosure include administering a binding agent to a subject having cancer to promote regression of one or more tumors or tumor lesions. Thus, administration of the binding agent of the present disclosure can result in at least partial regression of one or more tumors or tumor lesions.

[0015] In other embodiments, the methods of the present disclosure include administering a binding agent to a subject having cancer to reduce the size of one or more tumors or tumor lesions. Thus, administration of a binding agent of the present disclosure can result in a reduction in the size of one or more tumors or tumor lesions.

[0016] In other embodiments, the methods of the present disclosure include administering a binding agent to a subject having cancer to reduce the growth rate of one or more tumors or tumor lesions. Thus, administration of a binding agent of the present disclosure can result in slower growth of one or more tumors or tumor lesions.

[0017] In some embodiments, a binding agent of the present disclosure is administered to a subject having a tumor that expresses dopamine receptor D2 (DR2). In other embodiments, a binding agent of the present disclosure is administered to a subject having a tumor that expresses cluster of differentiation 47 (CD47).

[0018] In some embodiments, the binding agents of the present disclosure are multispecific and comprise at least one antigen-binding domain capable of binding to dopamine receptor D2 (DR2), at least one antigen-binding domain capable of binding to programmed cell death protein 1 (PD-1), and / or at least one antigen-binding domain capable of binding to cluster of differentiation 47 (CD47). The antigen-binding domains of the binding agent may bind to cells expressing DR2, cells expressing PD-1, and / or cells expressing CD47.

[0019] Thus, in some embodiments, the binding agents of the present disclosure are capable of binding to dopamine receptor D2 (DR2), programmed cell death protein 1 (PD-1), and / or cluster of differentiation 47 (CD47). In other embodiments of the present disclosure, the binding agents may bind to cells expressing DR2, cells expressing PD-1, and / or cells expressing CD47.

[0020] In exemplary embodiments, a binding agent or antigen-binding domain of the disclosure has the ability to bind to a DR2 antigen comprising a sequence at least 80% identical to a human DR2 protein, a PD-1 antigen comprising a sequence at least 80% identical to human PD-1, and / or a CD47 antigen comprising a sequence at least 80% identical to human CD47.

[0021] In exemplary embodiments, the binding agents or antigen-binding domains of the disclosure are capable of binding to human DR2, human PD-1, and / or human CD47. In other embodiments of the disclosure, the binding agents or antigen-binding domains may bind to human cells expressing DR2, human cells expressing PD-1, and / or human cells expressing CD47.

[0022] According to the present disclosure, the binding agent may also comprise an additional antigen-binding domain.

[0023] In some embodiments, the binding agent comprises at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2), at least one antigen-binding domain 2 (ABD2) that binds to an immunomodulator, and at least one antigen-binding domain 3 (ABD3) that binds to an immune cell.

[0024] In some embodiments, antigen-binding domain 2 (ABD2) is an antigen-binding domain capable of binding to PD-1 or to a cell expressing PD-1.

[0025] In some embodiments, antigen binding domain 3 (ABD3) is an antigen binding domain capable of binding to CD47 or to cells expressing CD47.

[0026] In some embodiments, a binding agent may comprise an antigen-binding domain that targets an epitope other than those covered by ABD1, ABD2 and ABD3, and thus may comprise an antigen-binding domain with the same or different specificity as that of ABD1, ABD2 and / or ABD3.

[0027] In some embodiments, the binding agent may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, ten or more antigen-binding domains.

[0028] In some embodiments, the binding agent may comprise between 2 and 12 antigen-binding domains.

[0029] In some embodiments, binding agents of the present disclosure may be multispecific.

[0030] In some embodiments, binding agents of the present disclosure may be multivalent.

[0031] In some embodiments, the binders of the present disclosure may be in monomeric form.

[0032] In some embodiments, binding agents of the present disclosure may be in the form of dimers or higher order forms such as trimers, tetramers, pentamers, and the like (eg, multimers).

[0033] In some cases, the antigen-binding domain of the binding agent originates from a heavy chain antibody. In some cases, the heavy chain antibody may be obtained by immunization of a camelid or a transgenic animal.

[0034] In some embodiments, the binding agent comprises at least one antigen-binding domain that binds to DR2 and comprises a complementarity determining region as described herein.

[0035] In some embodiments, the binding agent comprises at least one antigen-binding domain that binds to PD-1 and comprises a complementarity-determining region described herein.

[0036] In some embodiments, the binding agent comprises at least one antigen-binding domain that binds to CD47 and comprises a complementarity-determining region as described herein.

[0037] In some embodiments, the antigen binding domains are on one or more polypeptide chains.

[0038] In some embodiments, the antigen binding domains are on the same polypeptide chain.

[0039] In some embodiments, the binding agent comprises a single polypeptide chain.

[0040] In some embodiments, the binding agent comprises two polypeptide chains, and thus may be in the form of a dimer.

[0041] In some embodiments, a binding agent comprises more than two polypeptide chains, e.g., three or more polypeptide chains, four or more polypeptide chains, five or more polypeptide chains, six or more polypeptide chains, seven or more polypeptide chains, eight or more polypeptide chains, nine or more polypeptide chains, ten or more polypeptide chains. Thus, a binding agent may be in the form of a multimer. For example, a binding agent comprising three polypeptide chains is referred to herein as a trimer, a binding agent comprising four polypeptide chains is referred to herein as a tetramer, etc.

[0042] In some embodiments, the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises one or more antigen-binding domains.

[0043] In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen-binding domain.

[0044] In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen-binding domain.

[0045] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1).

[0046] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 2 (ABD2).

[0047] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 3 (ABD3).

[0048] In some exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1) and an identical antigen-binding domain 2 (ABD2).

[0049] In another exemplary embodiment, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1) and an identical antigen-binding domain 3 (ABD3).

[0050] In a further exemplary embodiment, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 2 (ABD2) and an identical antigen-binding domain 3 (ABD3).

[0051] In still further exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1), an identical antigen-binding domain 2 (ABD2), and an identical antigen-binding domain 3 (ABD3).

[0052] In some cases, antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and antigen-binding domain 3 (ABD3) may occupy the same position in each of the two polypeptide chains.

[0053] In some cases, antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and antigen-binding domain 3 (ABD3) may occupy different positions in each of the two polypeptide chains.

[0054] In some embodiments, each polypeptide chain of the binding agent is the same.

[0055] In some embodiments, each polypeptide chain of the binding agent is different.

[0056] In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.

[0057] In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.

[0058] In some embodiments, the antigen binding domains are separated by an amino acid sequence.

[0059] In some embodiments, the amino acid sequence is a linker. In some embodiments, the amino acid sequence is an antibody hinge or a fragment thereof.

[0060] Binding agents of the disclosure include, for example, an antigen-binding domain disclosed herein, a single polypeptide chain disclosed herein, a dimer of polypeptide chains disclosed herein, or a multimer of polypeptide chains disclosed herein.

[0061] Thus, binding agents of the present disclosure may have the format of antibodies and antigen-binding fragments thereof, antibody-like molecules (Fc, CH3 fusions, etc.), protein scaffolds and fusions with immune cell modulating agents, etc.

[0062] Advantageously, the binders of the present disclosure may have a format as disclosed in PCT / CA2020 / 051753, filed on December 18, 2020, and published under WO / 2021 / 119832 on June 24, 2021 (the entire content of which is incorporated herein by reference), such as Formula I, Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII, etc., or a format as disclosed herein.

[0063] In some embodiments, the binding agent is comprised of a polypeptide chain comprising one or more antigen-binding domains and a dimerization domain that allows at least two polypeptide chains to form a dimer.

[0064] In some embodiments, the binding agents of the present disclosure have the structure of Formula I in N-terminal to C-terminal fashion: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y and may comprise one or more polypeptide chains each independently comprising the amino acid sequence of m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain.

[0065] In some embodiments, the polypeptide chains comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, etc.

[0066] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen-binding domains.

[0067] In some embodiments, the binding agent comprises one polypeptide chain.

[0068] In some embodiments, the binding agent comprises two polypeptide chains, three polypeptide chains, four polypeptide chains, five polypeptide chains, six polypeptide chains, seven polypeptide chains, eight polypeptide chains, nine polypeptide chains, ten polypeptide chains, or more than ten polypeptide chains.

[0069] In some embodiments, the polypeptide chains may be covalently linked.

[0070] In some embodiments, the polypeptide chains may be non-covalently linked.

[0071] In some embodiments, the polypeptide chains may associate through electrostatic interactions.

[0072] In some embodiments, where m is an integer greater than or equal to 2, [(Ab a )-(L b )] units may be the same or different.

[0073] In some embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units may be the same or different.

[0074] In some embodiments where m is an integer greater than or equal to 2, each Ab a are the same or different.

[0075] In some embodiments where n is an integer greater than or equal to 2, each Ab d are the same or different.

[0076] In some embodiments, Ab a represents ABD1, ABD2 or ABD3.

[0077] In some embodiments, Ab d represents ABD1, ABD2 or ABD3.

[0078] In some embodiments, Ab drepresents ABD1, ABD2 or ABD3.

[0079] In embodiments, one or more polypeptide chains further comprise a hinge region of an antibody or antigen-binding fragment thereof. For example, in some embodiments, L b is the hinge region of an antibody or antigen-binding fragment thereof.

[0080] In some embodiments, the hinge region is a native hinge region from an IgG1, IgG2, IgG3, or IgG4.

[0081] In some embodiments, the hinge region is a variant hinge region having at least 70% identity to a native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0082] In some embodiments, each of the one or more linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0083] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0084] In some embodiments, the flexible linker is a GS linker. In some embodiments, the flexible linker comprises one or more GGGGS units as described herein.

[0085] In some embodiments, the rigid linker comprises multiple PA repeats as described herein.

[0086] In some embodiments, the helical linker comprises one or more EAAAK units as described herein.

[0087] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula II: X-(Ab a1 )-(Lb1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0088] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0089] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0090] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0091] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(Lc3 )-(Ab d3 )-Y (Formula VI).

[0092] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0093] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VIII).

[0094] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently comprises an antigen-binding domain.

[0095] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently represents an antigen-binding domain.

[0096] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 At least one of them is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0097] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 At least one of the domains is not antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0098] In some embodiments, L b1 comprises one or more linkers and / or a hinge region of an antibody or antigen-binding fragment thereof. In some embodiments, the hinge region is a native hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.

[0099] In some embodiments, L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers as disclosed herein.

[0100] In some embodiments, the polypeptide chain comprises an antigen binding domain ABD1 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0101] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD2 and an antigen binding domain that binds to a tumor cell as disclosed herein.

[0102] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD3 and an antigen binding domain that binds to a tumor cell as disclosed herein.

[0103] In some embodiments, the polypeptide chain comprises an antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0104] In some embodiments, the polypeptide chain comprises at least one antigen-binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0105] In some embodiments, the polypeptide chain comprises at least two antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0106] In some embodiments, the polypeptide chain comprises at least three antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0107] In some embodiments, the dimerization domain comprises an immunoglobulin dimerization domain. Other dimerization domains known to those of skill in the art are contemplated herein, including leucine zippers and the like.

[0108] In some embodiments, the dimerization domain comprises an IgG, IgM, IgA, IgD, or IgE dimerization domain (from human or animal IgG, IgM, IgA, IgD, or IgE).

[0109] In some embodiments, the dimerization domain comprises a CH3 domain of an antibody. The dimerization domain may also comprise a CH2 domain of an antibody.

[0110] In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.

[0111] In some exemplary embodiments, the dimerization domain comprises a mutated CH3 domain.

[0112] In some embodiments, the dimerization domain comprises a native CH2 and a native CH3 domain.

[0113] In some embodiments, the dimerization domain comprises a native CH2 and a variant CH3 domain.

[0114] In some embodiments, the dimerization domain comprises a variant CH2 and a variant CH3 domain.

[0115] In some embodiments, the dimerization domain comprises a variant CH2 and a native CH3 domain.

[0116] In some embodiments, the dimerization domain does not include a CH1 domain.

[0117] In some embodiments, the dimerization domain does not comprise a CH4 domain.

[0118] In some embodiments, the dimerization domain comprises an Fc region of an antibody or portion thereof, hi some embodiments, the dimerization domain comprises a constant region of an antibody heavy chain or portion thereof.

[0119] In embodiments, the antigen binding domain comprises or consists of the antigen binding domain of a single domain antibody (sdAb).

[0120] In embodiments, the antigen-binding domain comprises a heavy chain variable region (VH or VHH).

[0121] In some embodiments, the VHH is derived from a human, mouse, rat, etc.

[0122] In some embodiments, the VHH is from a transgenic mouse or rat capable of expressing a camelized mouse or rat VHH, a VHH from another species (such as a human), or a camelized VHH from another species (such as a camelized human VHH).

[0123] In some embodiments, the binding agent does not comprise a light chain variable region (VL or VLL).

[0124] In embodiments, the binding agent comprises a light chain variable region (VL or VLL).

[0125] In embodiments, the antigen-binding domain is a single-chain variable fragment (ScFv).

[0126] In embodiments, the antigen-binding domain is a V NAR It is from a fragment.

[0127] In other embodiments, the antigen-binding domain of the polypeptide chain is a single domain antibody (sdAb), a heavy chain variable region (VH or VHH), a light chain variable region (VL or VLL), a single chain variable fragment (ScFv), and / or a V NAR The fragments may include any combination of antigen-binding domains.

[0128] In some embodiments, the sdAb or VHH is from a camelid antibody.

[0129] In embodiments, the camelid antibody is from a dromedary, camel, llama, alpaca, etc.

[0130] In other embodiments, the sdAb or VHH is from a cartilaginous fish antibody.

[0131] In an embodiment, the cartilaginous fish antibody is a shark antibody.

[0132] In some embodiments, each antigen-binding domain specifically binds to a different epitope.

[0133] In other embodiments, each antigen-binding domain specifically binds to a different antigen.

[0134] In yet other embodiments, each antigen-binding domain specifically binds to a different protein.

[0135] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that modulates a function of CD47.

[0136] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to CD47 and enhances macrophage function by blocking the SIRPα / CD47 interaction.

[0137] In some embodiments, the antigen-binding domain that specifically binds to a tumor antigen is N-terminal to the dimerization domain, and the antigen-binding domain that specifically binds to an immunomodulator is C-terminal to the dimerization domain.

[0138] In some embodiments, the immunomodulator is an immune checkpoint protein, a cytokine, a chemokine, or an immune receptor or co-receptor.

[0139] It should be understood herein that a given antigen-binding domain may bind to epitopes present in different proteins. Thus, in some embodiments, an antigen-binding domain, or a binding agent comprising the same, may bind to more than one protein. In some embodiments, an antigen-binding domain or a binding agent may have affinity for more than one protein.

[0140] In some embodiments, the binding agents are bispecific, trispecific, or tetraspecific.

[0141] In some embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof.

[0142] In some embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

[0143] In some embodiments, one or more of the antigen-binding domains are humanized.

[0144] In some embodiments, one or more of the antigen-binding domains are partially humanized.

[0145] In some embodiments, the antigen-binding domain comprises one or more human frameworks.

[0146] In some embodiments, X or Y are independently selected from a linker, a cytokine, a chemokine, a tag, a masking domain, a phage coat protein (pIII, pVI, pV, pVII, or pIX), an antigen-binding domain, or a combination thereof.

[0147] In some embodiments, a binding agent of the present disclosure may have two polypeptide chains comprising an amino acid sequence identical to that of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027, or KC028.

[0148] In some embodiments, a binding agent of the disclosure may have a VHH moiety of any one of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027 or KC028, and a dimerization domain and / or linker different from those in Table 6 or Table 7.

[0149] In some embodiments, a binding agent of the present disclosure may have a VHH moiety of any one of KC011, KC012, KC013, KC014, KC015, KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027 or KC028, and a dimerization domain, e.g., a CH2-CH3 domain, that allows for the formation of a homodimer as described herein.

[0150] In some embodiments, a binding agent of the disclosure may have a VHH portion set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 and / or 266, and a dimerization domain, e.g., a CH2-CH3 domain, that allows for the formation of homodimers as described herein.

[0151] In some embodiments, the dimerization domain is as set forth in SEQ ID NO:168, or is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to SEQ ID NO:168.

[0152] In some embodiments, the binding agent is conjugated to a therapeutic moiety.

[0153] In some embodiments, the binding agent is conjugated to a detectable moiety.

[0154] In some embodiments, the binding agent is conjugated to a protein allowing for an extended half-life.

[0155] In some embodiments, the binding agent is attached to the nanoparticles.

[0156] Other aspects and embodiments of the present disclosure relate to compositions comprising at least one of the binding agents disclosed herein.

[0157] In some embodiments, the composition comprises monomers, dimers, and mixtures thereof.

[0158] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as dimers in the composition.

[0159] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as homodimers in the composition.

[0160] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as heterodimers in the composition.

[0161] Other aspects and embodiments of the present disclosure relate to pharmaceutical compositions comprising a binding agent disclosed herein and a pharmaceutically acceptable carrier.

[0162] Still other aspects and embodiments of the present disclosure relate to nucleic acids or sets of nucleic acids that encode the polypeptide chains and / or binding agents disclosed herein.

[0163] The nucleic acid may be in the form of a DNA segment as disclosed herein.

[0164] Additional aspects and embodiments of the present disclosure relate to a vector comprising a nucleic acid disclosed herein or a set of vectors each comprising a nucleic acid disclosed herein.

[0165] Further aspects and embodiments of the present disclosure relate to cells expressing the polypeptide chains or binding agents disclosed herein.

[0166] Additional aspects and embodiments of the present disclosure relate to cells comprising the nucleic acids or vectors disclosed herein.

[0167] Further aspects and embodiments of the present disclosure relate to kits that include the binding agents disclosed herein.

[0168] Still further aspects and embodiments of the present disclosure relate to kits comprising the nucleic acids disclosed herein.

[0169] Still further aspects and embodiments of the present disclosure relate to kits comprising the vectors disclosed herein.

[0170] Still further aspects and embodiments of the present disclosure relate to kits comprising the cells disclosed herein.

[0171] In additional aspects and embodiments, the present disclosure relates to methods of treating a disorder or disease comprising administering a binding agent disclosed herein.

[0172] In further aspects and embodiments, the present disclosure relates to methods of treating a disorder or disease comprising administering a composition disclosed herein.

[0173] In further aspects and embodiments, the present disclosure relates to methods of treating a disorder or disease comprising administering a pharmaceutical composition disclosed herein.

[0174] In some embodiments, the disorder or disease is cancer.

[0175] In some embodiments, the disorder or disease is a solid tumor. In some embodiments, the solid tumor is a neuroendocrine solid tumor.

[0176] In some embodiments, the disorder or disease is an advanced metastatic solid cancer.

[0177] In some embodiments, the disorder or disease is neuroblastoma.

[0178] In some embodiments, the disorder or disease is gastric cancer.

[0179] In some embodiments, the disorder or disease is a hematogenous cancer.

[0180] In some embodiments, the disorder or disease is lung cancer.

[0181] In some embodiments, the lung cancer is metastatic.

[0182] In some embodiments, the disorder or disease is small cell lung cancer.

[0183] In some embodiments, the disorder or disease is non-small cell lung cancer.

[0184] In some embodiments, the disorder or disease is myeloma.

[0185] In some embodiments, the disorder or disease is prostate cancer.

[0186] In some embodiments, the disorder or disease is ovarian cancer.

[0187] In some embodiments, the disorder or disease is breast cancer.

[0188] In some embodiments, the breast cancer is triple-negative breast cancer.

[0189] In some embodiments, the disorder or disease is rectal cancer.

[0190] In some embodiments, the disorder or disease is colorectal cancer.

[0191] In some embodiments, the disorder or disease is pancreatic cancer.

[0192] In some embodiments, the disorder or disease is glioblastoma.

[0193] In some embodiments, the disorder or disease is an infectious disease.

[0194] In some embodiments, the disorder or disease is an immune dysregulation.

[0195] In other aspects and embodiments, the present disclosure relates to methods of producing the binding agents disclosed herein, comprising transforming a cell with one or more vectors comprising a nucleic acid disclosed herein.

[0196] In some embodiments, the method may further comprise isolating and / or purifying the binding agent from impurities.

[0197] In other embodiments, the method may further comprise isolating and / or purifying the heterodimer from the monomer and / or homodimer.

[0198] In other embodiments, the method may further comprise isolating and / or purifying the homodimer from the monomer and / or heterodimer.

[0199] In some embodiments, the method further comprises conjugating the binding agent to a therapeutic moiety, a detectable moiety, or a protein that allows for extended half-life, or to a nanoparticle.

[0200] Further scope, applicability, and advantages of the present disclosure will become apparent from the non-limiting detailed description given below. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the present disclosure, is given by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0201] [Figure 1] Graph showing the binding curve of a D2-specific binder (VHH-hinge-CH2-CH3) for D2 proteoliposomes. Mock proteoliposomes were included as a negative control. [Figure 2]Table listing the EC50 of D2-specific binders (VHH-hinge-CH2-CH3) in GPCR BRET assays transfected with various G protein sensors. Dopamine was used as a positive control to demonstrate GPCR responses with G proteins including Gαz, Gαi2, GαoA, and Barr2. The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain set forth in SEQ ID NO: 114. [Figure 3] Heatmap showing changes in gene expression in NCI-H69 cells upon treatment with a binder (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO: 73) containing the D2-specific antigen-binding domain. The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain set forth in SEQ ID NO: 114. [Figure 4] Graph representing downregulated signaling pathways and protein classes from PANTHER analysis of the RNAseq results in Figure 3 . [Figures 5A-5C] Graphs showing downregulated genes verified by qRT-PCR in NCI-H69 (Figure 5A), NCI-H82 (Figure 5B), and HEK-D2 (Figure 5C) upon treatment with a binder containing a D2-specific antigen-binding domain (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO: 73). [Figures 6A-6B] Graphs showing the pharmacokinetics of a binder comprising a D2-specific antigen-binding domain (VHH-hinge-CH2-CH3) (KC013; SEQ ID NO: 73) and trastuzumab in SCID mice using a single dose injection (FIG. 6A: IV injection) (FIG. 6B: IP injection). [Figure 7A-7C]Graphs showing tumor kinetics and in vivo efficacy of a binder (VHH-hinge-CH2-CH3, solid line) (KC013; SEQ ID NO: 73) containing a D2-specific antigen-binding domain in NCG mice bearing small cell lung cancer (SCLC) xenograft models compared to an isotype control (dashed line) in the NCI-H727 tumor preventive model (FIG. 7A), the NCI-H69 tumor preventive model (FIG. 7B), or the NCI-H510A tumor preventive model (FIG. 7C). The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen-binding domain set forth in SEQ ID NO: 114. [Figure 8] 1 is a table listing the percentage of tumor growth inhibition in various tumor prevention SCLC tumor models in SCID mice upon treatment with a D2-specific binding agent (VHH-hinge-CH2-CH3) comprising the amino acid sequence set forth in SEQ ID NO: 71 (KC011), SEQ ID NO: 72 (KC012), SEQ ID NO: 73 (KC013), or SEQ ID NO: 74 (KC014). [Figure 9A] Graph showing binding curves of a PD-1-specific binder (KC036; SEQ ID NO: 94) for human, cynomolgus monkey, and rat recombinant PD1 protein or a control protein (BSA). The negative control is a binder containing a HEWL-specific antigen-binding domain (KC035; SEQ ID NO: 93). [Figure 9B] Top panel: Graph showing the binding curve of a PD-1-specific binding agent (KC036; SEQ ID NO: 94) to human recombinant PD1 protein compared to the anti-PD-1 antibody pembrolizumab (positive control). The negative control is a binding agent (KC035; SEQ ID NO: 93) comprising a HEWL-specific antigen-binding domain. Bottom panel: Graph showing the binding curve of PD-1-specific binding agents (KC036; SEQ ID NO: 94, KC058; and KC067) to human recombinant PD-1 protein compared to the anti-PD-1 antibody pembrolizumab (positive control). The negative control is a binding agent (KC035; SEQ ID NO: 93) comprising a HEWL-specific antigen-binding domain. [Figure 9C]Graph showing the binding curve of a CD47-specific binder (KC015; SEQ ID NO: 75) for human recombinant CD47 protein. The negative control for KC015 is a binder containing a HEWL-specific antigen-binding domain (KC016; SEQ ID NO: 76). The positive control is monoclonal antibody clone B6H12. The isotype control of clone B6H12 is used as a mouse IgG isotype control antibody. [Figure 9D] Graph showing the binding curve of CD47-specific binder KC015 for cynomolgus monkey CD47 protein. Negative control KC016 is a binder containing a HEWL-specific antigen-binding domain. The positive control is monoclonal antibody clone B6H12. The isotype control of clone B6H12 is used as a mouse IgG isotype control antibody. [Figure 9E] Graph depicting binding curves of the CD47-specific binding agent KC015 for various blood cell subtypes, including T cells, monocytes, granulocytes, red blood cells (RBCs), and platelets. [Figure 10] Graphs showing the cell-binding activity of a PD-1-specific binder (KC036; SEQ ID NO: 94) on CHO-PD1 cells (FIG. 10B) and parental CHO cells (FIG. 10A) obtained by flow cytometry. A commercially available PD-1 antibody was used as a positive control. The negative control was a binder containing a HEWL-specific antigen-binding domain (KC035; SEQ ID NO: 93). [Figure 11] Graph showing cytotoxicity data of PBMC-mediated THP-1 death induced by a PD-1-specific binding agent (KC036; SEQ ID NO: 94) in the presence of a CD33 BiTE or a control BiTE 48 hours after incubation, which was used to activate T cells via the CD3 subunit. The negative control is a binding agent containing a HEWL-specific antigen-binding domain (KC031; SEQ ID NO: 89). Positive controls include pembrolizumab and nivolumab. [Figure 12A]Graph showing the checkpoint inhibitory activity of a PD-1-specific binder (KC036; SEQ ID NO: 94) using a PD-1 / PD-L1 blockade bioassay system. The system is an engineered Jurkat cell line expressing a luciferase reporter under the NFAT promoter. The negative control is a binder containing a HEWL-specific antigen-binding domain (KC035; SEQ ID NO: 93). The anti-PD-1 antibody pembrolizumab is used as a positive control. [Figure 12B] Graph showing the checkpoint inhibitory activity of PD-1-specific binders (KC036; SEQ ID NO: 94, KC059, KC060, and KC067) using a PD-1 / PD-L1 blockade bioassay system. This system is an engineered Jurkat cell line expressing a luciferase reporter under the NFAT promoter. The negative control is a binder containing a HEWL-specific antigen-binding domain (KC035; SEQ ID NO: 93). The anti-PD-1 antibody pembrolizumab is used as a positive control. [Figure 13] Graph showing binding data for a trispecific binder (KC020; SEQ ID NO: 77) containing anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains, as well as a monospecific binder (KC015; SEQ ID NO: 75, VHH-hinge-CH2-CH3) containing the same anti-CD47 antigen-binding domain, to HEK293T cells and HEK293T-CD47 KO cells (CD47 knockout cells). Negative controls include the corresponding formats containing the anti-HEWL antigen-binding domain (KC016 (SEQ ID NO: 76) and KC039 (SEQ ID NO: 97)). A commercially available anti-human CD47 antibody (clone CC2C6) was used as a positive control. [Figure 14] Graph showing binding of a binder comprising anti-D2 and anti-CD47 antigen-binding domains (KC040; SEQ ID NO: 84) to human T cells compared to a control binder comprising an anti-HEWL antigen-binding domain (KC035; SEQ ID NO: 93) or a control binder comprising anti-CD3 and anti-HEWL antigen-binding domains (KC034; SEQ ID NO: 92). [Figures 15A-15B]Graphs showing T cell activation (FIG. 15A) and T cell-dependent cytotoxicity (FIG. 15B) induced by a binder containing anti-D2 and anti-CD47 antigen-binding domains (KC040; SEQ ID NO: 84) on human T cells at an E:T ratio of 5:1. Controls included medium alone (negative control), a binder containing an anti-HEWL antigen-binding domain and the same anti-D2 antigen-binding domain (negative control KC032; SEQ ID NO: 90), and OKT3 (positive control). [Figure 16] Graph depicting T cell activation upon treatment with a binder comprising D2 and CD47-specific antigen-binding domains (KC040: SEQ ID NO: 84) in the presence or absence of concanavalin A. Controls include a binder comprising a HEWL-specific antigen-binding domain (KC035: SEQ ID NO: 93) or a binder comprising anti-CD3 and anti-HEWL antigen-binding domains (positive control KC034; SEQ ID NO: 92), or OKT3 (positive control). [Figure 17] Graph showing checkpoint inhibitory activity of a CD47-specific binding agent (KC015; SEQ ID NO: 75) using the Promega CD47 / SIRPα Blockade Bioassay System, an engineered THP-1 cell line expressing a luciferase reporter that is induced by FcγR proteins engaging FcγR. The negative control is a binding agent (KC016; SEQ ID NO: 76) containing a HEWL-specific antigen-binding domain. The positive control is monoclonal antibody clone B6H12. The isotype control for clone B6H12 is a mouse IgG isotype control antibody. [Figure 18] Graph showing tumor volume upon treatment with a trispecific binding agent (KC021: SEQ ID NO: 78) comprising anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains in an established NCI-H82 xenograft model in NCG mice. The negative control includes a binding agent (KC031: SEQ ID NO: 89) comprising a HEWL-specific antigen-binding domain. [Figures 19A-19B]Plots showing T cell infiltration into NCI-H69 cell clusters upon treatment with a binder comprising anti-D2 and anti-PD-1 antigen-binding domains (KC037; SEQ ID NO: 95), a binder comprising anti-PD-1 and anti-CD47 antigen-binding domains (KC038; SEQ ID NO: 96), or trispecific binders comprising the same anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains (KC020; SEQ ID NO: 77 and KC021; SEQ ID NO: 78) (Figure 19A) or T cell activation with the same binders (Figure 19B). The control included a binder comprising a HEWL-specific antigen-binding domain (KC035; SEQ ID NO: 93). [Figures 20A-20B] Graph (Figure 20A) depicting tumor volume and table (Figure 20B) depicting tumor growth inhibition upon treatment with various anti-D2 and trispecific binding agents comprising the same anti-PD-1 and anti-CD47 antigen-binding domains (KC020; SEQ ID NO:77, KC022; SEQ ID NO:79, KC023; SEQ ID NO:80, KC024; SEQ ID NO:81, KC025; SEQ ID NO:82, KC026; SEQ ID NO:83). [Figure 21A] Graph showing tumor volume upon treatment with a CD47-specific binding agent (KC015; SEQ ID NO: 75) in a PBMC-pre-engrafted NCI-H82 xenograft model in NCG mice. Ten million PBMCs were implanted into NCG mice three days prior to tumor cell implantation. Treatment began 10 days after PBMC implantation, twice per week for a total of eight doses. The negative control used in the study was PBS. [Figure 21B] Table showing the in vivo efficacy of KC020 (SEQ ID NO: 77) using various tumor cells including NCI-H69, NCI-H82, SCLC-21H and RKO in different models. [Figure 21C] Graph showing tumor volume upon treatment with the anti-CD47 specific binder KC015 in an established MDA-MB-231 xenograft model in SCID mice. The negative control used in the study was PBS. Five million MDA-MB-231 cells were injected subcutaneously (sc) into the right flank of SCID mice. Mice were given weekly intraperitoneal (ip) treatment with the binder (8 mg / kg) for a total of eight doses. [Figure 22] Graph showing tumor volume following treatment with IgG1 or IgG4 isoform binders containing anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains (KC028; SEQ ID NO: 86; KC029; SEQ ID NO: 87; KC027: SEQ ID NO: 85). Controls include a binder containing an anti-HEWL antigen-binding domain (KC031: SEQ ID NO: 89). [Figures 23A-23B] Schematic (FIG. 23A) and photograph (FIG. 23B) of a biodistribution experiment showing the accumulation of a trispecific binder (KC021; SEQ ID NO: 78) comprising anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains in NCI-H82 (upper panel) and NCI-H69 (lower panel) xenograft models in NCG mice. [Figures 24A-24B] Graph showing liver metastases from hCD34+ humanized NCG mice bearing established NCI-H69 tumors treated with either PBS or KC020 (SEQ ID NO: 77) (Figure 24A) and H&E stained photographs of tumors indicating disseminated tumor cells from the PBS group but not from the KC020 (SEQ ID NO: 77) group as indicated by the black boxes (Figure 24B). [Figure 24C] Graph showing tumor kinetics and in vivo efficacy of KC020 in an NCI-H82 / PBMC co-transplant model at an E:T ratio of 1:5 in NCG mice. [Figures 25A-25C] Graphs depicting the binding of KC020 (SEQ ID NO: 77) subjected to stress conditions to D2 proteoliposomes (FIG. 25A), human recombinant PD-1 protein (FIG. 25B), and human recombinant CD47 protein (FIG. 25C) as measured by ELISA assay. Stress conditions included 3 days of agitation, 5 rounds of freezing and thawing, and 2 weeks of storage at 40°C. [Figures 26A-26C] Graphs depicting the binding of low pH stressed KC020 (SEQ ID NO: 77) to D2 proteoliposomes (FIG. 26A), human recombinant PD-1 protein (FIG. 26B), and human recombinant CD47 protein (FIG. 26C) as measured by ELISA assay. Stress conditions include 4 and 48 hour treatments at pH 3.5. [Figure 27] Graph showing the survival curve of KC020-treated animals implanted with the NCI-H82 SCLC tail vein metastasis model. NCG mice were inoculated with 50,000 NCI-H82 cells, followed by KC020 treatment the next day. Treatment with KC020 was administered biweekly. The negative control used in the study was PBS. [Figure 28A] Graph of mean tumor volume (mm3) days post-implantation in an MDA-MB-468 xenograft SCID mouse model treated weekly with 26 mg / kg KC020 compared to a PBS negative control. [Figure 28B] Graph of mean tumor volume (mm3) days post-implantation in an MDA-MB-231 xenograft SCID mouse model treated twice weekly with 1 mg / ml, 3 mg / ml or 10 mg / kg KC020 compared to a PBS negative control. [Figure 29A] Graph of mean tumor volume (mm3) days post-implantation in an MDA-MB-453 xenograft model in SCID mice treated weekly with 1 mg / ml, 10 mg / ml, or 28 mg / kg KC020 compared to a PBS negative control. [Figure 29B] Graph of mean tumor volume (mm3) days post-implantation in the T.Tn xenograft model in SCID mice treated weekly with 1 mg / ml, 10 mg / ml or 26 mg / kg KC020 compared to a PBS negative control. [Figure 30A] Alignment of selected anti-D2 VHHs in Table 3. [Figure 30B] Alignment of selected anti-PD-1 VHHs in Table 4 (Table 5). DETAILED DESCRIPTION OF THE INVENTION

[0202] Detailed Description definition Unless otherwise indicated, the amino acid numbering indicated for the dimerization domain is according to the EU numbering system.

[0203] The use of the terms "a," "an," and "the" and similar reference words in the context of describing embodiments (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0204] Unless otherwise stated or apparent from the context, as used herein, the term "or" is understood to be inclusive, covering both "or" and "and."

[0205] The term "and / or", when used herein, should be interpreted as an exclusive disclosure of each of the specified features or components with or without the other.

[0206] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise stated. The term "consisting of" should be construed as closed-ended.

[0207] The term "treatment," for purposes of this disclosure, refers to both therapeutic treatment and prophylactic or tumor preventative measures. Subjects in need of treatment include those already with the disorder, as well as those predisposed to having the disorder or those in whom the disorder is to be prevented. Subjects in need of treatment therefore include those already with cancer, as well as those predisposed to having cancer or those in whom cancer is to be addressed.

[0208] The term "about" or "approximately" in reference to a given value means that variation in the value is expected. In some embodiments, the term "about" or "approximately" generally refers to a range within + / - 10 percent, + / - 5 percent, + / - 4 percent, + / - 3 percent, + / - 2 percent, or + / - 1 percent of the given value or range.

[0209] As used herein, the term "DR2" or "D2" refers to a dopamine receptor D2 protein or a polypeptide comprising a DR2 amino acid sequence. The term "DR2" encompasses human DR2 protein and DR2 antigens.

[0210] As used herein, the term "PD-1" refers to the programmed cell death protein 1 protein or a polypeptide containing the PD-1 amino acid sequence. The term "PD-1" encompasses the human PD-1 protein and the PD-1 antigen.

[0211] As used herein, the term "CD47" refers to the cluster of differentiation 47 protein or a polypeptide comprising the CD47 amino acid sequence. The term "CD47" encompasses human CD47 protein and CD47 antigen.

[0212] The term "binding agent" refers to a molecule that comprises a polypeptide portion (such as a polypeptide chain) and that has the ability to specifically bind to an antigen or that comprises a domain that has the ability to specifically bind to an antigen, e.g., an antigen-binding domain.

[0213] The term "antibody" is used in the broadest sense and encompasses a variety of antibody formats and structures, including any immunoglobulin, monoclonal antibody, polyclonal antibody, bivalent antibody, monovalent antibody, diabody, multispecific (multi-specific) antibody, conventional antibody, single domain antibody, single chain antibody, heavy chain-only antibody, nanobody, full length antibody, humanized antibody, chimeric antibody that binds to a specific antigen, and any antigen-binding fragment that exhibits the desired antigen-binding activity. Antibodies may be naturally occurring (naturally occurring) or the result of recombinant technology.

[0214] As used herein, the expressions "single domain antibody" and "heavy chain-only antibody" are used interchangeably. The term "single domain antibody" includes naturally occurring single domain antibodies or heavy chain-only antibodies, as well as single domain antibodies in which the naturally occurring constant region has been replaced by another dimerization domain, such as the constant region of a human immunoglobulin (e.g., IgG), and variants such as humanized or chimeric single domain antibodies.

[0215] "Naturally occurring single domain antibodies" include antibodies produced by camelids (camelid antibodies) or sharks. "Naturally occurring single domain antibodies" also encompass antibodies produced by transgenic animals engineered to express heavy chain-only antibodies. Exemplary embodiments of transgenic animals are provided in International Application PCT / CA2021 / 050951, filed July 21, 2021, and published January 20, 2022 under WO2022 / 011457, the entire contents of which are incorporated herein by reference.

[0216] As used herein, the term "subject in need" refers to a subject having or suspected of having a disorder or disease associated with DR2, DR2 expression (e.g., expression in tissues, cells, or serum), or DR2 overexpression or upregulation. The term "subject in need" also refers to a subject having or suspected of having a disorder or disease that would benefit from treatment with a binding agent that targets DR2. A "subject in need" includes a subject having or suspected of having cancer.

[0217] As used herein, the term "humanized" means that a binding agent, such as an antibody or antigen-binding fragment, contains CDRs or CDR amino acid residues derived from a non-human animal antibody, FR regions derived from a human antibody, and, where applicable, constant regions derived from a human antibody.

[0218] In some cases, a humanized binding agent may comprise a CDR or CDR amino acid residues of a human antibody variable region (e.g., a variable region sequence of a human germline antibody).

[0219] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between a binding agent or its antigen-binding domain and an antigen. "Affinity" can be, for example, K D value, i.e., the ratio of the dissociation rate to the association rate when binding between an antigen and a binding agent (e.g., an antibody such as a single domain antibody) reaches equilibrium (k off / k on Affinity may be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). Antibodies are generally soluble in at least 10 -6 M or less (e.g., 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or 10 -9 M or less, and 10 -6 K (any value less than or equal to M) D Preferably, the antibody has a denaturing activity in the nanomolar range or below (e.g., 9 x 10 -9 M or less, 8×10 -9 M or less, 7×10 -9 M or less, 6×10 -9 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or less, 1×10 -9 M or less, e.g., 1 x 10 -10 M or less, 1×10 -11 M or less, 1×10 -12M or less) K D Even more preferably, the antibody has a titer in the picomolar range or below (e.g., 9 x 10 -12 M or less, 8×10 -12 M or less, 7×10 -12 M or less, 6×10 -12 M or less, 5×10 -12 M or less, 4×10 -12 M or less, 3×10 -12 M or less, 2×10 -12 M or less, 1×10 -12 M or less) D It has a value.

[0220] As used herein, the terms "specific binding" or "specifically binds" refer to a non-random binding reaction between two molecules, for example, between an antibody and an antigen. Specific binding can be characterized by binding affinity. -6 M or less (e.g., 5 × 10 -7 M or less, 2×10 -7 M or less, 10 -7 M or less, 5×10 -8 M or less, 2×10 -8 M or less, 10 -8 M or less, 5×10 -9 M or less, 4×10 -9 M or less, 3×10 -9 M or less, 2×10 -9 M or 10 -9 M or less, and 10 -6 K (any value less than or equal to M) D The value may indicate specific binding between a binding agent (e.g., an antibody such as a single domain antibody) and DR2 (e.g., human DR2).

[0221] As used herein, the terms "competing for binding," "competing for binding," "competing with," or "competing with" refer to the ability of a first antibody or antigen-binding fragment thereof (e.g., a single domain antibody or antigen-binding fragment thereof) to inhibit, to any detectable extent, the binding interaction between DR2 and a second anti-DR2 antibody, between PD-1 and a second anti-PD-1 antibody, or between CD47 and a second anti-CD47 antibody (e.g., a single domain antibody or antigen-binding fragment thereof). Means for reducing tumor size, inhibiting tumor growth, and / or inducing tumor regression may be achieved using competitive binding agents.

[0222] As used herein, the term "epitope" refers to a specific group of atoms or amino acid residues on an antigen to which an antibody (e.g., a single domain antibody) binds. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding to the antigen. Epitopes may be linear or conformational (i.e., comprising spaced amino acid residues). For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody (e.g., a single domain antibody) to an antigen by at least 85%, or at least 90%, or at least 95%, then the antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as the reference antibody. For example, a monoclonal, chimeric, human, or humanized antibody, or antigen-binding fragment thereof, may compete with a single domain antibody of the present disclosure for binding to human DR2.

[0223] As used herein, the term "sequence identity" of the present invention refers to the degree of identity between two nucleic acid sequences or two amino acid sequences when compared at their best and when mutations such as substitutions, insertions, or deletions are considered appropriate. The sequence identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0224] The term "antigen-binding fragment" as used in the expressions "antibody or antigen-binding fragment thereof" or "single-domain antibody or antigen-binding fragment thereof" refers to a fragment of an antibody or single-chain antibody that includes the antigen-binding domain and may or may not incorporate other portions of an antibody or single-domain antibody, such as amino acid residues of the hinge region, amino acid residues of the constant region, or portions of the Fc region. Regardless of structure, an antigen-binding fragment binds to the same antigen that is recognized by the intact antibody (e.g., a single-domain antibody).

[0225] An antigen-binding fragment of a single domain antibody includes, for example, the CDRs and sequences encompassing the CDRs, such as an entire variable region or portion thereof, or an entire heavy chain or portion thereof, and may or may not include other portions of the antibody.

[0226] The term "antigen-binding domain" relates to the part of an antibody involved in antigen binding, comprising, for example, one or more CDRs, one or more framework regions (FRs), or the entire variable region. The term "antigen-binding domain" in the context of a single domain antibody relates to the part of a single domain antibody involved in antigen binding, comprising, for example, one or more of CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more framework regions FR1, FR2, FR3, FR4, or the entire variable region (VH or VHH). The term "antigen-binding domain" in the context of a natural antibody relates to the part of a natural antibody involved in antigen binding, comprising, for example, one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light or heavy chain framework regions FR1, FR2, FR3, FR4, or the entire variable region or regions (heavy chain variable region (VH) and / or light chain variable region (VL)). In some embodiments, framework regions are not generally considered to be part of the antigen-binding domain, although some amino acid residues in one or more framework regions may contribute to antigen binding.

[0227] The term "functionally active" in reference to an antigen-binding domain means that the antigen-binding domain has the ability to bind to its target and optionally that the antigen-binding domain has one or more biological activities.

[0228] As used herein, the term "flexible linker" refers to a peptide that includes at least a portion made up of flexible amino acid residues that allows adjacent modules to move relative to one another.

[0229] As used herein, the term "rigid linker" refers to a peptide that includes at least a portion made up of amino acids that presents a rigid structure and maintains the distance between two modules.

[0230] As used herein, the term "helical linker" means a linker composed of amino acid residues that adopt an α-helical conformation.

[0231] As used herein, the term "cleavable linker" refers to a peptide that contains an enzymatic cleavage site that is sensitive to a protease selected from ADAMS, ADAMTS, aspartic proteases, caspases, cysteine ​​cathepsins, cysteine ​​proteinases, metalloproteinases, serine proteases, coagulation factor proteases, type II transmembrane serine proteases (TTSPs), and combinations thereof.

[0232] As used herein, the term "monospecific" with respect to a polypeptide chain or binding agent refers to a polypeptide chain or binding agent that binds to a single antigen or epitope. A monospecific polypeptide chain or binding agent may therefore have one antigen-binding domain or more than one binding domain (the same or different) that have the same specificity for a given antigen or epitope.

[0233] As used herein, the term "multispecific" with respect to a polypeptide chain or binding agent refers to a polypeptide chain or binding agent that binds to more than one antigen or epitope.

[0234] The term "multispecific" encompasses "bispecific," "trispecific," "tetraspecific," "pentaspecific," and "hexaspecific," among others.

[0235] As used herein, in the context of binding agents, the term "bispecific" refers to a binding agent that binds to two different antigens or proteins or two epitopes of the same antigen or protein.

[0236] As used herein, a phrase such as "at least two polypeptide chains" in a phrase such as "the binding agent comprises at least two polypeptide chains" refers to a certain number of polypeptide chain species, and does not refer to an absolute numerical value.

[0237] It should be understood herein that expressions referring to ranges of values ​​in the form "A to B," etc., include each individual value and any subranges within and comprising such range. For example, the expression "1 to 10" includes, without limitation, subranges such as "2 to 10," "2 to 9," "3 to 6," "5 to 7," and any individual value within 1 to 10, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0238] It should be understood herein that the term "at least" in reference to a given value is intended to include that value and greater values. For example, the term "at least 80%" includes "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%, "at least 99.1%, "at least 99.2%, "at least 99.3%, "at least 99.4%, "at least 99.5%, "at least 99.6%, "at least 99.7%, "at least 99.8%, "at least 99.9%" and 100%.

[0239] The term "codon-optimized" refers to a sequence in which a codon has been changed to another codon that encodes the same amino acid but is preferred or functions better in a given organism (increases expression, minimizes secondary structure in RNA, etc.). "Codon-optimized" sequences can be obtained using publicly available software or through service providers, including GenScript, Inc. (OptimumGene™, U.S. Pat. No. 8,326,547).

[0240] The term "single-chain format" in reference to antigen-binding domains means that the domains necessary for antigen binding are present on a single polypeptide chain.

[0241] As used herein, the term "regression" refers to the partial or complete disappearance of a tumor or tumor lesion, and such disappearance may be temporary or permanent.

[0242] As used herein, the term "tumor" includes "primary tumors" and "secondary tumors."

[0243] As used herein, the term "tumor lesion" refers to a lesion that originates from or is caused by a tumor.

[0244] Any of the terms "KC011", "KC012", "KC013", "KC014", "KC015", "KC016", "KC017", "KC018", "KC019", "KC020", "KC021", "KC022", "KC023", "KC024", "KC025", "KC026", "KC027", "KC028", "KC029", "KC030", "KC031", "KC032", "KC033", "KC034", "KC035", "KC036", "KC037", "KC038", "KC039", "KC040", "KC058", "KC059", "KC060", or "KC067" refers to any of the compounds listed in Table 6 or Table 7. It should be understood herein that the term "binding agent" refers to a binding agent comprising two chains, each having the corresponding amino acid sequence as shown in Table 7 (Table 8) or as described herein.

[0245] Dopamine receptor D2 antigen The anti-DR2 antibody or antigen-binding fragment thereof of the present disclosure may be obtained, for example, by immunizing an animal with a DR2 antigen. More specifically, the anti-DR2 single domain antibody or antigen-binding fragment thereof may be obtained by immunizing an animal with D2 proteoliposomes (i.e., proteoliposomes containing a D2 antigen).

[0246] Alternatively, anti-DR2 antibodies or antigen-binding fragments thereof may be obtained from a library of antibodies.

[0247] In one embodiment, the DR2 antigen is human DR2 protein (eg, Uniprot Accession No. P14416-1, the entire contents of which are incorporated herein by reference) or a fragment thereof.

[0248] Binding agents that bind to DR2 may be identified by in silico, in vitro, and / or in vivo methods. Some in vitro methods rely on binding of candidate antibodies or antigen-binding fragments thereof to antigen. Thus, anti-DR2 antibodies or antigen-binding fragments thereof may be identified by methods involving binding to DR2 antigen or D2 proteoliposomes disclosed herein and / or using the assays disclosed herein.

[0249] In one embodiment, the DR2 antigen is a human DR2 protein or a fragment thereof. In some embodiments, the DR2 antigen is a DR2 homolog having at least 80% sequence identity to the human DR2 or a fragment thereof. In some embodiments, the DR2 antigen is a DR2 variant having at least 80% sequence identity to the human DR2 or a fragment thereof.

[0250] In an exemplary embodiment, the DR2 homologue is from a primate.

[0251] In an exemplary embodiment, the DR2 homolog is cynomolgus monkey DR2.

[0252] In another exemplary embodiment, the DR2 homolog is rhesus monkey DR2.

[0253] According to the present disclosure, the DR2 antigen may comprise at least 10 amino acid residues of human DR2.

[0254] According to the present disclosure, the DR2 antigen may be a human DR2 antigen having 10 to 400 amino acid residues, 10 to 390 amino acid residues, 10 to 380 amino acid residues, 10 to 370 amino acid residues, 10 to 360 amino acid residues, 10 to 350 amino acid residues, 10 to 340 amino acid residues, 10 to 330 amino acid residues, 10 to 320 amino acid residues, 10 to 310 amino acid residues, 10 to 300 amino acid residues, 10 to 290 amino acid residues, 10 to 280 amino acid residues, or the like. amino acid residues, 10 to 270 amino acid residues, 10 to 260 amino acid residues, 10 to 250 amino acid residues, 10 to 240 amino acid residues, 10 to 230 amino acid residues, 10 to 220 amino acid residues, 10 to 210 amino acid residues, 10 to 200 amino acid residues, 10 to 190 amino acid residues, 10 to 180 amino acid residues, 10 to 170 amino acid residues, 10 to 160 amino acid residues, 10 to 150 amino acid residues, 10 to 140 amino acid residues, 10 to It contains 130 amino acid residues, 10 to 120 amino acid residues, or 10 to 110 amino acid residues, for example, 10 to 100 amino acid residues, 10 to 90 amino acid residues, 10 to 80 amino acid residues, 10 to 70 amino acid residues, 10 to 60 amino acid residues, 10 to 50 amino acid residues, 10 to 40 amino acid residues, 10 to 30 amino acid residues, 10 to 29 amino acid residues, 10 to 28 amino acid residues, 10 to 27 amino acid residues, or 10 to 26 amino acid residues. residues, 10 to 25 amino acid residues, 10 to 24 amino acid residues, 10 to 23 amino acid residues, 10 to 22 amino acid residues, 10 to 21 amino acid residues, 10 to 20 amino acid residues, 10 to 19 amino acid residues, 10 to 18 amino acid residues, 10 to 17 amino acid residues, 10 to 16 amino acid residues, 10 to 15 amino acid residues, 10 to 14 amino acid residues, 10 to 13 amino acid residues, 10 to 12 amino acid residues, and 10 to 11 amino acid residues.

[0255] An exemplary, non-limiting embodiment of a DR2 antigen is provided in SEQ ID NO:165.

[0256] Programmed cell death protein 1 antigen The anti-PD-1 antibodies or antigen-binding fragments of the disclosure may be obtained, for example, by immunizing an animal with a PD-1 antigen.

[0257] Alternatively, the antibodies or antigen-binding fragments of the present disclosure may be obtained from a library of anti-PD-1 antibodies, such as an anti-PD-1 single domain antibody library.

[0258] In one embodiment, the PD-1 antigen is a human PD-1 protein (e.g., Uniprot Accession No. Q15116, the entire contents of which are incorporated herein by reference) or a fragment thereof. In some embodiments, the PD-1 antigen is a PD-1 homolog having at least 80% sequence identity to the human PD-1 protein or a fragment thereof. In some embodiments, the PD-1 antigen is a PD-1 variant having at least 80% sequence identity to the human PD-1 protein or a fragment thereof. An exemplary embodiment of a PD-1 antigen is provided in SEQ ID NO: 166.

[0259] An exemplary embodiment of a PD-1 antigen includes the extracellular domain of PD-1 or a portion thereof.

[0260] According to the present disclosure, the PD-1 antigen comprises 10 to 250 amino acid residues, 10 to 200 amino acid residues, or 10 to 165 amino acid residues of the human PD-1 amino acid sequence, including, for example, 10 to 160 amino acid residues, 10 to 150 amino acid residues, 10 to 140 amino acid residues, 10 to 130 amino acid residues, 10 to 120 amino acid residues, 10 to 110 amino acid residues, 10 to 100 amino acid residues, 10 to 90 amino acid residues, 10 to 80 amino acid residues, 10 to 70 amino acid residues, 10 to 60 amino acid residues, 10 to 50 amino acid residues, 10 to 40 amino acid residues, 10 to 30 amino acid residues, , 10 to 29 amino acid residues, 10 to 28 amino acid residues, 10 to 27 amino acid residues, 10 to 26 amino acid residues, 10 to 25 amino acid residues, 10 to 24 amino acid residues, 10 to 23 amino acid residues, 10 to 22 amino acid residues, 10 to 21 amino acid residues, 10 to 20 amino acid residues, 10 to 19 amino acid residues, 10 to 18 amino acid residues, 10 to 17 amino acid residues, 10 to 16 amino acid residues, 10 to 15 amino acid residues, 10 to 14 amino acid residues, 10 to 13 amino acid residues, 10 to 12 amino acid residues, and 10 to 11 amino acid residues.

[0261] Differentiation antigen group 47 antigens The anti-CD47 antibodies or antigen-binding fragments of the disclosure may be obtained, for example, by immunizing an animal with a CD47 antigen.

[0262] Alternatively, the anti-CD47 antibodies or antigen-binding fragments thereof of the present disclosure may be obtained from a library of anti-CD47 antibodies (e.g., an anti-CD47 single domain antibody library).

[0263] In one embodiment, the CD47 antigen is a human CD47 protein (e.g., Uniprot Accession No. Q08722-1, which represents the canonical sequence, the entire contents of which are incorporated herein by reference) or a fragment thereof. In some embodiments, the CD47 antigen is a CD47 homolog having at least 80% sequence identity to human CD47 or a fragment thereof. In some embodiments, the CD47 antigen is a CD47 variant having at least 80% sequence identity to human CD47 or a fragment thereof.

[0264] According to the present disclosure, the CD47 antigen may be any of the following amino acid residues of the human CD47 amino acid sequence: 10 to 140 amino acid residues, 10 to 130 amino acid residues, 10 to 120 amino acid residues, 10 to 110 amino acid residues, 10 to 100 amino acid residues, 10 to 90 amino acid residues, 10 to 80 amino acid residues, 10 to 70 amino acid residues, 10 to 60 amino acid residues, 10 to 50 amino acid residues, 10 to 40 amino acid residues, 10 to 30 amino acid residues, 10 to 29 amino acid residues, 10 to 28 amino acid residues, 10 to 27 amino acid residues, 10 to 30 ... amino acid residues, 10 to 26 amino acid residues, 10 to 25 amino acid residues, 10 to 24 amino acid residues, 10 to 23 amino acid residues, 10 to 22 amino acid residues, 10 to 21 amino acid residues, 10 to 20 amino acid residues, 10 to 19 amino acid residues, 10 to 18 amino acid residues, 10 to 17 amino acid residues, 10 to 16 amino acid residues, 10 to 15 amino acid residues, 10 to 14 amino acid residues, 10 to 13 amino acid residues, 10 to 12 amino acid residues, or 10 to 11 amino acid residues.

[0265] An exemplary embodiment of the CD47 antigen comprises the extracellular domain of CD47 or a portion thereof. An exemplary embodiment of the CD47 antigen comprises a soluble form of the CD47 extracellular domain. An exemplary embodiment of the CD47 antigen is provided in SEQ ID NO: 167.

[0266] Antigen-binding domain (Ab) As disclosed herein, a binding agent may comprise one or more antigen-binding domains. The antigen-binding domains of the present disclosure may be selected for their ability to bind to a specific target. The antigen-binding domains may also be selected for their functional properties or biological effects in vivo and / or in vitro, including, for example, their ability to modulate cellular processes, such as gene expression, signal transduction, cell proliferation, and cell survival.

[0267] For example, a binding agent of the disclosure comprises one or more antigen-binding domains, each independently comprising one or more complementarity-determining regions (CDRs) of an antibody.

[0268] The specificity of the binding agents of the present disclosure may therefore be conferred by their antigen-binding domain.

[0269] A binding agent of the disclosure may comprise an antigen-binding domain capable of binding to DR2 or a cell expressing DR2, an antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1, and / or an antigen-binding domain capable of binding to CD47 or a cell expressing CD47.

[0270] In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to DR2 or a cell expressing DR2, including, for example, antigen binding domain 1 (ABD1).

[0271] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1, for example, comprising antigen-binding domain 2 (ABD2).

[0272] In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to CD47 or a cell expressing CD47, for example, comprising antigen binding domain 3 (ABD3).

[0273] In some embodiments, the antigen-binding domain targets the same epitope or antigen as ABD1, ABD2 and / or ABD3. In some embodiments, the antigen-binding domain competes with ABD1, ABD2 and / or ABD3 for binding to their respective epitope or antigen.

[0274] In some embodiments, the antigen-binding domain binds to at least one antigen that is not selected from among DR2, PD-1, or CD47.

[0275] In some embodiments, the binding agent may comprise more than one antigen-binding domain.

[0276] For example, in some embodiments, the binding agent may comprise two or more antigen-binding domains.

[0277] In some embodiments, the binding agent may comprise three or more antigen-binding domains.

[0278] In some embodiments, the binding agent may comprise four or more antigen-binding domains.

[0279] In some embodiments, the binding agent may comprise five or more antigen-binding domains.

[0280] In some embodiments, the binding agent may comprise six or more antigen-binding domains.

[0281] In some embodiments, the binder may be 1 to 12, e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 ... to 9, 4 to 10, 4 to 11, 4 to 12, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 9 to 10, 9 to 11, 9 to 12, 10 to 11, 10 to 12, or 11 to 12 antigen-binding domains.

[0282] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is capable of binding to a tumor cell.

[0283] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is capable of binding to an immune modulator. In some embodiments, at least one of the antigen-binding domains is capable of binding to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is PD-1.

[0284] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is capable of binding to an immune cell, hi some embodiments, at least one of the antigen-binding domains is capable of binding to a protein expressed on the surface of an immune cell (e.g., T cells, NK cells, monocytes, macrophages, etc.).

[0285] In some embodiments, the binding agents of the present disclosure may be multivalent and may comprise at least one antigen-binding domain that binds to a tumor cell and at least one antigen-binding domain that binds to an immune cell.

[0286] In some embodiments, the protein expressed on the surface of an immune cell is selected from CD47 or CD3.

[0287] In some embodiments, the protein expressed on the surface of an immune cell is CD47.

[0288] In some embodiments, the protein expressed on the surface of an immune cell is CD3.

[0289] The antigen-binding domain may be derived from a natural antibody (of human or animal origin) or a synthetic antibody.

[0290] In some embodiments, the antigen-binding domain of a native antibody is engineered to form a single chain.

[0291] In some embodiments, the antigen-binding domain may be derived from an IgG, such as IgG1, IgG2, IgG3, or IgG4. In particular embodiments, the antigen-binding domain is derived from a human IgG heavy chain.

[0292] In some embodiments, the antigen-binding domain may be derived from a heavy chain-only antibody (HCAb).

[0293] Exemplary embodiments of antigen-binding domains include, but are not limited to, single domain antibodies (sdAbs), heavy chain variable regions (VH or VHH), light chain variable regions (VL or VLL), single chain variable fragments (scFv), V NAR fragments, and combinations thereof.

[0294] In some embodiments, the antigen-binding domain of a binding agent disclosed herein is a VHH.

[0295] In some embodiments, the antigen-binding domain of a binding agent disclosed herein is a VHH comprising the complementarity determining regions of a VHH disclosed herein.

[0296] According to the present disclosure, CDRs may be identified using the Kabat numbering scheme (e.g., Kabat, J Immunol., 147:1709-19 (1991); Chothia C, Lesk AM, J Mol Biol. Aug 20;196(4):901-17 (1987)).

[0297] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to Kabat CDRs.

[0298] In some cases, CDR1, CDR2, CDR3 and / or FR2 correspond to Kabat CDR1, CDR2, CDR3 and / or FR2.

[0299] Alternatively, CDRs may be identified using the IMGT numbering scheme (see, for example, Lefranc, M.-P., The Immunologist, 7, pp. 132-136 (1999)).

[0300] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to the IMGT CDRs.

[0301] In other cases, CDR1, CDR2, CDR3 and / or FR2 correspond to IMGT CDR1, CDR2, CDR3 and / or FR2.

[0302] In some embodiments, the binding agent comprises two polypeptide chains, each comprising, in N-terminal to C-terminal fashion, a) an antigen-binding domain comprising an antigen-binding fragment of a single-domain antibody, b) a linker, and c) a dimerization domain.

[0303] In some embodiments, the binding agent comprises two polypeptide chains, each comprising, in N-terminal to C-terminal fashion, a) an antigen-binding domain of a single domain antibody, b) a linker, and c) a dimerization domain.

[0304] In some embodiments, the binding agent comprises two polypeptide chains, each comprising, in N-terminal to C-terminal fashion, a) a dimerization domain, b) a linker, and c) an antigen-binding domain comprising an antigen-binding fragment of a single-domain antibody.

[0305] In some embodiments, the binding agent comprises two polypeptide chains, each comprising, in N-terminal to C-terminal fashion, a) a dimerization domain, b) a linker, and c) an antigen-binding domain of a single domain antibody.

[0306] In other embodiments, the binding agent comprises two polypeptide chains, each comprising, from N-terminal to C-terminal, a) an antigen-binding domain of a single domain antibody, b) a linker, c) a dimerization domain, d) a linker, and e) an antigen-binding domain of a single domain antibody. In some embodiments, the antigen-binding domain in a) and the antigen-binding domain in e) are different. In some embodiments, the antigen-binding domain in a) and the antigen-binding domain in e) are the same. In some embodiments, the linker in b) and the linker in d) are the same. In some embodiments, the linker in b) and the linker in d) are different.

[0307] According to the present disclosure, a binder may comprise an additional amino acid sequence or moiety (small molecule, label, etc.) at the N-terminus and / or C-terminus. For example, according to the present disclosure, a binder may comprise an additional antigen-binding domain of a single domain antibody at the N-terminus or C-terminus. The additional antigen-binding domain may be separated from the core by one or more linkers.

[0308] In certain embodiments, a binding agent of the present disclosure may comprise an antigen-binding domain VHH derived from IgG1, IgG2a, IgG2b, IgG2c, or IgG3, or a combination thereof, of human, mouse, or rat, or a transgenic mouse or rat in which the mouse or rat VHH has been camelized, a human VHH, a camelized human VHH. The antibody may be obtained by immunizing a mouse or rat or transgenic mouse or rat lacking a functional CH1 domain in its heavy chain, IgG1, IgG2a, IgG2b, IgG2c, or IgG3, or a combination thereof, or any of the above VHH combinations, with an antigen of interest.

[0309] In certain embodiments, the polypeptide chains of the disclosure may comprise an antigen-binding domain of a camelid antibody, such as an IgG2 or IgG3 VHH. Camelid antibodies may be obtained by immunizing a dromedary, camel, llama, or alpaca with an antigen of interest.

[0310] In some embodiments, camelid antibodies may originate from so-called Old World camelids, such as the Bactrian camel (Camelus bactrianus), the dromedary (Camelus dromedarius), or New World camelids, such as the alpaca (Lama pacos), the llama (Lama glama), and the vicuna (Lama vicugna).

[0311] In another specific embodiment, the polypeptide chains of the present disclosure comprise an antigen-binding domain of a cartilaginous fish, such as the V of an IgNAR. NAR May contain fragments. V NAR The fragment may originate from a shark antibody.

[0312] If desired, the antigen-binding domain of a non-human antibody may be humanized. For example, the framework regions of a non-human VH, VHH, or HCAb may be modified to make them more human-like. Humanization of camelid antibodies is discussed, for example, in Vincke C. et al. (J. Biol Chem. 2009, 284(5):3273-3284), the entire contents of which are incorporated herein by reference. Humanized camelid antibodies can be prepared, for example, using universal humanized nanobody scaffolds (e.g., h-NbBcII10 disclosed in Vincke C. et al.). FGLA ) may be obtained by CDR grafting onto V NAR Antibodies may be humanized by converting non-CDR residues to those of the human germline Vκ1 sequence DPK9 as discussed in Kovalenko OV et al. (J Biol Chem. 2013, 288:17408-17419), the entire contents of which are incorporated herein by reference. The polypeptide chains of the present disclosure thus include humanized antigen-binding domains.

[0313] In yet another specific embodiment, the antigen-binding domain may comprise a human VH (modified or not). The human VH may, for example, be obtained from a synthetic human VH library. Modified human VHs include those in which some amino acid residues have been modified to make them more camel-like (i.e., by camelization).

[0314] The antigen-binding domain may be a single domain antibody (sdAb), a VHH, a conventional antibody or an antigen-binding fragment thereof, a bispecific antibody, a single-chain Fv-CH3 (scFv-CH3) fusion, a tandem-scFv-CH3 (TaFv-CH3) fusion, a diabody-CH3 (Db-CH3) fusion, a tandem Db-CH3 (TaDb-CH3) fusion, a single-chain Db-CH3 fusion (scDb-CH3), a Fab-CH3 fusion, a single-chain Fab-CH3 fusion, a Fab-scFv-CH3 fusion, a dual affinity retargeting (DART)-CH3 fusion, a Fab- Those skilled in the art will understand that the antibody formats may be incorporated into antibodies, antigen-binding fragments, or antibody-like molecules, including, without limitation, DART-CH3 fusions, single-chain Fv-Fc (scFv-Fc) fusions, tandem-scFv-Fc (TaFv-Fc) fusions, diabody-Fc (Db-Fc) fusions, tandem Db-Fc (TaDb-Fc) fusions, single-chain Db-Fc fusions (scDb-Fc), Fab-Fc fusions, single-chain Fab-Fc fusions, Fab-scFv-Fc fusions, dual affinity retargeting (DART)-Fc fusions, Fab-DART-Fc fusions, etc. These antibody formats may have a native CH3, a variant CH3 domain, a native CH2-CH3 domain, or a variant CH2-CH3 domain as disclosed herein.

[0315] Exemplary Embodiments of ABD1 Antigen-binding domain 1 (ABD1) may, for example, be selected from antigen-binding domains that bind to DR2, some non-limiting exemplary embodiments of which are provided herein.

[0316] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), comprising heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:1, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:2, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:3.

[0317] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6.

[0318] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10.

[0319] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13.

[0320] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0321] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20.

[0322] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0323] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:25, CDRH2 having the amino acid sequence set forth in SEQ ID NO:26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:27.

[0324] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31.

[0325] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34.

[0326] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38.

[0327] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 39, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 41.

[0328] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0329] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48.

[0330] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52.

[0331] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55.

[0332] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 280, and wherein X 1a ~X 1k any one of which is any (e.g., naturally occurring) amino acid residue, or

[0333] X 1a is H or Q, and X 1b is L or R, and X 1c is F or L, and X 1d is A or T, and X 1e is V or I, and X 1f is R or K, and X1g is D or E, and X 1h is R or K, and X 1j is V or L and / or X 1k is A or V.

[0334] [ka]

[0335] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:7.

[0336] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and CDRs identical to the Kabat CDRs of SEQ ID NO:7.

[0337] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and CDRs identical to the IMGT CDRs of SEQ ID NO:7.

[0338] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:7.

[0339] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 14.

[0340] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14 and CDRs identical to the Kabat CDRs of SEQ ID NO:14.

[0341] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:14.

[0342] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0343] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 21.

[0344] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21 and CDRs identical to the Kabat CDRs of SEQ ID NO:21.

[0345] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21 and a CDR that is identical to the IMGT CDR of SEQ ID NO:21.

[0346] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:21.

[0347] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 28.

[0348] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28 and CDRs identical to the Kabat CDRs of SEQ ID NO:28.

[0349] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28 and a CDR that is identical to the IMGT CDR of SEQ ID NO:28.

[0350] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:28.

[0351] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 35.

[0352] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35 and CDRs identical to the Kabat CDRs of SEQ ID NO:35.

[0353] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:35.

[0354] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:35.

[0355] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 42.

[0356] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42 and CDRs identical to the Kabat CDRs of SEQ ID NO:42.

[0357] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42 and a CDR that is identical to the IMGT CDR of SEQ ID NO:42.

[0358] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:42.

[0359] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 49.

[0360] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49 and CDRs identical to the Kabat CDRs of SEQ ID NO:49.

[0361] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49 and CDRs identical to the IMGT CDRs of SEQ ID NO:49.

[0362] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:49.

[0363] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:56.

[0364] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56 and CDRs identical to the Kabat CDRs of SEQ ID NO:56.

[0365] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56 and a CDR that is identical to the IMGT CDR of SEQ ID NO:56.

[0366] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:56.

[0367] Exemplary embodiments of ABD2 Antigen-binding domain 2 (ABD2) may be selected, for example, from antigen-binding domains that bind to PD-1, some non-limiting exemplary embodiments of which are provided herein.

[0368] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), comprising heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:57, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:58, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:59.

[0369] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62.

[0370] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 176, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 177, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 178.

[0371] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 179, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 180, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 181.

[0372] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 183, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 184, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 185.

[0373] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 186, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 187, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 188.

[0374] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 190, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 191, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 192.

[0375] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), comprising CDRH1 having the amino acid sequence set forth in SEQ ID NO: 193, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 194, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 195.

[0376] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 197, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 198, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 199.

[0377] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 200, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 201, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 202.

[0378] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 204, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 206.

[0379] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 207, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 209.

[0380] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:211, CDRH2 having the amino acid sequence set forth in SEQ ID NO:212, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:213.

[0381] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 214, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 215, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 216.

[0382] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 218, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 219, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 220.

[0383] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 221, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 222, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 223.

[0384] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 225, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 226, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 227.

[0385] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230.

[0386] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234.

[0387] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237.

[0388] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241.

[0389] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244.

[0390] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248.

[0391] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 249, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 250, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 251.

[0392] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 253, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 254, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 255.

[0393] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 256, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 257, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 258.

[0394] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 260, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 261, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 262.

[0395] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 263, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 264, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 265.

[0396] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 281, and wherein X 1a ~X 1kany one of X is any (e.g., naturally occurring) amino acid residue, or 1a is L or E, and X 1b is A or P, and X 1c is D or G, and X 1d is N or S, and X 1e is S or T, and X 1f is K or R, and X 1g is D or G, and X 1h is K or Q, and X 1j is T or A and / or X 1k is L or V.

[0397] [ka]

[0398] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 63.

[0399] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63 and CDRs identical to the Kabat CDRs of SEQ ID NO:63.

[0400] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63 and a CDR that is identical to the IMGT CDR of SEQ ID NO:63.

[0401] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63.

[0402] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 182. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 182.

[0403] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:182 and CDRs identical to the Kabat CDRs of SEQ ID NO:182.

[0404] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:182 and a CDR identical to the IMGT CDR of SEQ ID NO:182.

[0405] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 182.

[0406] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 189. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 189.

[0407] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:189 and CDRs identical to the Kabat CDRs of SEQ ID NO:189.

[0408] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:189 and a CDR that is identical to the IMGT CDR of SEQ ID NO:189.

[0409] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 189.

[0410] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 196. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 196.

[0411] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:196 and CDRs identical to the Kabat CDRs of SEQ ID NO:196.

[0412] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:196 and CDRs identical to the IMGT CDRs of SEQ ID NO:196.

[0413] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 196.

[0414] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 203. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:203.

[0415] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:203 and CDRs identical to the Kabat CDRs of SEQ ID NO:203.

[0416] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:203 and a CDR that is identical to the IMGT CDR of SEQ ID NO:203.

[0417] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:203.

[0418] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 210. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 210.

[0419] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:210 and CDRs identical to the Kabat CDRs of SEQ ID NO:210.

[0420] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:210 and a CDR identical to the IMGT CDR of SEQ ID NO:210.

[0421] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:210.

[0422] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 217. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 217.

[0423] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:217 and CDRs identical to the Kabat CDRs of SEQ ID NO:217.

[0424] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:217 and a CDR that is identical to the IMGT CDR of SEQ ID NO:217.

[0425] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:217.

[0426] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 224. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 224.

[0427] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:224 and CDRs identical to the Kabat CDRs of SEQ ID NO:224.

[0428] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:224 and a CDR that is identical to the IMGT CDR of SEQ ID NO:224.

[0429] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 224.

[0430] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 231. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 231.

[0431] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:231 and CDRs identical to the Kabat CDRs of SEQ ID NO:231.

[0432] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:231 and a CDR that is identical to the IMGT CDR of SEQ ID NO:231.

[0433] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:231.

[0434] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 238. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 238.

[0435] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:238 and CDRs identical to the Kabat CDRs of SEQ ID NO:238.

[0436] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:238 and a CDR identical to the IMGT CDR of SEQ ID NO:238.

[0437] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:238.

[0438] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 245. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 245.

[0439] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:245 and CDRs identical to the Kabat CDRs of SEQ ID NO:245.

[0440] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:245 and CDRs identical to the IMGT CDRs of SEQ ID NO:245.

[0441] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:245.

[0442] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 252. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 252.

[0443] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:252 and CDRs identical to the Kabat CDRs of SEQ ID NO:252.

[0444] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:252 and a CDR identical to the IMGT CDR of SEQ ID NO:252.

[0445] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:252.

[0446] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 259. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 259.

[0447] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:259 and CDRs identical to the Kabat CDRs of SEQ ID NO:259.

[0448] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:259 and CDRs identical to the IMGT CDRs of SEQ ID NO:259.

[0449] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:259.

[0450] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 266. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 266.

[0451] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:266 and CDRs identical to the Kabat CDRs of SEQ ID NO:266.

[0452] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:266 and a CDR identical to the IMGT CDR of SEQ ID NO:266.

[0453] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:266.

[0454] Exemplary Embodiments of ABD3 Antigen-binding domain 3 (ABD3) may be selected, for example, from antigen-binding domains that bind to CD47, some non-limiting exemplary embodiments of which are provided herein.

[0455] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), comprising a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66.

[0456] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 3 (ABD3), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69.

[0457] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70. In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:70.

[0458] In some embodiments, the heavy chain comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70 and CDRs identical to the Kabat CDRs of SEQ ID NO:70.

[0459] In some embodiments, the heavy chain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:70.

[0460] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[0461] Exemplary Embodiments of Other Antigen-Binding Domains In some embodiments, the binding agent comprises one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3), and at least one antigen-binding domain binds to a different antigen.

[0462] In some embodiments, the binding agent may therefore comprise an antigen-binding domain that binds to CD3. Such antigen-binding domains include those known to those skilled in the art. An exemplary embodiment of an antigen-binding domain that binds to CD3 is provided in SEQ ID NO: 115.

[0463] Thus, in some embodiments, the binding agent may comprise an antigen-binding domain that binds to DR2, PD-1, and / or CD3. In other embodiments, the binding agent may comprise an antigen-binding domain that binds to DR2 and / or CD3. In yet other embodiments, the binding agent may comprise an antigen-binding domain that binds to PD-1 and / or CD3.

[0464] In some embodiments, the binding agent comprises one or more antigen-binding domains, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3), and at least one of the antigen-binding domains may be selected from the group consisting of, but not limited to, CD3, CD36, DRD1, DRD2, DRD3, DRD4, DRD5, PD-L1, TROP2, CD147, MCT1, IL1RAP, AMIGO2, PTK7, MCT2, MCT4, NHE1, H+ / K+-ATPase, LAP, HLA-I A2, CD73, CD98, CEACAM5 / 6, ICAM-1, MCSP, fibronectin, beta1 integrin, tetraspanin 8, CD164, CD59, CD63, CD44, CD166, cWF, TNF, IL-17A, IL17-F, IL-6R, BCMA, TNF, RANKL, ADAMTS5, VEGF, Ang2, CX3CR1, CXCR4, TfR1 (CD71), CXCR2, CD3, PD1, PDL-1, CTLA-4, CD8, LAG-3, OX40, CD27, CD122 / IL2RB, TLR8 / CD288, TIM-3, ICOS / CD278, NKG2A, A2AR, B7-H the antigen-binding domain is selected from the group consisting of antigen-binding domains that specifically bind to 3, B7-H4, GITR / TNFRSF18, 4-IBB / CD137, KIR2DL1, KIR3DL2, SIRPα, CD47, VISTA, CD40, CD112, CD96, TOGOT, BTLA, TIGIT, CD4, VEGFR2, CD19, IGFR1, EpCAM, EGFR, DLL3, CGRP, CD79b, CD28, CCR5, ErbB3, ErbB2, TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, IDO1, IDO2, TLR-4, TLR-7, TLR-8, TLR-9, NOX2, and SIGLEC-7.

[0465] Polypeptide chain The binding agents of the present disclosure comprise one or more polypeptide chains.

[0466] A segment of DNA encoding a desired polypeptide chain sequence may be synthesized in vitro. Different DNA modules are assembled in an organized and oriented manner into a single piece that is then cloned into an expression vector. The resulting polypeptide chain is thus composed of the different modules that form a single chain.

[0467] Polypeptide chains of the present disclosure include, for example and without limitation, antigen binding domains, linkers, and dimerization domains that promote assembly of at least two polypeptide chains.

[0468] In some embodiments, the polypeptide chains of the present disclosure may be monospecific.

[0469] In some embodiments, the polypeptide chains of the present disclosure may be multispecific.

[0470] In some embodiments, the polypeptide chains of the present disclosure may be monovalent.

[0471] In some embodiments, the polypeptide chains of the present disclosure may be multivalent.

[0472] In some cases, the polypeptide chain does not comprise a dimerization domain.

[0473] In some cases, the polypeptide chain comprises a dimerization domain.

[0474] In some aspects of the present disclosure, the polypeptide chains may be monospecific.

[0475] An exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising one antigen-binding domain. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but where the antigen-binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but where the antigen-binding domains have the same CDRs and different framework regions. A further exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs (e.g., conservative substitutions in one or more CDRs) without affecting their ability to bind to the same antigen or epitope.

[0476] In some aspects of the present disclosure, the polypeptide chains may be multispecific, including, for example, bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, biparatopic, and multiparatopic polypeptide chains.

[0477] In exemplary configurations, one or more antigen-binding domains may be located N-terminal, C-terminal, or on each side of the dimerization domain.

[0478] In another exemplary configuration, the polypeptide chain may comprise at least one antigen binding domain N-terminal to the dimerization domain and at least one antigen binding domain C-terminal to the dimerization domain.

[0479] In a further exemplary configuration, the polypeptide chain may comprise one antigen binding domain N-terminal to the dimerization domain and at least two antigen binding domains C-terminal to the dimerization domain.

[0480] In a still further exemplary configuration, the polypeptide chain may comprise two antigen-binding domains N-terminal to the dimerization domain and two antigen-binding domains C-terminal to the dimerization domain.

[0481] In some embodiments, the polypeptide chain may comprise Formula I, Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IIIa and IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII, etc.

[0482] The polypeptide chains of the present disclosure comprise an antigen-binding domain that is functionally active as a single chain or when part of a binding agent disclosed herein.

[0483] For example, the antigen-binding domain of a polypeptide chain may bind to its target and may be biologically active.

[0484] In some embodiments, the biological activity of an antigen-binding domain includes, for example and without limitation, blocking the binding of a target to its natural receptor or ligand. Alternatively, the biological activity of an antigen-binding domain includes its ability to sequester a target. Further, the biological activity of an antigen-binding domain includes its ability to induce signal transduction.

[0485] A polypeptide chain that contains more than one antigen-binding domain is characterized as being multivalent.

[0486] The polypeptide chains of the present disclosure may include additional amino acid sequences (defined by X and Y, respectively, in the formulas disclosed herein) at their N- or C-terminus or both termini.

[0487] In some embodiments, the amino acid sequence at the N-terminus (defined by X) may include a signal peptide, an exemplary embodiment of which is provided in SEQ ID NO:133.

[0488] In some embodiments, the amino acid sequence at the N-terminus (defined by X) or the amino acid sequence at the C-terminus (defined by Y) may independently comprise a linker, a cytokine, a chemokine, a tag (e.g., a His tag (e.g., SEQ ID NO: 134)), a masking domain, a phage coat protein, an antigen-binding domain, or a combination thereof.

[0489] Exemplary embodiments of multispecific polypeptide chains include polypeptide chains comprising at least two antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs, leading to different binding specificities.

[0490] A polypeptide chain may more particularly be characterized as bispecific if it binds to two different epitopes or antigens. A polypeptide chain may be characterized as trispecific if it binds to three different epitopes or antigens. A polypeptide chain may be characterized as tetraspecific if it binds to four different epitopes or antigens. A polypeptide chain may be characterized as pentaspecific if it binds to five different epitopes or antigens. A polypeptide chain may be characterized as hexaspecific if it binds to six different epitopes or antigens.

[0491] A polypeptide chain containing two antigen-binding domains that bind to two non-overlapping epitopes on the same target is characterized as being biparatopic. A polypeptide chain containing three, four, or more epitopes on the same target is characterized as being multiparatopic.

[0492] The antigen-binding domains of a given polypeptide chain are selected based on the intended use, e.g., detection, diagnostic, and / or therapeutic use. Each of the antigen-binding domains of a particular polypeptide chain may be selected to produce an additive or synergistic effect.

[0493] In some embodiments, the antigen-binding domain may be selected for its ability to specifically bind to a protein involved in a disease or condition.

[0494] For example, the polypeptide chains of the present disclosure may comprise at least one antigen-binding domain that specifically binds to an antigen expressed by a tumor cell or the tumor cell environment (ie, a tumor-specific antigen-binding domain).

[0495] In other aspects and embodiments of the present disclosure, the polypeptide chain may comprise at least one antigen-binding domain that specifically binds to an immunomodulator.

[0496] For example, the polypeptide chain may include one or more antigen-binding domains (e.g., immunospecific antigen-binding domains) that bind to an immune checkpoint protein, a cytokine, a chemokine, or an immune receptor or co-receptor, etc.

[0497] In some exemplary embodiments, the antigen-binding domain may bind to dopamine receptor D2 (DR2).

[0498] In some exemplary embodiments, the antigen-binding domain may bind to PD-1.

[0499] In some exemplary embodiments, the antigen-binding domain may bind to CD47.

[0500] In exemplary embodiments, the polypeptide chains of the present disclosure may comprise at least one tumor-specific antigen-binding domain and at least one immunospecific antigen-binding domain.

[0501] In some embodiments, the tumor-specific antigen-binding domain may be located N-terminal to the dimerization domain.

[0502] In some embodiments, the tumor-specific antigen-binding domain may be located C-terminal to the dimerization domain.

[0503] In some embodiments, the tumor-specific antigen-binding domain may be located both N-terminal and C-terminal to the dimerization domain.

[0504] In some embodiments, multiple immunospecific antigen-binding domains may be located N-terminal to the dimerization domain.

[0505] In some embodiments, the immunospecific antigen-binding domain may be located C-terminal to the dimerization domain.

[0506] In some embodiments, the immunospecific antigen-binding domains may be located both N-terminal and C-terminal to the dimerization domain.

[0507] In exemplary, non-limiting embodiments, a polypeptide chain or binding agent may comprise two immunospecific antigen-binding domains C-terminal to a dimerization domain, hi some embodiments, the immunospecific antigen-binding domains immediately adjacent to the C-terminal portion of the dimerization domain may be linked via a non-cleavable linker.

[0508] Dimerization domain (DD) In some embodiments, the polypeptide chains of the present disclosure comprise a dimerization domain, such that two polypeptide chains may assemble to form a binding agent. Exemplary embodiments of binding agents include homodimers and heterodimers.

[0509] The dimerization domain may comprise, for example, without limitation, an immunoglobulin constant region, including, for example, the Fc, CH2 and / or CH3 domains of a heavy chain immunoglobulin.

[0510] In certain embodiments and aspects of the present disclosure, the dimerization domain may have a sequence identical to that of a native IgG1, IgG2, IgG3, or IgG4 constant region or their corresponding CH2 and / or CH3 domains.

[0511] Dimerization domains having sequences identical to those of natural human antibodies are specifically encompassed by the present disclosure. Exemplary embodiments of dimerization domains include, for example, the CH2-CH3 domains of a natural human antibody heavy chain.

[0512] Thus, in some embodiments, the dimerization domain comprises a native constant region of an antibody, such as a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, or a native human IgG4 constant region.

[0513] Thus, in some embodiments, the dimerization domain comprises a native CH3 domain.

[0514] In an exemplary embodiment, the dimerization domain comprises a native human CH3 domain.

[0515] In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.

[0516] In some embodiments, the native CH3 domain is a native IgG1 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG1 CH3 (e.g., SEQ ID NO: 116).

[0517] In some embodiments, the native CH3 domain is a native IgG2 CH3 domain, hi other embodiments, the native CH3 domain is a native human IgG2 CH3 domain.

[0518] In some embodiments, the native CH3 domain is a native IgG3 CH3 domain, hi other embodiments, the native CH3 domain is a native human IgG3 CH3 domain.

[0519] In some embodiments, the native CH3 domain is a native IgG4 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG4 CH3 domain.

[0520] When two polypeptide chains of a binding agent are composed of the same amino acid sequence, the binding agent forms a homodimer. However, co-expression of polypeptide chains having the CH2-CH3 domains of a natural antibody but different amino acid sequences can result in a mixture of homodimers and heterodimers. Different binding agents present in the mixture can be separated by methods known in the art, including, for example, size exclusion chromatography.

[0521] Exemplary heterodimers of the present disclosure therefore include those formed by two polypeptide chains having the CH3 domain or CH2-CH3 domains of a natural antibody and having different sequences or configurations.

[0522] In some embodiments, the polypeptide chains may have a variant dimerization domain that includes, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 amino acid substitutions compared to the native or wild-type sequence.

[0523] In exemplary embodiments, the variant dimerization domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. The amino acid substitutions may be conservative or non-conservative as outlined in Table 2 (Table 2).

[0524] In exemplary embodiments, the polypeptide chain may have a variant dimerization domain with a sequence that is 80% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domain. Polypeptide chains encompassed by the present disclosure include those that contain a variant dimerization domain that is 85% to 99% identical, 90% to 99% identical, or 95% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domain.

[0525] In some embodiments, polypeptide chains of the present disclosure may comprise mutant dimerization domains comprising amino acid substitutions that favor heterodimer formation. Heterodimers of the present disclosure may therefore be formed by polypeptide chains comprising such mutations.

[0526] Thus, in some embodiments, the dimerization domain comprises a variant constant region of an antibody, such as a variant human IgG1 constant region, a variant human IgG2 constant region, a variant human IgG3 constant region, or a variant human IgG4 constant region, etc. The variant constant region may have one or more amino acid substitutions, insertions, or deletions compared to the native constant region.

[0527] Thus, in some embodiments, the dimerization domain comprises a variant CH3 domain, which may have one or more amino acid substitutions, insertions, or deletions compared to the native CH3 domain.

[0528] In some embodiments, the dimerization domain comprises a native CH2 and a variant CH3 domain.

[0529] In some embodiments, the dimerization domain comprises a variant CH2 and a variant CH3 domain. The variant CH2 domain may have one or more amino acid substitutions, insertions, or deletions compared to the native CH2 domain.

[0530] In some embodiments, the variant CH3 domain is a variant IgG1 CH3 domain. In other embodiments, the variant CH3 domain is a variant human IgG1 CH3 domain.

[0531] In some embodiments, the variant CH3 domain is a variant IgG2 CH3 domain. In other embodiments, the variant CH3 domain is a variant human IgG2 CH3 domain.

[0532] In some embodiments, the variant CH3 domain is a variant IgG3 CH3 domain. In other embodiments, the variant CH3 domain is a variant human IgG3 CH3 domain.

[0533] In some embodiments, the variant CH3 domain is a variant IgG4 CH3 domain. In other embodiments, the variant CH3 domain is a variant human IgG4 CH3 domain.

[0534] In some embodiments, the variant CH2 domain is a variant IgG1 CH2 domain. In other embodiments, the variant CH2 domain is a variant human IgG1 CH2 domain.

[0535] In some embodiments, the variant CH2 domain is a variant IgG2 CH2 domain. In other embodiments, the variant CH2 domain is a variant human IgG2 CH2 domain.

[0536] In some embodiments, the variant CH2 domain is a variant IgG3 CH2 domain, hi other embodiments, the variant CH2 domain is a variant human IgG3 CH2 domain.

[0537] In some embodiments, the variant CH2 domain is a variant IgG4 CH2 domain, hi other embodiments, the variant CH2 domain is a variant human IgG4 CH2 domain.

[0538] In some embodiments, the Fc region may be modified to prevent glycosylation, increase its half-life, or modulate receptor binding or effector function. Exemplary mutations are discussed in Saunders KO (Front. Immunol. 10:1296, 2019; the entire contents of which are incorporated herein by reference), including, for example, mutation of asparagine 297 (e.g., N297).

[0539] In some embodiments, the dimerization domain comprises an Fc region or portion thereof of an antibody.

[0540] In some embodiments, the dimerization domain comprises an Fc region or portion thereof, with or without Fc modifications.

[0541] In other embodiments, the dimerization domain comprises an altered or mutated Fc region or portion thereof. For example, the Fc region or portion thereof may be altered or mutated to modify one or more characteristics of the binding agent. In exemplary embodiments, the Fc region or portion thereof is aglycosylated. In other exemplary embodiments, the Fc region or portion thereof comprises one or more of the Fc modifications in Table 1.

[0542] In other exemplary embodiments, the Fc region or portion thereof is altered or mutated to increase ADCC activity. For example, defucosylation (e.g., N297 according to the EU numbering system) may result in increased FcRgII binding on NK cells, potentially increasing ADCC. Thus, in some embodiments, the Fc region or portion thereof may have a reduced number of fucose residues. In other embodiments, the binding agent comprises a defucosylated Fc region or portion thereof. In yet other embodiments, the Fc region or portion thereof of the binding agent lacks fucose residues. In additional embodiments, the Fc region or portion thereof of the binding agent lacks core fucose residues. In further embodiments, the Fc region or portion thereof of the binding agent completely lacks core fucose residues.

[0543] Exemplary, non-limiting embodiments of mutations in the constant region (e.g., Fc region) that improve one or more effector functions are encompassed by the present disclosure, exemplary embodiments of which are provided in Table 1 (List of Mutations Antibodies (Basel). 2020 Nov 17;9(4):64, the entire contents of which are incorporated herein by reference).

[0544] [Table 1]

[0545] According to the present disclosure, the variant CH3 domain comprises one or more mutations compared to a native CH3 domain. Thus, in some embodiments, the dimerization domain comprises a variant CH3 domain comprising one or more mutations compared to a native CH3 domain.

[0546] For example, the variant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG1. In another example, the variant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG2. In yet another example, the variant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG3. In a further example, the variant CH3 domain may comprise one or more mutations compared to the native CH3 domain of human IgG4.

[0547] In accordance with the present disclosure, a binding agent may be composed of two different polypeptide chains that associate to form a dimer, referred to herein as a heterodimer.

[0548] Heterodimers can be produced by co-expressing two different polypeptide chains (eg, chain A and chain B).

[0549] In some embodiments, the polypeptide chains may comprise a dimerization domain comprising a wild-type human CH2 and a mutant human CH3 that favors heterodimer formation. In some cases, the polypeptide chains have the potential to form homodimers when expressed alone or to form heterodimers (or a mixture of homodimers and heterodimers) when expressed with a complementary chain.

[0550] Thus, in addition to the monomers, heterodimers and homodimers containing the CH3 mutations disclosed herein, mixtures of such monomers, heterodimers and / or homodimers are encompassed by the present disclosure.

[0551] In other embodiments, the polypeptide chains disclosed herein may comprise a mutant dimerization domain that contains mutations known in the art to favor heterodimer formation.

[0552] For example, polypeptide chains of the present disclosure may include the structures set forth in Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII disclosed herein, as well as mutations known in the art to favor heterodimer formation.

[0553] Exemplary embodiments of CH3 mutations are disclosed, for example, in Ha, JH et al. (Front Immunol, 2016;7:394) or Godar M et al. (Expert Opinion on Therapeutic patents, 2018;28(3):251-276), the entire contents of which are incorporated by reference, and include, for example, knobs-into-holes (mutation T366Y in the first CH3 domain and mutation Y407T in the second CH3 domain; mutation T366W in the first CH3 domain and mutations T366S, L368A, Y407V in the second CH3 domain; or mutations S354C, T366W in the first CH3 domain and mutations S354C, T366W in the second CH3 domain). The following mutations were used: DD / KK mutations (mutations K409D, K392D in the first CH3 domain, and mutations D399K, E356K in the second CH3 domain); asymmetric re-engineering techniques (mutations E356K, E357K, D399K in the first CH3 domain and mutations K439E, K370E, K409D in the second CH3 domain); BiMAb mutations (mutations K249E, K288E in the first CH3 domain, and mutations K249E, K288E in the second CH3 domain); In-line mutations E236K, D278K), XmAb mutations (mutations S364H, F405A in the first CH3 domain, mutations Y349T, T394F in the second CH3 domain), DuoBody mutations (mutations F405L in the first CH3 domain, mutation K409R in the second CH3 domain), Azymetric mutations (mutations T350V, L351Y, S400E, F405A, Y407V in the first CH3 domain, mutations T350V, T3 66L, N390R, K392M, T394W), Biclonics mutations (mutation T366K (+L351K) in the first CH3 domain, mutation L351D in the second CH3 domain, or E or D at Y349, L368, or Y349 + R355), ZW1 mutations (mutations T350V, L351Y, F405A, Y407V in the first CH3 domain, mutations T350V, T366L, K392L, T394W in the second CH3 domain), 7.8.60 mutations (mutations in the first CH3 domain: K360D, D399M, Y407A, and mutations in the second CH3 domain: E345R, Q347R, T366V, K409V), EW-RVT mutations (mutations in the first CH3 domain: K360E, K409W, and mutations in the second CH3 domain: Q347R, D399V, F405T), EW-RVTs-s mutations (mutations in the first CH3 domain: K360E, K409W, Y349C, and mutations in the second CH3 domain: Q347R, D399V, F405T, S354C), SEED mutations (mutations in the first CH3 domain: 45 IgA-derived residues on the IgG1 CH3 and mutations in the second CH3 domain: IgA Examples of CH3 mutations include the IgG1-derived 57 residues in CH3, the A107 mutation (mutations K370E, K409W in the first CH3 domain, and mutations E357N, D399V, F405T in the second CH3 domain). Other exemplary embodiments of CH3 mutations are disclosed in International Application PCT / CA2020 / 051753, filed December 18, 2020, and published June 24, 2021, under the publication number WO2021 / 119832.

[0554] Linker (L) The different modules of the polypeptide chains disclosed herein may be associated with each other via linkers.

[0555] In some embodiments, the linker used to join one or more modules of the polypeptide chain is not a cleavable linker.

[0556] In an exemplary embodiment, the linker (Lc) located immediately adjacent to the C-terminus of the dimerization domain does not comprise a cleavable linker.

[0557] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains does not comprise a cleavable linker.

[0558] In other embodiments, the linker used to join one or more modules of the polypeptide chain may comprise a non-cleavable linker.

[0559] In an exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.

[0560] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains is a non-cleavable linker.

[0561] In a further exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain and the linker joining the first two antigen-binding domains located C-terminal to the dimerization domain are non-cleavable linkers.

[0562] In some embodiments, the linker immediately adjacent to the N-terminus of the dimerization domain may preferably comprise the hinge region of an antibody.

[0563] In some embodiments, the hinge region is a native hinge region.

[0564] In some embodiments, the native hinge region is a native IgG1 hinge region. In some embodiments, the native hinge region is a native human IgG1 hinge region.

[0565] In some embodiments, the native hinge region is a native IgG2 hinge region, hi other embodiments, the native hinge region is a native human IgG2 hinge region.

[0566] In some embodiments, the native hinge region is a native IgG3 hinge region, hi other embodiments, the native hinge region is a native human IgG3 hinge region.

[0567] In some embodiments, the native hinge region is a native IgG4 hinge region. In some embodiments, the native hinge region is a native human IgG4 hinge region.

[0568] In some embodiments, the hinge region is a mutant hinge region.

[0569] In some embodiments, the mutant hinge region is a mutant IgG1 hinge region. In some embodiments, the mutant hinge region is a mutant human IgG1 hinge region.

[0570] In some embodiments, the mutant hinge region is a mutant IgG2 hinge region, hi other embodiments, the mutant hinge region is a mutant human IgG2 hinge region.

[0571] In some embodiments, the mutant hinge region is a mutant IgG3 hinge region, hi other embodiments, the mutant hinge region is a mutant human IgG3 hinge region.

[0572] In some embodiments, the mutant hinge region is a mutant IgG4 hinge region. In some embodiments, the mutant hinge region is a mutant human IgG4 hinge region.

[0573] In some embodiments, all modules of the polypeptide chain are linked via a non-cleavable linker.

[0574] Exemplary embodiments of non-cleavable linkers include those that remain substantially intact during protein expression or manufacturing processes. As used herein, "substantially intact" means that linker cleavage occurs in 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the total polypeptide content of a given solution or composition.

[0575] Other exemplary embodiments of non-cleavable linkers also include linkers that do not contain specific cleavage sites for one or more proteases present in human or animal blood or serum.

[0576] Additional exemplary embodiments of non-cleavable linkers further include linkers that retain their integrity upon administration of the binding agent in an individual for at least 1, 2, 3, 4, 5, 6, 12, 24, 48 hours or longer following administration.

[0577] In a further exemplary embodiment, the linker comprises both a non-cleavable linker and a cleavable linker.

[0578] In some embodiments, the linker is not cleavable.

[0579] In some cases, cleavable linkers may be used for in vivo release of a drug (e.g., a cytostatic molecule, a cytotoxic molecule, a chemotherapeutic agent, etc.) or label attached to a polypeptide chain of the present disclosure.

[0580] Exemplary embodiments of cleavable linkers are provided, for example, in U.S. Patent Application Publication No. 2019 / 0010242, and include linkers that are susceptible to cleavage by proteases, typically extracellular proteases, such as proteases produced by tumors or activated immune effector cells, such as ADAMS, ADAMTS, e.g., ADAMS; ADAMS; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine ​​cathepsins, e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, e.g., cruzipain; legumain; otubain-2; KLK, e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinase, e.g., meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoatase, HtrAl, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA;and those having a site for specific cleavage by a protease selected from a type II transmembrane serine protease (TTSP), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof. In some embodiments, the polypeptide chains of the present disclosure do not contain such a linker at a position corresponding to Lc.

[0581] Exemplary embodiments of linkers include flexible linkers, rigid linkers, helical linkers, and combinations thereof. Linkers are discussed, for example, in Chen X et al. (Adv Drug Deliv Rev. 2013;65(10):1357-1369), the entire contents of which are incorporated herein by reference.

[0582] In some embodiments, a hinge region or a portion thereof may be used to link a module to a dimerization domain and is considered herein as a linker. The hinge region may be derived from a natural antibody (of human or animal origin) or a synthetic antibody. The hinge region may be obtained, for example, from an IgG, such as IgG1, IgG2, IgG3, or IgG4. Exemplary embodiments of hinge regions are provided in SEQ ID NO:98, SEQ ID NO:121, SEQ ID NO:125, and SEQ ID NO:129.

[0583] In some cases, the hinge region may have one or more amino acid substitutions, insertions, and / or deletions compared to the native hinge region. A mutant hinge region may, for example, comprise a sequence (mutant hinge region) that is 80% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 hinge region. An exemplary, non-limiting example of a mutant hinge region is the hinge region of IgG4 in which S228 is replaced with P (EU numbering) (Angal, S. et al., Mol Immunol 30, 105-108, 1993). Other exemplary embodiments of mutant hinges are provided in SEQ ID NOs: 118-120, 122-124, 126-128, and 130-132.

[0584] Flexible linkers are typically composed of small polar amino acids, such as threonine or serine, and glycine. An exemplary, non-limiting embodiment of a flexible linker is a GS linker (glycine / serine repeat), such as (GGGS) n (GGGGS) m (SEQ ID NO: 172), (GS) n , (GGS) n , (GGGS) n (SEQ ID NO: 173), (GGGGS) n (SEQ ID NO: 104), (GGSG) n (SEQ ID NO: 174), (GGGSS) n (SEQ ID NO: 175), and the like, where n and m can independently be an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or greater, such as 15, 20, or 25.

[0585] Specific exemplary, non-limiting embodiments of flexible linkers include those comprising or consisting of the amino acid sequence set forth in SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103 or SEQ ID NO:104.

[0586] Other exemplary, non-limiting embodiments of linkers include linkers comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 172, 173, 174, or 175.

[0587] SEQ ID NO: 104 has the formula (GGGGS) n (n is an integer selected from 1 to 10) or alternatively represented by the formula GGGGSX1 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 104).

[0588] SEQ ID NO: 172 is a sequence of the formula (GGGS) n (GGGGS) mwherein n and / or m are independently an integer selected from 1 to 10, or may alternatively be represented by the formula GGGSX1GGGGSX2, where it is to be understood that X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 172, and X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 6 to 10 of SEQ ID NO: 172.

[0589] SEQ ID NO: 173 is a sequence of the formula (GGGS) n where n is an integer selected from 1 to 10, or alternatively may be represented by the formula GGGSX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 173.

[0590] SEQ ID NO: 174 is a sequence of the formula (GGSG) n where n is an integer selected from 1 to 10, or alternatively may be represented by the formula GGSGX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 174.

[0591] SEQ ID NO: 175 is a sequence of the formula (GGGSS) n where n is an integer selected from 1 to 10, or alternatively may be represented by the formula GGGSSX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 175.

[0592] According to the present disclosure, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater, for example, 15, 20 or 25.

[0593] According to the present disclosure, m can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater, for example, 15, 20 or 25.

[0594] The rigid linkers of the present disclosure typically comprise a proline-rich sequence (XP) n wherein X represents any amino acid, preferably Ala, Lys, or Glu, and n is an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. (Chen X et al., 2013).

[0595] Specific exemplary, non-limiting embodiments of rigid linkers include those comprising or consisting of the amino acid sequence set forth in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107 or SEQ ID NO:108.

[0596] SEQ ID NO: 108 is a compound of the formula (X(PAPAP)) n It should be understood that SEQ ID NO:108 may be represented by the formula (XPAPAP)X2KA, where X may be present or absent and, if present, is A, and X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 6 of SEQ ID NO:108.

[0597] Helical linkers may be characterized as rigid, but are separated into distinct linker families herein. Exemplary embodiments of helical linkers are discussed in Chen X et al., 2013, and include, for example, repeats of alanine residues flanked by positively and negatively charged amino acid residues.

[0598] Specific exemplary, non-limiting embodiments of helical linkers include those comprising or consisting of the amino acid sequences set forth in SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111 and SEQ ID NO:112.

[0599] SEQ ID NO: 112 is of the formula X(EAAAK) nIt should be understood that X and X2 may be independently present or absent and, when present, preferably A. Alternatively, SEQ ID NO: 112 may be represented by the formula X(EAAAK)X3X2, where X and X2 are independently present or absent and, when present, preferably A; and X3 is absent or, if present, is 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO: 112.

[0600] In an exemplary embodiment, the linker (L in Formulas II-VIII) immediately adjacent to the C-terminus of the dimerization domain c1 The linker (identified as n) may comprise either a flexible linker, a rigid linker, or a helical linker. Linkers that may be specifically selected to occupy this position include, without limitation, linkers comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 100-109, SEQ ID NO: 111, or SEQ ID NO: 112 (where n is 1).

[0601] In an exemplary embodiment, a linker (L in Formulas II-VIII) joining the first two antigen-binding domains located C-terminal to the dimerization domain is c2 The linker (identified as n) may comprise either a flexible linker, a rigid linker, or a helical linker. Linkers that may be specifically selected to occupy this position include, but are not limited to, linkers comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 100-110 or SEQ ID NO: 112 (where n is 1).

[0602] The present disclosure also provides linkers having the addition of 1 to 10 amino acids (and any range or value between 1 and 10, e.g., 1 to 5, etc.) at one or both of the N-terminus or C-terminus of any of SEQ ID NOs: 100-112. These additional amino acid residues may each be independently selected from any amino acid residue. These additional amino acid residues preferably form a non-cleavable sequence.

[0603] The present disclosure also provides linkers having deletions of 1, 2, 3, 4, or 5 amino acids (and any value between 1 and 5) at one or both of the N-terminus or C-terminus of any of SEQ ID NOs: 100-112.

[0604] Suitable linkers may comprise, for example, an amino acid sequence containing about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 25, about 3 to about 20, about 3 to about 15, or about 3 to about 10 amino acid residues.

[0605] In exemplary embodiments, the length of each linker can independently be in the range of about 5 to about 50 amino acid residues, including, for example, about 5 to about 40 amino acid residues, about 10 to about 40 amino acid residues, about 20 to about 40 amino acid residues, about 20 to about 35 amino acid residues, about 25 to about 30 amino acid residues, and any subranges within and including such ranges.

[0606] In some embodiments, a linker comprising the amino acid sequence set forth in SEQ ID NO:104, SEQ ID NO:108 or SEQ ID NO:112 may have an "n" value of preferably 1 to 10, more preferably 2 to 5, including 2, 3, 4 or 5.

[0607] Binder Binding agents of the disclosure include, for example, the antigen-binding domains disclosed herein.

[0608] Binding agents of the present disclosure include, for example, the polypeptide chains disclosed herein.

[0609] Binding agents of the present disclosure include, for example, dimers of the polypeptide chains disclosed herein.

[0610] Binding agents of the present disclosure include, for example, multimers of the polypeptide chains disclosed herein.

[0611] Binding agents of the present disclosure may have the format of antibodies and antigen-binding fragments thereof, antibody-like molecules (Fc, CH3 fusions, etc.), protein scaffolds and fusions with immune cell modulating agents, etc.

[0612] In some embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof.

[0613] In some embodiments, the binding agent comprises an antibody-like molecule.

[0614] In some embodiments, the binding agent may be fused to the protein scaffold.

[0615] In some embodiments, the binding agent comprises an immune cell modulating agent.

[0616] Thus, in some embodiments, binding agents include antibodies and antigen-binding fragments thereof, such as, but not limited to, single domain antibodies from camelids or sharks, human antibodies including IgG (including human IgG1, human IgG2, human IgG3, human IgG4), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD, animal antibodies including, for example, IgG (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE, and IgD, and the like.

[0617] In other embodiments, the binding agent comprises an antigen-binding fragment, such as, but not limited to, Fab, Fab', F(ab')2, complementarity determining regions, and variable regions, including VH, VHH, and VL.

[0618] Binding agents also include immune cell modulators, such as dual affinity retargeting molecules (DARTs), chimeric antigen receptor (CAR) constructs, bispecific T cell engager constructs (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs) containing scFvs or VHHs, etc.

[0619] Binding agents also include fusions with protein scaffolds, including ankyrin repeat proteins, the Z-domain of Staphylococcus protein-A, fibronectin type III, and knottins, among others. Exemplary embodiments of binding agents include monospecific, bispecific (symmetric or asymmetric), trispecific, or multispecific antibodies, as well as monovalent, bivalent, trivalent, or multivalent antibodies, single chain FVs (scFVs) and derivatives such as diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs, scFV-Fc, minibodies (scFV-CH3), tandem diabodies, didiabodies, and bibodies, VHs or VHHs and derivatives such as tandem bispecific or multispecific VHHs, bivalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, bivalent CH3 fusions, VHH pentabodies, and decabodies.

[0620] In some embodiments, the binding agents of the present disclosure may have a format as disclosed in PCT / CA2020 / 051753, filed December 18, 2020, such as Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII, or as disclosed herein as Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII.

[0621] The binding agents of the present disclosure may be formed by the assembly of two polypeptide chains having the same composition (having the same or different amino acid sequences) or different compositions, including compositions set forth in Formula I, Formula II, Formula III, Formula IIIa and IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII disclosed herein.

[0622] Binding agents of the present disclosure can be, for example, N-terminal to C-terminally represented by Formula I: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )]n -Y and m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain.

[0623] In some embodiments, L b In some embodiments, L b2 and / or L b3 does not have to exist.

[0624] In some embodiments, L c In some embodiments, L c1 , L c2 , and / or L c3 does not have to exist.

[0625] In some embodiments, L b and L c Both do not have to be present.

[0626] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0627] In some embodiments, the polypeptide chain comprises two or more antigen-binding domains.

[0628] In some embodiments, the polypeptide chain comprises three or more antigen-binding domains.

[0629] In some embodiments, the polypeptide chain comprises four or more antigen-binding domains.

[0630] In some embodiments, the polypeptide chain comprises five or more antigen-binding domains.

[0631] In some embodiments, the polypeptide chain comprises six or more antigen-binding domains.

[0632] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen-binding domains.

[0633] In some embodiments, the binder may be 1 to 12, e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 ... to 9, 4 to 10, 4 to 11, 4 to 12, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 9 to 10, 9 to 11, 9 to 12, 10 to 11, 10 to 12, or 11 to 12 antigen-binding domains.

[0634] In some embodiments, the binding agent comprises one polypeptide chain.

[0635] In some embodiments, the binding agent comprises two polypeptide chains.

[0636] In some embodiments, the binding agent comprises three polypeptide chains.

[0637] In some embodiments, the binding agent comprises four polypeptide chains.

[0638] In some embodiments, the binding agent comprises five polypeptide chains.

[0639] In some embodiments, the binding agent comprises six polypeptide chains.

[0640] In some embodiments, the binding agent comprises seven polypeptide chains.

[0641] In some embodiments, the binding agent comprises eight polypeptide chains.

[0642] In some embodiments, the binding agent comprises nine polypeptide chains.

[0643] In some embodiments, the binding agent comprises 10 polypeptide chains.

[0644] In some embodiments, the binding agent comprises more than 10 polypeptide chains.

[0645] In some embodiments, the binding agent has Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0646] In some embodiments, the binding agent has Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0647] In some embodiments, the binding agent has Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1)-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0648] In some embodiments, the binding agent has formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0649] In some embodiments, the binder has formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0650] In some embodiments, the binder has Formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0651] In some embodiments, the binder has Formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(Lb1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VIII).

[0652] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1).

[0653] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2).

[0654] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1)-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 3 (ABD3).

[0655] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 3 (ABD3).

[0656] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 represents antigen-binding domain 1 (ABD1), and Ab d1represents antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3).

[0657] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in N-terminal to C-terminal fashion, the amino acid sequence of Formula III, and Ab a1 is the antigen-binding domain that binds to DR2, and Ab d1 is the antigen-binding domain that binds to PD-1, and Ab d2 is the antigen-binding domain that binds to CD47.

[0658] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in N-terminal to C-terminal fashion, the amino acid sequence of Formula III, and Ab a1 is the antigen-binding domain that binds to DR2, and Ab d1 is the antigen-binding domain that binds to CD47, and Ab d2 is the antigen-binding domain that binds to PD-1.

[0659] In some embodiments, the binding agent has the formula IIIa in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa) The polypeptide chains comprise one or more polypeptide chains each independently comprising the amino acid sequence of

[0660] In some embodiments, the binding agent has the formula IIIb in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb) The polypeptide chains comprise one or more polypeptide chains each independently comprising the amino acid sequence of

[0661] In some embodiments, the binding agent comprises two identical polypeptide chains.

[0662] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula II (with the same or different amino acid sequences).

[0663] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula III (with the same or different amino acid sequences).

[0664] In some embodiments, both polypeptide chains of the binding agent may have the structure set forth in Formula IIIa (with the same or different amino acid sequences).

[0665] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula IIIb (with the same or different amino acid sequences).

[0666] In some embodiments, one polypeptide chain may have the structure set forth in Formula IIIa and one polypeptide chain may have the structure set forth in Formula IIIb (with the antigen-binding domains having the same or different amino acid sequences).

[0667] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula II, and the other may have the configuration shown in Formula III.

[0668] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula II, and the other has the configuration shown in Formula IV.

[0669] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula III, and the other has the configuration shown in Formula IV.

[0670] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula IV, and the other has the configuration shown in Formula IV.

[0671] Multispecific binding agents include binding agents that are specific for more than one epitope or more than one antigen (of the same or different antigens). For example, a multispecific polypeptide chain or binding agent may thus have more than one antigen-binding domain, at least two of which bind to different antigens or epitopes.

[0672] Binding agents of the present disclosure may therefore be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc. In some embodiments, each antigen-binding domain may be specific for a given antigen. In some embodiments, two or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, three or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, four or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, five or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, six or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. Specificity may depend on the number of antigen-binding domains present in a given binding agent.

[0673] Exemplary, non-limiting embodiments of multispecific binding agents include those composed of multispecific polypeptide chains. Other exemplary, non-limiting embodiments of multispecific binding agents include those with two antigen-binding domains of different specificities. Still other exemplary, non-limiting embodiments of multispecific binding agents include those with more than two antigen-binding domains that bind to two different antigens, proteins, or two different epitopes on the same antigen or protein.

[0674] In some embodiments, the binding agents of the present disclosure are monovalent or multivalent.

[0675] Exemplary, non-limiting embodiments of multivalent binding agents include binding agents comprised of multivalent polypeptide chains. Other non-limiting exemplary embodiments of multivalent binding agents include binding agents comprised of more than one monovalent polypeptide chain.

[0676] According to the present disclosure, a bispecific binding agent may be bivalent or multivalent depending on the number of antigen-binding domains it contains. Exemplary non-limiting embodiments of bispecific binding agents include those that comprise two identical bispecific polypeptide chains that form a dimer.

[0677] Exemplary Embodiments of Multispecific Binding Agents As indicated herein, the binding agents of the present disclosure may be multispecific.

[0678] Exemplary, non-limiting exemplary embodiments of multispecific binding agents include those provided herein.

[0679] For example, in some embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein.

[0680] In other embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to a protein expressed on the surface of an immune cell.

[0681] In still other embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to a protein expressed on the surface of a tumor cell.

[0682] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 2 (ABD2).

[0683] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 3 (ABD3).

[0684] In some embodiments, the binding agent comprises at least one antigen binding domain 2 (ABD2) and at least one antigen binding domain 3 (ABD3).

[0685] In some embodiments, the binding agent comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

[0686] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:56, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63, SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0687] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 7, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0688] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0689] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0690] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0691] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0692] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0693] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 49, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0694] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0695] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 7, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0696] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 14, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0697] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 21, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0698] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 28, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0699] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 35, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0700] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 42, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0701] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO:49, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO:63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO:70.

[0702] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 56, at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0703] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 77. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 77. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 77.

[0704] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 78. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 78. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 78.

[0705] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 79. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 79.

[0706] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 80. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 80. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 80.

[0707] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 81. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 81. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 81.

[0708] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 82. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 82. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 82. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 82.

[0709] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 83. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 83.

[0710] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 85. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 85. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 85.

[0711] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 86. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 86. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 86. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 86.

[0712] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 87. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 87. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 87.

[0713] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 88. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 88. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 88.

[0714] Other exemplary embodiments of multispecific binding agents are provided herein, including those in Table 7.

[0715] In yet other embodiments, the anti-PD-1 VHH portion of KC020, KC021, KC022, KC023, KC024, KC025, KC026, KC027, KC028, KC029, or KC030 (SEQ ID NO:63) may be replaced with another anti-PD-1 VHH portion, such as SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266.

[0716] In some embodiments, a binding agent of the present disclosure comprises an antigen-binding domain that binds to DR2, an antigen-binding domain that binds to PD1, and an antigen-binding domain that binds to CD47. In some embodiments, a binding agent of the present disclosure may be capable of bringing T cells into proximity with DR2-expressing tumor cells, may be capable of restoring T cell function, and / or may be capable of enhancing macrophage function through blockade of the SIRPα / CD47 interaction. In some embodiments of the present disclosure, a binding agent may exhibit its anti-tumor activity through antibody-dependent cell-mediated cytotoxicity (ADCC).

[0717] variant Variants of the sequences disclosed herein are also encompassed by the present disclosure.

[0718] Variants encompassed by the present disclosure include those that may include an insertion of one or more amino acid residues at one or more positions, a deletion of one or more amino acid residues at one or more positions, or a substitution (conservative or non-conservative substitution) of one or more amino acid residues at one or more positions.

[0719] For example, naturally occurring residues are divided into groups based on common side chain properties. Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (Groups 1-6) for another amino acid from the same group. Non-conservative substitutions involve exchanging a member of one of these groups for a member of another group. (Group 1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile) (Group 2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), (Group 3) Acidic: Aspartic acid (Asp), glutamic acid (Glu) (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg) (Group 5) residues that affect chain orientation: glycine (Gly), proline (Pro); and (Group 6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0720] Other exemplary embodiments of conservative substitutions are shown in Table 2 (Table 2) under the heading of "preferred substitutions." If such substitutions result in undesirable properties, more substantial changes such as those designated "exemplary substitutions" in Table 2 (Table 2), or as further described below with respect to amino acid classes, may be introduced and the products screened.

[0721] One of ordinary skill in the art will recognize that certain amino acids are more positively charged, neutral, negatively charged, or have a reduced charge compared to other amino acids. Amino acids can be categorized based on their net charge as indicated by the isoelectric point of the amino acid. The isoelectric point is the pH at which the average net charge of the amino acid molecule is zero. When pH > pI, the amino acid has a net negative charge, and when pH < pI, the amino acid has a net positive charge. In some embodiments, the pI value measured for an antibody is from about 3 to 9 (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, and 9) and any value in between. In some embodiments, the pI value measured for an antibody is between about 4 and 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), and any value in between. Exemplary isoelectric points of amino acids are shown in Table 2 below (Table 2). Generally, amino acids having a positively charged side chain include, for example, arginine (R), histidine (H), and lysine (K). Amino acids having a negatively charged side chain include, for example, aspartic acid (D) and glutamic acid (E). Amino acids having polar characteristics include, for example, serine (S), threonine (T), asparagine (N), glutamine (Q), and cysteine (C), tyrosine (Y) and tryptophan (W). Nonpolar amino acids include, for example, alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), glycine (G) and proline (P).

[0722] In some embodiments, the isoelectric point of an antibody is modified via amino acid substitution. See, e.g., U.S. Patent Application Publication No. 20110076275. In some embodiments, modification of the isoelectric point of the polypeptide constituting the antibody results in a change in the half-life of the antibody.

[0723] [Table 2]

[0724] In general, the degree of similarity and identity between variable chains is determined herein using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) using default settings, i.e., the blastp program, the BLOSUM62 matrix (open gap penalty of 11 and extension gap penalty of 1; gap dropoff of 50, expectation value of 10.0, word size of 3) and activated filters.

[0725] The percent identity thus provides an indication of amino acids that may occupy identical, the same or similar positions in comparison to the original peptide.

[0726] The percent similarity is a measure of identical amino acids and amino acids that have been replaced with conservative amino acid substitutions in comparison to the original peptide at the same or similar positions.

[0727] A variant of the present disclosure may therefore comprise a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to an original or reference sequence or portion of an original sequence.

[0728] In some embodiments, polypeptide chains having amino acid sequences that are at least 75%, 80%, 85%, 90%, 95%, 99% identical, or less than 100% identical to a given amino acid sequence may have amino acid substitutions, additions, or deletions that are generally located outside the complementarity determining regions.

[0729] In some embodiments, variants may have at least 80% sequence identity with the sequences disclosed herein. In other embodiments, variants may have at least 85% sequence identity with the sequences disclosed herein. In further embodiments, variants may have at least 90% sequence identity with the sequences disclosed herein. In further embodiments, variants may have at least 95% sequence identity with the sequences disclosed herein. In other embodiments, variants may have at least 99% sequence identity with the sequences disclosed herein.

[0730] Exemplary embodiments of variants include polypeptide chains or binding agents comprising hinge, Fc, CH3, CH2 / CH3 regions derived from a native antibody but containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid differences (including amino acid substitutions, insertions or deletions).

[0731] In some embodiments, the polypeptide chains of the disclosure may therefore comprise a variant hinge region that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the hinge region of a native antibody.

[0732] In some embodiments, the polypeptide chains of the disclosure may therefore comprise a variant Fc portion that is at least 80% identical to the Fc of a native antibody.

[0733] In some embodiments, the polypeptide chains of the disclosure may therefore comprise a variant CH2 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the CH2 domain of a native antibody.

[0734] In some embodiments, the polypeptide chains of the disclosure may therefore comprise a variant CH3 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the CH3 domain of a native antibody.

[0735] In some embodiments, the polypeptide chains of the disclosure may therefore comprise a variant CH2 / CH3 domain that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the CH2 / CH3 domain of a native antibody.

[0736] Competitive Binders Binding agents of the present disclosure also include those that compete with binding agents in Table 6 or Table 7, or with binding agents having CDRs or variable domains (VHH) shown in any one of Table 3, Table 4, or Table 5.

[0737] A binding agent of the disclosure may comprise one or more antigen-binding domains of a single domain antibody that competes with a binding agent of Table 6 or Table 7, or with a single domain antibody having CDRs or variable domains (VHHs) shown in any one of Tables 3, 4 or 5.

[0738] In exemplary embodiments, competition between binders (e.g., between single domain antibodies) may be determined based on any type of binding assay (FACS, ELISA, SPR, etc.) using the DR2 antigen or cells expressing the DR2 antigen or D2 proteoliposomes (expressing the D2 antigen).

[0739] In exemplary embodiments, competition between binding agents (e.g., between single domain antibodies) may be determined based on any type of binding assay (FACS, ELISA, SPR, etc.) using PD-1 antigen or cells expressing PD-1 antigen.

[0740] In exemplary embodiments, competition between binding agents (e.g., between single domain antibodies) may be determined based on any type of binding assay (FACS, ELISA, SPR, etc.) using the CD47 antigen or cells expressing the CD47 antigen.

[0741] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single domain antibody (a competitive single domain antibody) that has the ability to compete with a single domain antibody comprising antigen-binding domain 1 (ABD1) as described herein.

[0742] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single domain antibody (a competitive single domain antibody) that has the ability to compete with a single domain antibody comprising antigen-binding domain 2 (ABD2) as described herein.

[0743] In some embodiments, the competitive binder comprises the CDRs of a single domain antibody.

[0744] In some embodiments, the competitive binder comprises a variable region of a single domain antibody.

[0745] In some embodiments, the competitive binding agent is a single domain antibody or antigen-binding fragment thereof.

[0746] In some embodiments, the competitive binding agent is an antibody or an antigen-binding fragment thereof.

[0747] In some embodiments, the binding agent comprises one or more antigen-binding domains of a single domain antibody (a competitive single domain antibody) that has the ability to compete with a single domain antibody comprising antigen-binding domain 3 (ABD3) as described herein.

[0748] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single domain antibody comprises the CDRH1, CDRH2 and / or CDRH3 amino acid sequence of an anti-DR2 single domain antibody in Table 3.

[0749] In some embodiments, at least one of the one or more antigen-binding domains of a competitive single domain antibody comprises the CDRH1, CDRH2, and / or CDRH3 amino acid sequence of an anti-PD-1 single domain antibody in Table 4.

[0750] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single domain antibody comprises the CDRH1, CDRH2 and / or CDRH3 amino acid sequence of an anti-CD47 single domain antibody in Table 5.

[0751] In some embodiments, at least one of the one or more antigen-binding domains of the competitive single domain antibody comprises a variable region that is at least 80% identical to a variable region in Table 3.

[0752] In some embodiments, the competitive binding agent has a binding affinity of 10 to the DR2 antigen. -6 K below M D It may have a value.

[0753] In some embodiments, the competitive binding agent has a binding affinity of 10 to the PD-1 antigen. -6 K below M D It may have a value.

[0754] In some embodiments, the competitive binding agent has a binding affinity of 10 to the CD47 antigen. -6 K below M D It may have a value.

[0755] In some embodiments, the method includes administering a competitive binding agent.

[0756] In some embodiments, the competitive binding agent is capable of competing with KC001, KC013, KC020, KC021 and / or KC027 for binding to DR2, PD-1 and / or CD47.

[0757] In some embodiments, the methods comprise administering a competitive binding agent capable of competing with KC001, KC013, KC020, KC021 and / or KC027 for binding to DR2, PD-1 and / or CD47.

[0758] In some embodiments, the methods comprise administering a competitive binding agent capable of competing with KC020 for binding to DR2, PD-1 and / or CD47.

[0759] In some embodiments, the methods comprise administering a competitive binding agent capable of competing with KC020 for binding to DR2, PD-1 and / or CD47.

[0760] Nucleic acids, vectors, kits, cells and methods for producing polypeptide chains The nucleic acid molecules of the present disclosure may be single-stranded or double-stranded. The nucleic acid molecules disclosed herein may comprise deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, or modified ribonucleotides. The nucleic acid molecules of the present disclosure may comprise, for example, DNA.

[0761] DNA segments and vectors encoding one or more modules or entire polypeptide chains are specifically provided.

[0762] The DNA segments and / or vectors may be provided in separate vials and sold as a kit.

[0763] More particularly, the present disclosure relates to nucleic acid molecules encoding an antigen-binding domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 and / or 266.

[0764] In some embodiments, a nucleic acid molecule of the disclosure may encode a binding agent comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 and / or 266.

[0765] In other embodiments, a nucleic acid molecule of the present disclosure may encode a binding agent comprising the amino acid sequence set forth in any one of SEQ ID NOs: 71-75.

[0766] In yet other embodiments, a nucleic acid molecule of the present disclosure may encode a binding agent comprising the amino acid sequence set forth in any one of SEQ ID NOs: 77-83 or 85-86.

[0767] In exemplary embodiments, the nucleic acid molecule may comprise a sequence at least 50% identical to any one of SEQ ID NOs: 151-164 or any one of SEQ ID NOs: 267-279.

[0768] The present disclosure particularly relates to nucleic acid molecules having codon-optimized sequences, which in some embodiments may improve expression of polypeptide chains in mammalian cells (e.g., including but not limited to, human cells).

[0769] In exemplary embodiments, the nucleic acid molecule may comprise a sequence at least 50% identical to any one of SEQ ID NOs: 151-164 or any one of SEQ ID NOs: 267-279, and the sequence is codon optimized.

[0770] The present disclosure also relates to vectors comprising the nucleic acid molecules disclosed herein. In some embodiments, the vectors comprise codon-optimized sequences and express a polypeptide chain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70-75, 77-83, 85-86, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0771] The present disclosure also relates to methods of making multispecific binding agents. The methods involve transforming a cell with an expression vector containing a nucleic acid molecule disclosed herein. In some embodiments, the methods are for making the binding agent by transforming a cell with an expression vector containing a codon-optimized sequence that allows for expression of a polypeptide chain containing an amino acid sequence set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70-75, 77-83, 85-86, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259, and / or 266.

[0772] Due to the inherent degeneracy of the genetic code, DNA sequences encoding the same, substantially the same, or functionally equivalent amino acid sequences can be produced and used. The nucleotide sequences of the present disclosure may be manipulated using methods generally known in the art to alter nucleotide sequences for various purposes, including, but not limited to, modifying the cloning, processing, and / or expression of gene products. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides can be used to manipulate nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis can be used to introduce mutations such as creating new restriction sites, altering glycosylation patterns, shifting codon preferences, and generating splice variants. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

[0773] The polypeptide chains and binding agents disclosed herein may be produced by a variety of methods familiar to those of skill in the art, including by recombinant DNA methods or by in vitro transcription / translation.

[0774] Generally, the polypeptide chains described herein are expressed from nucleic acid sequences inserted into expression vectors, i.e., vectors that contain elements for the transcriptional and translational control of the inserted coding sequence in a particular host. These elements may include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions.

[0775] A variety of expression vector / host cell systems known to those skilled in the art can be used to express the polypeptide chains described herein. When a binding agent is composed of separate polypeptide chains, each such polypeptide chain may be provided by a separate expression vector or a unique expression vector. According to the present disclosure, the two chains of a binding agent may be encoded by a single vector or by separate vectors (vector sets).

[0776] Polypeptides are often expressed in mammalian cells. For long-term production of recombinant proteins, stable expression systems may be used, in which a DNA segment is integrated into the host cell genome with the use of a selectable marker or maintained in an episomal form. Host cell species may be selected for their ability to modulate the expression of inserted sequences or to process the expressed polypeptide in the desired manner. Different host cells (e.g., CHO, HeLa, MDCK, HEK293, and W138) with unique cellular machinery and characteristic mechanisms for post-translational activity are commercially available from the American Type Culture Collection (ATCC) and may be selected to ensure the correct modification and processing of the expressed polypeptide.

[0777] Other types of expression systems can be used, including, for example, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with baculovirus vectors, plant cell systems transformed with viral or bacterial expression vectors, or animal cell systems.

[0778] The present disclosure therefore relates to isolated cells transformed or transfected with a vector, nucleic acid, set of vectors or set of nucleic acids encoding at least one of the polypeptide chains described herein. The present disclosure therefore also relates to isolated cells capable of expressing the polypeptide chains or binding agents disclosed herein.

[0779] The present disclosure also relates to methods of making the binding agents, which may include providing to a cell (e.g., a mammalian cell) and expressing a vector or set of vectors encoding one or more of the polypeptide chains disclosed herein.

[0780] The method may also include purifying the binding agent from the cells or cell debris.

[0781] In some embodiments, the production method allows reaching purity levels (of the dimer) of at least 80%, at least 85%, at least 90%, at least 99%.

[0782] In some embodiments, the binder has a purity level of 80.0% to 99.9%.

[0783] In some embodiments, the binder has a purity level of 95.0% to 99.9%.

[0784] In some embodiments, the binder has a purity level of at least 90.0+ / -5.0%.

[0785] In some embodiments, the binder has a purity level of at least 95.0+ / -5.0%.

[0786] In some embodiments, the purity level of the binder is equal to or greater than 95%.

[0787] In some embodiments, the binder has a purity level of 97.0+ / -1.0%.

[0788] In some embodiments, the purity level of the binder is equal to or greater than 97%.

[0789] In some embodiments, the binder has a purity level of 99.0+ / -1.0%.

[0790] In some embodiments, the purity level of the binder is equal to or greater than 99%.

[0791] In some embodiments, the titer of the binding agent produced by the cells may be 0.1 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 0.5 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 1 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 2 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 3 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 4 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 5 g / L or higher. In some cases, the titer of the binding agent produced by the cells may be 6 g / L or higher. Typically, homodimers are generated by transfection of cells with a vector comprising a nucleic acid sequence encoding one of the polypeptide chains disclosed herein. The collected supernatant may contain homodimers or a mixture of monomers and / or homodimers.

[0792] Typically, heterodimers are generated by cotransfection of cells with at least two vectors (vector sets), each containing a nucleic acid sequence encoding two distinct polypeptide chains. The appropriate ratio of chain A to chain B generally depends on the level of protein expression obtained from each individual plasmid and can vary, for example, from about 1:10 to about 10:1. A DNA ratio of about 1:1 is particularly preferred for some of the constructs disclosed herein.

[0793] Heterodimers can also be made by transfecting cells with a single vector encoding both polypeptide chains, and the collected supernatant may contain heterodimers or a mixture of monomers, heterodimers, and / or homodimers.

[0794] Methods of making polypeptide chains of the disclosure can further include separating or isolating monomers, homodimers, and heterodimers from a mixture containing them. Homodimers or heterodimers can be purified and isolated, for example, by size exclusion chromatography or with the aid of tags or by other methods known to those of skill in the art.

[0795] The method may also include the step of isolating and / or purifying the binding agent from impurities.

[0796] The methods of the present disclosure therefore result in compositions comprising homodimers, heterodimers, or mixtures of monomers, heterodimers, and / or homodimers.

[0797] In some exemplary embodiments, the composition may comprise predominantly homodimers, hi exemplary embodiments, the composition may comprise at least about 80%, at least 85%, at least 90%, at least 99%, or 100% homodimers.

[0798] In other exemplary embodiments, the composition may comprise predominantly heterodimers, hi exemplary embodiments, the composition may comprise at least about 80%, at least 85%, at least 90%, at least 99%, or 100% heterodimers.

[0799] Conjugates The polypeptide chains or binding agents of the present disclosure may be conjugated to, for example, a therapeutic moiety (for therapeutic purposes) or a detectable moiety (i.e., for detection or diagnostic purposes) or a protein that allows for extended half-life, or attached to a nanoparticle. In some cases, the therapeutic or detectable moiety may be linked to at least one amino acid residue of the polypeptide chain.

[0800] In exemplary embodiments, the polypeptide chains or binding agents of the present disclosure are conjugated to a therapeutic moiety, such as, but not limited to, chemotherapeutic agents, cytokines, cytotoxic agents, and anti-cancer drugs (e.g., small molecules).

[0801] Therapeutic moieties include, but are not limited to, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and many others recognized by those skilled in the art (e.g., lutetium (e.g., Lu 177 ), bismuth (e.g., Bi 213 ), copper (e.g., Cu 67 )), 5-fluorouracil, adriamycin, irinotecan, taxanes, Pseudomonas endotoxin, ricin, auristatins (e.g., monomethylauristatin E, monomethylauristatin F), maytansinoids (e.g., mertansine), and other toxins.

[0802] In another exemplary embodiment, the polypeptide chains or binding agents of the present disclosure are conjugated to a detectable moiety, including, without limitation, moieties detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, and / or other physical means. The detectable moiety may be coupled either directly and / or indirectly (e.g., via a linkage, such as, without limitation, a DOTA or NHS linkage) to the polypeptide chain or binding agent using methods well known in the art. A variety of detectable moieties may be used, with the choice depending on the required sensitivity, ease of conjugation, stability requirements, and available equipment. Suitable detectable moieties include fluorescent labels, radioactive labels (e.g., without limitation, 125 I, In 111 , Tc 99 , I 131 and positron emitting isotopes for PET scanners, etc.), nuclear magnetic resonance active labels, luminescent labels, chemiluminescent labels, chromophore labels, enzyme labels (for example, without limitation, horseradish peroxidase, alkaline phosphatase, etc.), quantum dots, and / or nanoparticles. The detectable moiety may initiate and / or generate a detectable signal, thereby allowing the signal from the detectable moiety to be detected.

[0803] Pharmaceutical Composition Pharmaceutical compositions comprising the polypeptide chains or binding agents of the present disclosure are also encompassed by the present disclosure. The pharmaceutical composition may also include a pharmaceutically acceptable carrier.

[0804] In some embodiments, the pharmaceutical composition comprises a conjugated binding agent as disclosed herein. In some embodiments, the pharmaceutical composition comprises a binding agent conjugated to a therapeutic moiety. In some embodiments, the pharmaceutical composition comprises a binding agent conjugated to a detectable label.

[0805] In addition to the active ingredient, the pharmaceutical composition may contain pharmaceutically acceptable carriers including water, PBS, saline, gelatin, oil, alcohol, and other excipients and auxiliaries that facilitate processing of the active compound into a pharmaceutically usable preparation. In other cases, such preparations may be sterilized.

[0806] As used herein, "pharmaceutical composition" refers to a therapeutically effective amount of an agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers. A "therapeutically effective amount," as used herein, refers to an amount that provides a therapeutic effect for a given condition and administration regimen. Such compositions may be liquid or lyophilized or otherwise dried formulations, and may contain various buffer components (e.g., Tris-HCl, acetate, phosphate), diluents for pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, surfactants (e.g., Tween 20, Tween 80, Pluronic F68, bile salts), solubilizers (e.g., glycerol, polyethyleneglycerol), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), bulking agents or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers, e.g., polyethylene glycol, to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations such as polymeric compounds, e.g., polylactic acid, polyglycolic acid, hydrogels, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Particulate compositions coated with polymers (e.g., poloxamers or poloxamines) are also encompassed by the present disclosure. Other embodiments of the compositions of the present disclosure incorporate particulate protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, oral, vaginal, and rectal routes. In one embodiment, the pharmaceutical composition is administered parenterally, pericancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, and intratumorally.

[0807] Furthermore, as used herein, "pharmaceutically acceptable carrier" or "pharmaceutical carrier" is known in the art and includes, but is not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0808] For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or animal models, such as mice, rats, rabbits, dogs, or pigs. Animal models can also be used to determine concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes for administration in humans. These techniques are well known to those skilled in the art, and a therapeutically effective dose refers to that amount of active ingredient that ameliorates symptoms or conditions. Therapeutic efficacy and toxicity can be assessed by standard pharmaceutical procedures in cell cultures or using experimental animals, e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 The dose at which the patient is vaccinated may be determined by calculating and comparing statistics of the dose at which the patient is vaccinated (the dose that is lethal to 50% of the population). Any of the above pharmaceutical compositions may be applied to any subject in need of therapy, including, but not limited to, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and especially humans.

[0809] The pharmaceutical compositions described herein may be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0810] In some embodiments, the pharmaceutical composition is made from a binder having a purity level of 80.0% to 99.9%. Thus, in some embodiments, the pharmaceutical composition comprises a binder that is substantially free of impurities. For example, the pharmaceutical composition comprises a binder with a purity of at least 90.0 + / - 5.0%. In other examples, the pharmaceutical composition comprises a binder with a purity of at least 95.0 + / - 5.0%. In other embodiments, the pharmaceutical composition comprises a binder with a purity of at least 99.0 + / - 1.0%.

[0811] In some embodiments, the pharmaceutical composition comprises a binding agent that is stable in solution under one or more stress conditions as described herein.

[0812] In some embodiments, the pharmaceutical composition is formulated for intravenous administration, hi other embodiments, the pharmaceutical composition is formulated for intraperitoneal administration.

[0813] In some embodiments, the pharmaceutical composition is formulated at a concentration suitable for administration to a human at a dose of 0.01 to 150 mg / kg, 0.01 to 100 mg / kg, 0.01 to 50 mg / kg, 0.05 to 150 mg / kg, 0.05 to 100 mg / kg, 0.05 to 50 mg / kg, 0.05 to 30 mg / kg, 0.05 to 10 mg / kg, 0.1 to 50 mg / kg, 0.1 to 30 mg / kg, 0.1 to 10 mg / kg, or 1.0 to 10 mg / kg.

[0814] How to use The binding agents of the present disclosure may be used for the treatment of a disorder or disease.

[0815] The methods of the present disclosure may be used, inter alia, to treat or delay the progression of cancer in a subject in need thereof.

[0816] In one embodiment, the methods of the disclosure comprise administering a pharmaceutical composition comprising a means for reducing tumor size and a pharmaceutically acceptable carrier, wherein the means for reducing tumor size comprises a means for binding to dopamine receptor D2 (DR2) and / or a means for blocking the interaction between PD-1 and PD-L1 and / or a means for blocking the interaction between CD47 and SIRPα carrier.

[0817] In one embodiment, the methods of the disclosure comprise administering a pharmaceutical composition comprising a means for inhibiting tumor growth and a pharmaceutically acceptable carrier, wherein the means for inhibiting tumor growth comprises a means for binding to dopamine receptor D2 (DR2) and / or a means for blocking the interaction between PD-1 and PD-L1 and / or a means for blocking the interaction between CD47 and SIRPα carrier.

[0818] In one embodiment, the methods of the disclosure comprise administering a pharmaceutical composition comprising a means for inducing tumor regression and a pharmaceutically acceptable carrier, wherein the means for inducing tumor regression comprises a means for binding to dopamine receptor D2 (DR2) and / or a means for blocking the interaction between PD-1 and PD-L1 and / or a means for blocking the interaction between CD47 and SIRPα carrier.

[0819] Means for reducing tumor size, inhibiting tumor growth and / or inducing tumor regression may be achieved using a binding agent comprising one or more antigen-binding domains that specifically bind to dopamine receptor D2 (DR2) and / or one or more antigen-binding domains that specifically bind to programmed cell death protein 1 (PD-1) and / or one or more antigen-binding domains that specifically bind to cluster of differentiation 47 (CD47).

[0820] The amino acid sequence of the antigen-binding domain may correspond to that of an antibody or antigen-binding fragment thereof (including a single domain antibody or antigen-binding fragment thereof) that binds to the desired antigen and / or inhibits the interaction between the antigen and one of its ligands.

[0821] The amino acid sequence of the antigen-binding domain may be identical to the amino acid sequence of an original antibody isolated from a non-human animal (mouse, rat, rabbit, camel, etc.) capable of expressing human, humanized, or camelized antibodies, such as a transgenic animal. The amino acid sequence of the antigen-binding domain may also be modified by design to make it more human- or animal-like. Thus, the amino acid sequence of the antigen-binding domain may comprise the amino acid sequence of a non-humanized, human, humanized, or camel antibody.

[0822] In some embodiments, the amino acid sequence of one or more antigen-binding domains of the binding agent is in a single-chain format.

[0823] In some embodiments, the antigen-binding domain may be derived from a single domain antibody or antigen-binding fragment thereof capable of binding to D2 and inducing downstream signaling. In some embodiments, the antigen-binding domain may be derived from a single domain antibody or antigen-binding fragment thereof capable of binding to D2 and blocking downstream signaling. In some embodiments, the antigen-binding domain may be derived from a single domain antibody or antigen-binding fragment thereof capable of downregulating the expression of one or more cancer-associated pathways. D2 downstream signaling may be assessed, for example, but not limited to, using a GPCR BRET assay and / or a gene expression assay as described herein.

[0824] In some embodiments, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that has the ability to block the interaction between PD-1 and PD-L1. Thus, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that binds to PD-1. Alternatively, therefore, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that binds to PD-L1. PD-1 / PD-L1 blocking assays may be performed using a reporter system, such as, but not limited to, the luminescent reporter systems described herein.

[0825] In some embodiments, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that has the ability to block the interaction of CD47 with SIRPβ. Thus, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that binds to CD47. Alternatively, therefore, the antigen-binding domain may be derived from a single-domain antibody or antigen-binding fragment thereof that binds to SIRPα. The CD47 / SIRPα blocking assay may be performed using a reporter system, such as, but not limited to, the luminescence reporter system described herein.

[0826] Conventional binding assays include, but are not limited to, ELISA, flow cytometry, surface plasmon resonance, electrochemiluminescence, mesoscale detection, radioimmunoassay, fluorescence immunoassay, LC-MS detection, thermal shift assay, biolayer interferometry, etc. Binding assays may be performed between recombinant proteins (or moieties), using cell lines expressing the protein on their surface, or using liposomes or nanoparticles containing the protein. Generally, binding of a binding agent to its target results in the release or decrease of a detectable signal.

[0827] Binding may be assessed by competition assays, in which candidate binding agents can be identified that can inhibit the interaction of a previously identified binding agent (with desired binding and / or activity) with its target (DR2, PD-1, or CD47). Alternatively, binding may be assessed in silico using the three-dimensional structure of the antibody / antigen complex (Zhao J. et al., Antibodies 2018, 7, 22; doi 10.3390 / antib7030022).

[0828] The methods of the present disclosure include administering a binding agent disclosed herein to a subject in need thereof.

[0829] In some embodiments, a subject in need thereof has an established tumor.

[0830] In some embodiments, a subject in need thereof has failed prior treatment comprising an anti-PD-1 antibody or an anti-PD-L1 antibody. For example, the binding agent may be administered to a subject who has failed prior treatment with either pembrolizumab alone or in combination with chemotherapy.

[0831] In some embodiments, the binding agent is multispecific and comprises at least one antigen-binding domain capable of binding to dopamine receptor D2 (DR2), at least one antigen-binding domain capable of binding to programmed cell death protein 1 (PD-1), and at least one antigen-binding domain capable of binding to cluster of differentiation 47 (CD47).

[0832] In some embodiments, the subject in need thereof has a solid tumor.

[0833] In another embodiment, the subject in need thereof has a neuroendocrine solid tumor.

[0834] In a further embodiment, the subject in need thereof has cancer.

[0835] In still a further embodiment, a subject in need thereof has metastatic cancer.

[0836] In additional embodiments, the subject in need thereof has advanced cancer.

[0837] In some embodiments, the cancer comprises tumor cells that express dopamine receptor D2 (DR2).

[0838] In exemplary embodiments, the cancer is a neuroendocrine cancer, neuroblastoma, gastric cancer, blood cancer, lung cancer, myeloma, prostate cancer, ovarian cancer, breast cancer, rectal cancer, pancreatic cancer, or glioblastoma.

[0839] In some embodiments, the lung cancer is small cell lung cancer.

[0840] In other embodiments, the lung cancer is non-small cell lung cancer.

[0841] In some embodiments, the breast cancer is triple-negative breast cancer.

[0842] In some embodiments, the rectal cancer is colorectal cancer.

[0843] In exemplary embodiments, a subject in need thereof is treated with a binding agent disclosed herein. In some embodiments, the binding agent may be conjugated to a therapeutic moiety and used for therapeutic methods.

[0844] In exemplary embodiments, a subject in need thereof is treated with a multispecific binding agent disclosed herein. In some embodiments, the multispecific binding agent may be conjugated to a therapeutic moiety and used for therapeutic methods.

[0845] According to the present disclosure, the method comprises administering to a subject in need thereof a multispecific binding agent comprising at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2), at least one antigen-binding domain that binds to immunomodulatory factor 2 (ABD2), and at least one antigen-binding domain 3 (ABD3) that binds to an immune cell.

[0846] More specifically, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein ABD1 is an antigen-binding domain that binds to dopamine receptor D2 (DR2), ABD2 is an antigen-binding domain that binds to programmed cell death protein 1 (PD-1), and ABD3 is an antigen-binding domain that binds to cluster of differentiation 47 (CD47).

[0847] In some embodiments, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3); ABD1 is a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 3; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO:39, CDRH2 having the amino acid sequence set forth in SEQ ID NO:40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:41; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55; q. the amino acid sequence set forth in SEQ ID NO: 280; r. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7; s. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14; t. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21; u. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28; v. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35; w. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42; x. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49; or y. An amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 56 Including, ABD2 is a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 57, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 58, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 59; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 176, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 177, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 178; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 179, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 180, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 181; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 183, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 184, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 185; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 186, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 187, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 188; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 190, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 191, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 192; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 193, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 194, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 195; i. a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 197, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 199; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 200, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 201, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 202; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 204, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 206; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 207, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 209; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 211, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 213; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 214, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 215, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 216; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 218, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 219, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 220; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 221, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 222, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 223; q. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 225, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 226, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 227; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 249, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 250, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 251; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 253, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 254, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 255; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 256, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 257, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 258; aa. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 260, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 261, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 262; bb. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 263, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 264, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 265; cc. the amino acid sequence set forth in SEQ ID NO: 281; dd. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; ee. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 182; ff. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 189; gg. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 196; hh. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 203; ii. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 210; jj. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 217; kk. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 224; ll. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 231; mm. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 238; nn. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 245; oo. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 252; an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth on page SEQ ID NO: 259; or qq. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 266 Including, ABD3 is a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or c. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 Includes.

[0848] In an exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7. or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0849] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14. and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0850] In yet another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, to the amino acid sequence set forth in SEQ ID NO:21. They comprise identical or at least 99% identical amino acid sequences, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0851] In a further exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, to the amino acid sequence set forth in SEQ ID NO:28. They comprise identical or at least 99% identical amino acid sequences, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0852] In an exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 35. or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0853] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:42. and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0854] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:49. and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0855] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 56. and antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70, wherein antigen-binding domain 2 (ABD2) comprises an amino acid sequence that is at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0856] In some embodiments, the multispecific binding agent may comprise 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acid substitutions or 1 amino acid substitution or addition in any of the ABD1, ABD2 and / or ABD3 units.

[0857] In other embodiments, the multispecific binding agent may comprise 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acid deletions, or 1 amino acid deletion in any of the ABD1, ABD2 and / or ABD3 units.

[0858] In still other embodiments, the multispecific binding agent may comprise 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 amino acid additions, or 1 amino acid addition in any of the ABD1, ABD2 and / or ABD3 units.

[0859] In an exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:7.

[0860] In another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:14.

[0861] In a further exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:21.

[0862] In another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:28.

[0863] In yet another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:35.

[0864] In another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:42.

[0865] In another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:49.

[0866] In yet another exemplary embodiment, antigen-binding domain 1 (ABD1) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:56.

[0867] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:63.

[0868] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:182.

[0869] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:189.

[0870] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:196.

[0871] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:203.

[0872] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:210.

[0873] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:217.

[0874] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:224.

[0875] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:231.

[0876] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:238.

[0877] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:245.

[0878] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:252.

[0879] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:259.

[0880] In an exemplary embodiment, antigen-binding domain 2 (ABD2) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO:266.

[0881] In an exemplary embodiment, antigen-binding domain 3 (ABD3) may comprise an amino acid sequence having 1 to 10 amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 70. The amino acid substitutions, deletions and / or additions may or may not be consecutive.

[0882] In some embodiments, the amino acid substitutions, deletions, and / or additions may be outside of one or more of the CDRs.

[0883] In some embodiments, the amino acid substitutions, deletions and / or additions may be outside of CDR3.

[0884] In yet another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, or SEQ ID NO:56; antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO:63, SEQ ID NO:182, SEQ ID NO:189, SEQ ID NO:196, SEQ ID NO:203, SEQ ID NO:210, SEQ ID NO:217, SEQ ID NO:224, SEQ ID NO:231, SEQ ID NO:238, SEQ ID NO:245, SEQ ID NO:252, SEQ ID NO:259, or SEQ ID NO:266; and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO:70.

[0885] In yet another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 7, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[0886] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 14, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[0887] In an exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:21, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO:63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO:70.

[0888] In a further exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:28, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO:63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO:70.

[0889] In still a further exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 35, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[0890] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO: 42, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO: 70.

[0891] In another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:49, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO:63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO:70.

[0892] In yet another exemplary embodiment, the method comprises administering to a subject in need thereof a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3), wherein antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:56, antigen-binding domain 2 (ABD2) comprises the amino acid sequence set forth in SEQ ID NO:63, and antigen-binding domain 3 (ABD3) comprises the amino acid sequence set forth in SEQ ID NO:70.

[0893] In some embodiments, in binding agents comprising ABD3 (such as those comprising ABD1, ABD2 and ABD3) and methods or uses thereof, the amino acid sequence set forth in SEQ ID NO: 63 may be replaced with the amino acid sequence set forth in SEQ ID NO: 182, SEQ ID NO: 189, SEQ ID NO: 196, SEQ ID NO: 203, SEQ ID NO: 210, SEQ ID NO: 217, SEQ ID NO: 224, SEQ ID NO: 231, SEQ ID NO: 238, SEQ ID NO: 245, SEQ ID NO: 252, SEQ ID NO: 259, or SEQ ID NO: 266.

[0894] In some embodiments, the multispecific binding agents of the present disclosure may be used to target tumors in vivo.

[0895] In some embodiments, the polypeptide chains and multispecific binding agents may be used to promote or induce tumor regression and / or reduce tumor size and / or volume in vivo.

[0896] Regression may be assessed by measuring the size and / or volume of the tumor or tumor lesion, which is generally determined before treatment and monitored at least once during or after the treatment period.

[0897] A decrease in tumor size and / or volume compared to the initial tumor size and / or volume may be indicative of tumor regression.

[0898] In some embodiments, a decrease in tumor size and / or volume need only occur at least once (e.g., at a single time point) during a treatment or monitoring period to be considered tumor regression. For example, a decrease in tumor size and / or volume followed by an increase in tumor size or volume is considered tumor regression for purposes of the present disclosure.

[0899] In some embodiments, a reduction in tumor size and / or volume is observed at several time points (continuous or non-continuous) during the treatment or monitoring period.

[0900] A reduction in tumor size and / or volume of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at le...

Claims

1. 1. A method of treating a subject having cancer, comprising administering a multispecific binding agent comprising at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2), at least one antigen-binding domain 2 (ABD2) that binds to programmed cell death protein 1 (PD-1), and / or at least one antigen-binding domain 3 (ABD3) that binds to cluster of differentiation 47 (CD47), resulting in a reduction in tumor size and / or volume.

2. 10. The method of claim 1, wherein the size and / or volume of the tumor is reduced by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% compared to the initial size and / or volume of the tumor.

3. 3. The method of claim 1 or 2, wherein the cancer is neuroendocrine cancer, neuroblastoma, gastric cancer, blood cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, myeloma, prostate cancer, ovarian cancer, breast cancer, triple-negative breast cancer, rectal cancer, colorectal cancer, pancreatic cancer, or glioblastoma.

4. 1. A method of treating triple-negative breast cancer in a subject in need thereof, comprising administering a multispecific binding agent comprising at least one antigen-binding domain 1 (ABD1) that binds to dopamine receptor D2 (DR2) and at least one antigen-binding domain 2 (ABD2) that binds to programmed cell death protein 1 (PD-1) and / or at least one antigen-binding domain 3 (ABD3) that binds to cluster of differentiation 47 (CD47).

5. 5. The method of any one of claims 1 to 4, wherein the subject has metastatic or advanced cancer.

6. 6. The method of any one of claims 1 to 5, wherein the multispecific binding agent is administered once a week, once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, or once every six months.

7. 7. The method of any one of claims 1 to 6, wherein the multispecific binding agent is administered at a dose of about 1 mg / kg to about 50 mg / kg.

8. 8. The method of any one of claims 1 to 7, comprising administering more than eight doses of the multispecific binding agent.

9. 9. The method of any one of claims 1 to 8, comprising administering the multispecific binding agent until the size and / or volume of one or more tumors or tumor lesions is reduced.

10. 10. The method of any one of claims 1 to 9, comprising administering the multispecific binding agent until one or more tumors or tumor lesions show signs of regression.

11. 11. The method of any one of claims 1 to 10, wherein the administration results in a reduction in the size and / or volume of one or more tumors or tumor lesions.

12. 12. The method of any one of claims 1 to 11, wherein said administration results in regression of one or more tumors or tumor lesions.

13. 13. The method of any one of claims 1 to 12, wherein the multispecific binding agent comprises two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

14. ABD1, a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 3; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO:25, CDRH2 having the amino acid sequence set forth in SEQ ID NO:26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:27; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 39, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 41; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55; q. the amino acid sequence set forth in SEQ ID NO: 280; r. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7; s. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14; t. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21; u. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28; v. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35; w. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42; x. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49; or y. An amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 56 14. The method of any one of claims 1 to 13, comprising:

15. ABD2, a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 57, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 58, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 59; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 176, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 177, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 178; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 179, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 180, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 181; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 183, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 184, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 185; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 186, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 187, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 188; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 190, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 191, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 192; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 193, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 194, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 195; i. a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 197, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 198, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 199; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 200, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 201, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 202; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 204, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 205, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 206; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 207, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 209; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 211, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 213; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 214, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 215, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 216; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 218, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 219, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 220; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 221, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 222, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 223; q. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 225, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 226, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 227; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 249, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 250, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 251; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 253, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 254, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 255; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 256, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 257, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 258; aa. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 260, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 261, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 262; bb. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 263, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 264, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 265; cc. the amino acid sequence set forth in SEQ ID NO: 281; dd. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; ee. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 182; ff. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 189; gg. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 196; hh. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 203; ii. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 210; jj. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 217; kk. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 224; ll. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 231; mm. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 238; nn. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 245; oo. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 252; an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth on page SEQ ID NO: 259; or qq. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 266 15. The method of any one of claims 1 to 14, comprising:

16. ABD3, a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or c. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 16. The method of any one of claims 1 to 15, comprising:

17. the multispecific binding agent a. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 7, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; b. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; c. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; d. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; e. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; f. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; g. antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 49, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; or h. Antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

70.

17. The method of any one of claims 1 to 16, comprising:

18. the multispecific binding agent a. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 7, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; b. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 14, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; c. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 21, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; d. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 28, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; e. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 35, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; f. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 42, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; g. antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 49, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70; or h. Antigen-binding domain 1 (ABD1) comprising the amino acid sequence set forth in SEQ ID NO: 56, antigen-binding domain 2 (ABD2) comprising the amino acid sequence set forth in SEQ ID NO: 63, and antigen-binding domain 3 (ABD3) comprising the amino acid sequence set forth in SEQ ID NO: 70 18. The method of any one of claims 1 to 17, comprising:

19. 19. The method of any one of claims 13 to 18, wherein the polypeptide chains form dimers via a dimerization domain comprising a CH3 domain and optionally a CH2 domain and / or a hinge region.

20. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 one of which represents antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b1 , L c1 , L c2 each independently comprises one or more linkers; and DD represents the dimerization domain; 20. The method of any one of claims 13 to 19.

21. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 represents antigen-binding domain 1 (ABD1), and Ab d1 represents antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b1 , L c1 , L c2 each independently comprises one or more linkers; and DD represents the dimerization domain; 20. The method of any one of claims 13 to 19.

22. 22. The method of any one of claims 19 to 21, wherein the dimerization domain is the CH2-CH3 domain of a natural human antibody heavy chain.

23. 23. The method of any one of claims 19 to 22, wherein the two polypeptide chains are identical and assemble to form a homodimer.

24. 23. The method of any one of claims 19 to 22, wherein the two polypeptide chains are different and assemble to form a heterodimer.

25. the multispecific binding agent comprises two identical polypeptide chains, each of the polypeptide chains comprising: a. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 77; b. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 78; c. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 79; d. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 80; e. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 81; f. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 82; g. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 83; h. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 85; or i. an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 86 25. The method of any one of claims 1 to 24, comprising:

26. 26. The method of any one of claims 1 to 25, wherein the multispecific binding agent is humanized.

27. 27. The method of any one of claims 13 to 26, wherein the polypeptide chain comprises amino acid substitutions, additions or deletions outside the complementarity determining regions.

28. the multispecific binding agent a. the amino acid sequence set forth in SEQ ID NO: 77; b. The amino acid sequence set forth in SEQ ID NO: 78; c. The amino acid sequence set forth in SEQ ID NO: 79; d. the amino acid sequence set forth in SEQ ID NO: 80; e. the amino acid sequence set forth in SEQ ID NO: 81; f. the amino acid sequence set forth in SEQ ID NO: 82; g. The amino acid sequence set forth in SEQ ID NO: 83; h. the amino acid sequence set forth in SEQ ID NO: 85; or i. The amino acid sequence shown in SEQ ID NO: 86 28. The method of any one of claims 1 to 27, comprising two polypeptide chains each comprising:

29. 29. The method of any one of claims 1 to 28, wherein the multispecific binding agent or the antigen-binding domain of the multispecific binding agent specifically binds to human DR2.

30. 30. The method of any one of claims 1 to 29, wherein the multispecific binding agent or an antigen-binding domain of the multispecific binding agent specifically binds to human PD-1.

31. 31. The method of any one of claims 1 to 30, wherein the multispecific binding agent or the antigen-binding domain of the multispecific binding agent specifically binds to human CD47.

32. 32. The method of any one of claims 1 to 31, wherein the multispecific binding agent is conjugated to a therapeutic moiety, a detectable moiety, or a protein allowing for extended half-life, or is attached to a nanoparticle.

33. a binding agent comprising one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2); a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 176, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 177, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 178; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 179, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 180, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 181; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 183, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 184, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 185; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 186, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 187, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 188; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 190, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 191, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 192; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 193, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 194, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 195; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 197, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 198, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 199; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 200, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 201, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 202; i. a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 204, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 205, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 206; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 207, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 208, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 209; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 211, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 212, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 213; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 214, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 215, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 216; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 218, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 219, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 220; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 221, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 222, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 223; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 225, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 226, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 227; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 228, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 229, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 230; q. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 232, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 233, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 234; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 235, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 236, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 237; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 239, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 240, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 241; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 242, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 243, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 244; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 246, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 247, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 248; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 249, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 250, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 251; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 253, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 254, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 255; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 256, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 257, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 258; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 260, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 261, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 262; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 263, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 264, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 265; aa. the amino acid sequence set forth in SEQ ID NO: 281; bb. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 182; cc. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 189; dd. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 196; ee. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 203; ff. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 210; gg. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 217; hh. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 224; ii. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 231; jj. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 238; kk. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 245; ll. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 252; mm. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 259; or nn. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 266 A binder comprising:

34. a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 3; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO:39, CDRH2 having the amino acid sequence set forth in SEQ ID NO:40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:41; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55; q. the amino acid sequence set forth in SEQ ID NO: 280; r. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7; s. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14; t. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21; u. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28; v. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35; w. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42; x. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49; or y. An amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 56 34. The binding agent of claim 33, further comprising one or more antigen-binding domains 1 (ABD1) comprising:

35. a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or c. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 38. The binding agent of claim 36 or 37, further comprising one or more antigen-binding domains 3 (ABD3) comprising:

36. 36. The binding agent of any one of claims 33 to 35, wherein the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, each polypeptide chain comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2) and at least one antigen-binding domain 3 (ABD3).

37. 37. The binding agent of claim 36, wherein the polypeptide chains form dimers via a dimerization domain comprising a CH3 domain and optionally a CH2 domain and / or a hinge region.

38. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 one of which represents antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b1 , L c1 , L c2 each independently comprises one or more linkers; and DD represents the dimerization domain; 38. The binder of claim 36 or 37.

39. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 represents antigen-binding domain 1 (ABD1), and Ab d1 represents antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b1 , L c1 , L c2 each independently comprises one or more linkers; and DD represents the dimerization domain; 38. The binder of claim 36 or 37.

40. 40. The binding agent of any one of claims 36 to 39, wherein the dimerization domain is the CH2-CH3 domain of a native human antibody heavy chain.

41. 41. The binding agent of any one of claims 36 to 40, wherein the two polypeptide chains are identical and assemble to form a homodimer.

42. 41. The binding agent of any one of claims 36 to 40, wherein the two polypeptide chains are identical and assemble to form a heterodimer.

43. 43. A pharmaceutical composition comprising the binding agent of any one of claims 36 to 42.

44. A nucleic acid molecule encoding an antigen-binding domain comprising the amino acid sequence set forth in any one of SEQ ID NOs: 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 182, 189, 196, 203, 210, 217, 224, 231, 238, 245, 252, 259 or 266, wherein the nucleic acid sequence is at least 50% identical to the nucleic acid sequence set forth in any one of SEQ ID NOs: 141-150 or 267-279.

45. 45. The nucleic acid molecule of claim 44, comprising a nucleic acid sequence that is at least 50% identical to any one of SEQ ID NOs: 151 to 164.

46. 46. ​​The nucleic acid molecule of claim 44 or 45, wherein the nucleic acid sequence encodes a binding agent having an amino acid sequence as set forth in any one of SEQ ID NOs: 71 to 75.

47. 46. ​​The nucleic acid molecule of claim 44 or 45, wherein the nucleic acid sequence encodes a binding agent having an amino acid sequence as set forth in any one of SEQ ID NOs: 77 to 83 or 85 to 86.

48. 48. The nucleic acid molecule of any one of Claims 44 to 47, wherein the nucleic acid sequence is codon optimized.

49. 49. A vector comprising the nucleic acid molecule of any one of claims 44 to 48.

50. 49. A method of making a multispecific binding agent, the method comprising transforming a cell with an expression vector comprising the nucleic acid molecule of any one of claims 44 to 48.

51. 51. The method of claim 50, comprising isolating and / or purifying the multispecific binding agent.

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