Binding agents targeting tumor and / or immune cells

Abstract

The present disclosure generally relates to binding agents capable of targeting tumor cells and / or immune cells. The binding agents of the present disclosure comprise one or more antigen-binding domains of a heavy chain antibody (HcAb) capable of binding to DR2, PD-1 and / or CD47. The binding agents of the present disclosure may be in monomeric or multimeric form and may be monospecific or multispecific.

Description

[Technical field]

[0001] The present disclosure generally relates to binding agents capable of targeting tumor cells and / or immune cells. The binding agents of the present disclosure comprise one or more antigen-binding domains of a VHH antibody capable of binding to DR2, PD-1 and / or CD47. The binding agents of the present disclosure may be in monomeric or multimeric form and may be monospecific or multispecific. [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, which makes 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, fusion with HSA or other carrier proteins, have been exploited, but such constructs may introduce immunogenicity or may have limited success.

[0003] VHHs have been exploited as building blocks to generate bispecific and multispecific antibodies. 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] A number of VHH-based therapeutics are currently in late-stage clinical trials or approved by the FDA. These include the bivalent monospecific antibody caplacizumab against the antigen vWF, approved for thrombotic thrombocytopenic purpura (Duggan, 2018). A trivalent nanobody complex against RSV, ALX-0171, is in late-stage development for respiratory syncytial virus infection (Detallea et al., 2015). ALX-0061, which is monovalent against the antigen IL-6R but is 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 applicant has developed novel binding agents that target tumor cells and / or immune cells that contain one or more antigen-binding domains. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Publication No. 2019 / 0010242 [Patent Document 2] U.S. Patent Application Publication No. 20110076275

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[0007]

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[0008] The present disclosure relates generally to binding agents that have the ability to target, inter alia, tumor cells and / or immune cells.

[0009] In some embodiments, the binding agents of the disclosure have the ability to bind to DR2, PD-1 and / or CD47. In other embodiments of the disclosure, the binding agents may bind to cells expressing DR2, cells expressing PD-1 and / or cells expressing CD47.

[0010] In some embodiments, the cells expressing DR2 comprise tumor cells.

[0011] In some embodiments, the cells that express PD-1 comprise immune cells.

[0012] In some embodiments, the cell expressing CD47 comprises an immune cell or a tumor cell.

[0013] Thus, in some embodiments, the binding agent 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. In accordance with the present disclosure, the binding agent may also comprise additional antigen-binding domains.

[0014] In some embodiments, the antigen binding domain capable of binding to DR2 or a cell expressing DR2 is antigen binding domain 1 (ABD1) as described herein. In some embodiments, the antigen binding domain capable of binding to DR2 or a cell expressing DR2 is not limited to antigen binding domain 1 (ABD1).

[0015] Thus, in some embodiments, the binding agent may comprise at least one antigen-binding domain 1 (ABD1).

[0016] In some embodiments, a binding agent may comprise at least one antigen binding domain 1 (ABD1) and at least one other antigen binding domain.

[0017] In some cases, the binding agent may comprise at least one antigen binding domain 1 (ABD1) and may also bind to an immune cell. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain that binds to an immune cell.

[0018] In some embodiments, the antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1 is antigen-binding domain 2 (ABD2) as described herein. In some embodiments, the antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1 is not limited to antigen-binding domain 2 (ABD2).

[0019] Thus, in some embodiments, the binding agent may comprise at least one antigen-binding domain 2 (ABD2).

[0020] In some embodiments, the binding agent may comprise at least one antigen binding domain 2 (ABD2) and at least one other antigen binding domain.

[0021] In some cases, the binding agent may include at least one antigen binding domain 2 (ABD2) and may also bind to a tumor cell. Thus, in some embodiments, the binding agent includes at least one antigen binding domain 2 (ABD2) and at least one antigen binding domain that binds to a tumor cell.

[0022] In some embodiments, the antigen binding domain capable of binding to CD47 or a cell expressing CD47 is antigen binding domain 3 (ABD3) as described herein. In some embodiments, the antigen binding domain capable of binding to CD47 or a cell expressing CD47 is not limited to ABD3.

[0023] Thus, in some embodiments, the binding agent may comprise at least one antigen-binding domain 3 (ABD3).

[0024] In some embodiments, the binding agent may comprise at least one antigen binding domain 3 (ABD3) and at least one other antigen binding domain.

[0025] In some cases, the binding agent may comprise at least one antigen binding domain 3 (ABD3) and may also bind to tumor cells. Thus, in some embodiments, the binding agent comprises at least one antigen binding domain 3 (ABD3) and at least one antigen binding domain that binds to tumor cells. In other cases, the binding agent may comprise at least one antigen binding domain 3 (ABD3) and may also bind to immune cells. In some embodiments, the binding agent comprises at least one antigen binding domain 3 (ABD3) and at least one antigen binding domain that binds to immune cells.

[0026] In some instances, the binding agent has the ability to bind to DR2 and PD-1. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to DR2 and at least one antigen-binding domain that binds to PD-1.

[0027] In some cases, the binding agent has the ability to bind to DR2 and CD47. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to DR2 and at least one antigen-binding domain that binds to CD47.

[0028] In some instances, the binding agent has the ability to bind to DR2, PD-1, and CD47. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to DR2, at least one antigen-binding domain that binds to PD-1, and at least one antigen-binding domain that binds to CD47.

[0029] In some instances, the binding agent has the ability to bind to PD-1 and CD47. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to PD-1 and at least one antigen-binding domain that binds to CD47.

[0030] In some cases, the binding agent has the ability to bind to PD-1, CD47, and a tumor cell. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to PD-1, at least one antigen-binding domain that binds to CD47, and at least one antigen-binding domain that binds to a tumor cell.

[0031] In some cases, the binding agent has the ability to bind to PD-1 and to a tumor cell. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to PD-1 and at least one antigen-binding domain that binds to a tumor cell.

[0032] In some cases, the binding agent has the ability to bind to CD47 and to a tumor cell. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to CD47 and at least one antigen-binding domain that binds to a tumor cell.

[0033] In some cases, the binding agent has the ability to bind to CD47 and to an immune cell. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to CD47 and at least one antigen-binding domain that binds to an immune cell.

[0034] 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).

[0035] 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 2 (ABD2).

[0036] 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 3 (ABD3).

[0037] In some embodiments, the binding agent comprises one or more antigen-binding domains, and at least one of the antigen-binding domains comprises: Antigen-binding domain 1 (ABD1), Antigen-binding domain 2 (ABD2) or Antigen-binding domain 3 (ABD3) is selected from.

[0038] 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. A binding agent may therefore comprise an antigen-binding domain with the same or different specificity than that of ABD1, ABD2 and / or ABD3.

[0039] In some embodiments, a binding agent may comprise more than one antigen-binding domain.

[0040] For example, 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.

[0041] In some embodiments, the binding agent may comprise between 1 and 12 antigen-binding domains.

[0042] In some embodiments, the binding agents of the present disclosure may be monospecific.

[0043] In some embodiments, the binding agents of the present disclosure may be multispecific.

[0044] In some embodiments, the binding agents of the present disclosure may be monovalent.

[0045] In some embodiments, the binding agents of the present disclosure may be multivalent.

[0046] In some embodiments, the binding agents of the present disclosure may be in monomeric form.

[0047] 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).

[0048] 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.

[0049] 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.

[0050] In some embodiments, the binding agent comprises at least one antigen-binding domain that binds to PD-1 and comprises a complementarity determining region as described herein.

[0051] 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.

[0052] In some embodiments, the antigen binding domains are on one or more polypeptide chains.

[0053] In some embodiments, the antigen binding domains are on the same polypeptide chain.

[0054] In some embodiments, the binding agent comprises a single polypeptide chain.

[0055] In some embodiments, the binding agent comprises two polypeptide chains, and thus may be in the form of a dimer.

[0056] In some embodiments, the binding agent comprises more than two polypeptide chains, for example, 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, the 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1).

[0061] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 2 (ABD2).

[0062] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 3 (ABD3).

[0063] 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).

[0064] 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).

[0065] 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).

[0066] 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).

[0067] 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.

[0068] 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.

[0069] In some embodiments, each polypeptide chain of the binding agent is the same.

[0070] In some embodiments, each polypeptide chain of the binding agent is different.

[0071] In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.

[0072] In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.

[0073] In some embodiments, the antigen binding domains are separated by an amino acid sequence.

[0074] In some embodiments, the amino acid sequence is a linker.

[0075] Binding agents of the disclosure include, for example, an antigen-binding domain as disclosed herein, a single polypeptide chain as disclosed herein, a dimer of the polypeptide chains as disclosed herein, or a multimer of the polypeptide chains as disclosed herein.

[0076] Thus, the binding agents of the 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.

[0077] Advantageously, the binding agent of the present disclosure may have a format as disclosed in PCT / CA2020 / 051753, filed December 18, 2020, the entire contents of which are incorporated herein by reference, 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.

[0078] In some embodiments, the binding agent is composed of a polypeptide chain that includes one or more antigen binding domains and a dimerization domain that allows at least two polypeptide chains to form a dimer.

[0079] In some embodiments, the binding agents of the present disclosure have the formula I, in N-terminal to C-terminal fashion: 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 stands for dimerization domain.

[0080] 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.

[0081] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen binding domains.

[0082] In some embodiments, the binding agent comprises a single polypeptide chain.

[0083] In some embodiments, the binding agent comprises 2 polypeptide chains, 3 polypeptide chains, 4 polypeptide chains, 5 polypeptide chains, 6 polypeptide chains, 7 polypeptide chains, 8 polypeptide chains, 9 polypeptide chains, 10 polypeptide chains or more than 10 polypeptide chains.

[0084] In some embodiments, the polypeptide chains may be covalently linked.

[0085] In some embodiments, the polypeptide chains may be non-covalently linked.

[0086] In some embodiments, the polypeptide chains may associate via electrostatic interactions.

[0087] 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.

[0088] 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.

[0089] In some embodiments where m is an integer greater than 2, each Ab a are the same or different.

[0090] In some embodiments where n is an integer greater than 2, each Ab d are the same or different.

[0091] In some embodiments, Ab a represents ABD1, ABD2 or ABD3.

[0092] In some embodiments, Ab d represents ABD1, ABD2 or ABD3.

[0093] In some embodiments, Ab d represents ABD1, ABD2 or ABD3.

[0094] In embodiments, one or more of the polypeptide chains further comprises 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.

[0095] In some embodiments, the hinge region is a native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0096] In some embodiments, the hinge region is a variant hinge region having at least 70% identity to a naturally occurring hinge region from IgG1, IgG2, IgG3, or IgG4.

[0097] 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.

[0098] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0099] 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.

[0100] In some embodiments, the rigid linker comprises multiple PA repeats as described herein.

[0101] In some embodiments, the helical linker comprises one or more EAAAK units as described herein.

[0102] In some embodiments, the binding agent comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0103] In some embodiments, the binding agent comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0104] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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).

[0105] In some embodiments, the binding agent comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0106] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0107] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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).

[0108] In some embodiments, the binding agent comprises one or more polypeptide chains, and at least one of the polypeptide chains has the 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).

[0109] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently comprises an antigen-binding domain.

[0110] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently represents an antigen-binding domain.

[0111] 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).

[0112] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 At least one of them is not antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3).

[0113] In some embodiments, L b1comprises 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 naturally occurring hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.

[0114] In some embodiments, L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers as disclosed herein.

[0115] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD1 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0116] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD2 as disclosed herein and an antigen binding domain that binds to a tumor cell.

[0117] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to a tumor cell.

[0118] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0119] In some embodiments, the polypeptide chain comprises at least one antigen-binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0120] In some embodiments, the polypeptide chain comprises at least two antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0121] In some embodiments, the polypeptide chain comprises at least three antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0122] 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.

[0123] 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).

[0124] 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.

[0125] In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.

[0126] In some exemplary embodiments, the dimerization domain comprises a mutated CH3 domain.

[0127] In some embodiments, the dimerization domain comprises a native CH2 and a native CH3 domain.

[0128] In some embodiments, the dimerization domain comprises a native CH2 and a variant CH3 domain.

[0129] In some embodiments, the dimerization domain comprises a variant CH2 and a variant CH3 domain.

[0130] In some embodiments, the dimerization domain comprises a mutated CH2 and a native CH3 domain.

[0131] In some embodiments, the dimerization domain does not comprise a CH1 domain.

[0132] In some embodiments, the dimerization domain does not comprise a CH4 domain.

[0133] In some embodiments, the dimerization domain comprises an Fc region of an antibody or portion thereof.

[0134] In some embodiments, the polypeptide chain comprises at least two or at least three antigen-binding domains and a dimerization domain that allows assembly of two polypeptide chains to form a multivalent and / or multispecific binding agent.

[0135] In embodiments the antigen binding domain comprises or consists of the antigen binding domain of a single domain antibody (sdAb).

[0136] In embodiments, the antigen binding domain comprises a heavy chain variable region (VH or VHH).

[0137] In some embodiments, the VHH is derived from a human, a mouse, a rat, etc.

[0138] 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 human) or a camelized VHH from another species (such as a camelized human VHH).

[0139] In some embodiments, the binding agent does not comprise a light chain variable region (VL or VLL).

[0140] In embodiments, the binding agent comprises a light chain variable region (VL or VLL).

[0141] In embodiments, the antigen-binding domain is a single chain variable fragment (ScFv).

[0142] In embodiments, the antigen-binding domain is V NAR It is from a fragment.

[0143] 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 present invention includes any combination of antigen-binding domains of the fragments.

[0144] In some embodiments the sdAb or VHH is from a camelid antibody.

[0145] In embodiments, the camelid antibodies are from dromedaries, camels, llamas, alpacas, etc.

[0146] In another embodiment, the sdAb or VHH is from a cartilaginous fish antibody.

[0147] In an embodiment, the cartilaginous fish antibody is a shark antibody.

[0148] In some embodiments, each antigen-binding domain specifically binds to a different epitope.

[0149] In other embodiments, each antigen-binding domain specifically binds to a different antigen.

[0150] In yet other embodiments, each antigen-binding domain specifically binds to a different protein.

[0151] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to a protein expressed by the tumor, hi other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to and modulates the activity of a protein expressed by the tumor.

[0152] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to an immune checkpoint protein, hi other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to an immune checkpoint protein and modulates its activity.

[0153] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to an immune cell protein. In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to an immune cell protein and modulates its activity. In yet other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to or engages an immune cell and recruits or redirects the immune cell.

[0154] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to peripheral blood mononuclear cells (PBMCs). In other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to and modulates the activity of PBMCs. In yet other embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to and recruits or redirects PBMCs.

[0155] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to a T cell protein. In other embodiments, the binding agent may bind to a T cell protein and modulate its activity. In yet other embodiments, the binding agent may bind to a T cell protein and recruit or redirect T cells. In some embodiments, the binding agent may bind to an activated T cell and / or modulate the activity of an activated T cell.

[0156] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen binding domain that binds to a protein expressed by a tumor and at least one antigen binding domain that binds to and recruits or redirects an immune cell.

[0157] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to a protein expressed by a tumor, at least one antigen-binding domain that binds to an immune checkpoint protein, and at least one antigen-binding domain that binds to and recruits or redirects an immune cell.

[0158] In some embodiments, the binding agent comprises a polypeptide chain that includes at least one antigen-binding domain that modulates an immune checkpoint inhibitor.

[0159] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds to a tumor antigen, at least one antigen-binding domain that binds to an immune checkpoint protein, and at least one antigen-binding domain that binds to a T cell.

[0160] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that binds a tumor antigen, at least one antigen-binding domain that binds an immune checkpoint protein, and at least one antigen-binding domain that binds CD47.

[0161] In some embodiments, the binding agent comprises a polypeptide chain comprising at least one antigen-binding domain that modulates a function of CD47.

[0162] 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.

[0163] In some embodiments, the binding agent comprises a polypeptide chain that includes an antigen-binding domain that specifically binds to the receptor.

[0164] In some embodiments, the binding agent comprises a polypeptide chain that comprises an antigen-binding domain that specifically binds to a tumor antigen and an antigen-binding domain that specifically binds to an immune modulator.

[0165] 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.

[0166] In some embodiments, the immunomodulator is an immune checkpoint protein, a cytokine, a chemokine, or an immune receptor or co-receptor.

[0167] 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.

[0168] In some embodiments, the binding agent comprises at least two polypeptide chains capable of forming a dimer.

[0169] In some embodiments, the two polypeptide chains are the same.

[0170] In some embodiments, the two polypeptide chains are different.

[0171] In some embodiments, the polypeptide chains are the same and the binding agent is a homodimer.

[0172] In some embodiments, the polypeptide chains are different and the binding agent is a heterodimer.

[0173] In some embodiments, the binding agents are multispecific.

[0174] In some embodiments, the binding agents are bispecific, trispecific, or tetraspecific.

[0175] In some embodiments, a binding agent comprises one or more polypeptide chains that are multispecific.

[0176] In some embodiments, a binding agent comprises one or more polypeptide chains that are bispecific, trispecific, or tetraspecific.

[0177] In some embodiments, one or more polypeptide chains have the same valency and specificity.

[0178] In some embodiments, one or more polypeptide chains have different valencies and specificities.

[0179] In some embodiments, each of the one or more polypeptide chains is an antibody heavy chain.

[0180] 2. The binding agent of any one of the preceding claims which is an antibody or an antigen-binding fragment thereof.

[0181] In some embodiments, the binding agent is a bispecific antibody.

[0182] In some embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

[0183] In some embodiments, one or more of the antigen-binding domains are humanized.

[0184] In some embodiments, one or more of the antigen binding domains are partially humanized.

[0185] In some embodiments, the antigen-binding domain comprises one or more human frameworks.

[0186] 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.

[0187] In some embodiments, the polypeptide chain is conjugated to a therapeutic moiety.

[0188] In some embodiments, the polypeptide chain is conjugated to a detectable moiety.

[0189] In some embodiments, the polypeptide chain is conjugated to a protein that allows for extended half-life.

[0190] In some embodiments, the polypeptide chain is attached to a nanoparticle.

[0191] Other aspects and embodiments of the present disclosure relate to compositions comprising at least one of the binding agents disclosed herein.

[0192] In some embodiments, the composition comprises monomers, dimers, and mixtures thereof.

[0193] 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.

[0194] 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.

[0195] 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.

[0196] Other aspects and embodiments of the present disclosure relate to pharmaceutical compositions comprising a binding agent disclosed herein and a pharma- ceutically acceptable carrier.

[0197] Yet other aspects and embodiments of the present disclosure relate to a nucleic acid or set of nucleic acids encoding the polypeptide chains and / or binding agents disclosed herein.

[0198] The nucleic acid may be in the form of a DNA segment as disclosed herein.

[0199] 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.

[0200] Further aspects and embodiments of the present disclosure relate to cells expressing the polypeptide chains or binding agents disclosed herein.

[0201] Additional aspects and embodiments of the present disclosure relate to cells comprising the nucleic acids or vectors disclosed herein.

[0202] Further aspects and embodiments of the present disclosure relate to kits that include the binding agents disclosed herein.

[0203] Still further aspects and embodiments of the present disclosure relate to kits that include the nucleic acids disclosed herein.

[0204] Still further aspects and embodiments of the present disclosure relate to kits that include the vectors disclosed herein.

[0205] Still further aspects and embodiments of the present disclosure relate to kits comprising the cells disclosed herein.

[0206] In additional aspects and embodiments, the present disclosure relates to a method of treating a disorder or disease comprising administering a binding agent disclosed herein.

[0207] In further aspects and embodiments, the present disclosure relates to methods of treating a disorder or disease comprising administering a composition disclosed herein.

[0208] In further aspects and embodiments, the present disclosure relates to a method of treating a disorder or disease comprising administering a pharmaceutical composition disclosed herein.

[0209] In some embodiments, the disorder or disease is cancer.

[0210] In some embodiments, the disorder or disease is a solid tumor.

[0211] In some embodiments, the disorder or disease is progressive metastatic solid cancer.

[0212] In some embodiments, the disorder or disease is a hematogenous cancer.

[0213] In some embodiments, the disorder or disease is lung cancer.

[0214] In some embodiments, the lung cancer is metastatic.

[0215] In some embodiments, the disorder or disease is small cell lung cancer.

[0216] In some embodiments, the disorder or disease is non-small cell lung cancer.

[0217] In some embodiments, the disorder or disease is myeloma.

[0218] In some embodiments, the disorder or disease is prostate cancer.

[0219] In some embodiments, the disorder or disease is breast cancer.

[0220] In some embodiments, the disorder or disease is rectal cancer.

[0221] In some embodiments, the disorder or disease is pancreatic cancer.

[0222] In some embodiments, the disorder or disease is glioblastoma.

[0223] In some embodiments, the disorder or disease is an infectious disease.

[0224] In some embodiments, the disorder or disease is an immune dysregulation.

[0225] In other aspects and embodiments, the present disclosure relates to a method of producing a binding agent disclosed herein, comprising transforming a cell with one or more vectors comprising a nucleic acid disclosed herein.

[0226] In some embodiments, the method may further include isolating and / or purifying the binding agent from impurities.

[0227] In other embodiments, the method may further comprise isolating and / or purifying the heterodimer from the monomer and / or homodimer.

[0228] In other embodiments, the method may further comprise isolating and / or purifying the homodimers from the monomers and / or heterodimers.

[0229] In some embodiments, the method further comprises conjugating the binding agent to a therapeutic moiety, a detectable moiety, or a protein allowing for extended half-life, or to a nanoparticle.

[0230] 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 description of the drawings]

[0231] [Figure 1] Graph showing the binding curve of D2 specific binder (VHH-hinge-CH2-CH3) for D2 proteoliposomes. Mock proteoliposomes were included as a negative control. [Diagram 2] Table listing the EC50 of D2 specific binder (VHH-hinge-CH2-CH3) in GPCR BRET assays transfected with various G protein sensors. Dopamine was used as a positive control to demonstrate GPCR response with G proteins including Gαz, Gαi2, GαoA, and Barr2. The negative control is 4HEM specific binder (VHH-hinge-CH2-CH3) (KC018) containing the antigen binding domain shown in SEQ ID NO: 114. [Diagram 3] Heat map showing gene expression changes in NCI-H69 cells upon treatment with a binder (VHH-hinge-CH2-CH3) containing the D2 specific antigen binding domain (KC013; SEQ ID NO: 73). The negative control is a 4HEM specific binder (VHH-hinge-CH2-CH3) containing the antigen binding domain shown in SEQ ID NO: 114 (KC018). [Figure 4] Graph depicting downregulated signaling pathways and protein classes from PANTHER analysis of the RNAseq results in Figure 3 . [Diagram 5] 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; sequence number 73). [Figure 6]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 (Figure 6A: IV injection) (Figure 6B: IP injection). [Figure 7] Graphs showing tumor kinetics and in vivo efficacy of a binder comprising a D2-specific antigen-binding domain (VHH-hinge-CH2-CH3, solid line) (KC013; SEQ ID NO: 73) in NCG mice with small cell lung cancer (SCLC) xenograft models compared to an isotype control (dashed line) in a NCI-H727 (FIG. 7A), NCI-H69 (FIG. 7B) or NCI-H510A (FIG. 7C) tumor preventive model. The negative control is a 4HEM-specific binder (VHH-hinge-CH2-CH3) comprising the antigen-binding domain shown in SEQ ID NO: 114 (KC018). [Figure 8] 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, 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] Graph showing the binding curve of a PD-1 specific binder (KC036; SEQ ID NO: 94) for human recombinant PD1 protein compared to the anti-PD-1 antibody pembrolizumab (positive control). The negative control is a binder containing a HEWL specific antigen binding domain (KC035: SEQ ID NO: 93). [Figure 9C]Graph showing the binding curve of 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. Positive control is monoclonal antibody clone B6H12. Isotype control of clone B6H12 is used as 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] Graph showing the cell binding activity of a PD-1 specific binder (KC036; SEQ ID NO: 94) on CHO-PD1 cells (FIG. 10B) and CHO parental cells (FIG. 10A) obtained by flow cytometry. A commercially available PD-1 antibody is used as a positive control. The negative control is 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 PD-1 specific binder (KC036; SEQ ID NO: 94) in the presence of CD33 BiTE or control BiTE after 48 hours of incubation used to activate T cells via the CD3 subunit. Negative control is binder containing HEWL specific antigen binding domain (KC031: SEQ ID NO: 89). Positive controls include pembrolizumab and nivolumab. [Figure 12]Graph showing checkpoint inhibitory activity of PD-1 specific binder (KC036; SEQ ID NO: 94) using 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 13] Graphs depicting 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 (KC016 (SEQ ID NO: 76) and KC039 (SEQ ID NO: 97)) containing an anti-HEWL antigen binding domain. A commercially available anti-human CD47 antibody (clone CC2C6) is 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 an anti-CD3 and anti-HEWL antigen binding domain (KC034; SEQ ID NO: 92). [Figure 15] Graphs depicting 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 on human T cells (KC040; SEQ ID NO: 84) at an E:T ratio of 5:1. Controls include media 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 illustrating T cell activation upon treatment with a binder comprising D2 and a CD47-specific antigen-binding domain (KC040: SEQ ID NO: 84) in the presence or absence of concanavalin A. Controls include a binder comprising an HEWL-specific antigen-binding domain (KC035: SEQ ID NO: 93) or an anti-CD3 and an anti-HEWL antigen-binding domain (positive control KC034; SEQ ID NO: 92) or OKT3 (positive control). [Figure 17] Graph showing checkpoint inhibitory activity of CD47-specific binder (KC015; SEQ ID NO: 75) using the Promega CD47 / SIRPα blockade bioassay system, an engineered THP-1 cell line expressing a luciferase reporter induced by FcγR protein engaging FcγR. The negative control 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 for clone B6H12 is a mouse IgG isotype control antibody. [Figure 18] Graph showing tumor volume upon treatment with a trispecific binding agent comprising anti-D2, anti-PD-1 and anti-CD47 antigen binding domains (KC021: SEQ ID NO: 78) in an established NCI-H82 xenograft model in NCG mice. Negative control includes a binding agent comprising a HEWL specific antigen binding domain (KC031: SEQ ID NO: 89). [Figure 19] Plots depicting T cell infiltration into NCI-H69 cell clusters upon treatment with binding agents comprising anti-D2 and anti-PD-1 antigen binding domains (KC037; SEQ ID NO: 95), anti-PD-1 and anti-CD47 antigen binding domains (KC038; SEQ ID NO: 96), or trispecific binding agents 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) (FIG. 19A) or T cell activation with the same agents (FIG. 19B). Control includes binding agent comprising a HEWL-specific antigen binding domain (KC035: SEQ ID NO: 93). [Figure 20]Graphs depicting tumor volume (Figure 20A) and tables depicting tumor growth inhibition (Figure 20B) upon treatment with different 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 CD47 specific binding agent (KC015; SEQ ID NO: 75) in PBMC pre-engrafted NCI-H82 xenograft model in NCG mice. Ten million PBMCs were engrafted into NCG mice 3 days prior to tumor cell engraftment. Treatment began 10 days after PBMC engraftment, twice per week for a total of 8 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 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 binder (8 mg / kg) treatments intraperitoneally (ip) for a total of eight doses. [Figure 22] Graph showing tumor volume upon 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). Control includes binder containing anti-HEWL antigen binding domain (KC031: SEQ ID NO: 89). [Figure 23]Schematic (FIG. 23A) and photograph (FIG. 23B) of a biodistribution experiment showing accumulation of a trispecific binding agent comprising anti-D2, anti-PD-1, and anti-CD47 antigen-binding domains (KC021; SEQ ID NO: 78) in NCI-H82 (upper panel) and NCI-H69 (lower panel) xenograft models in NCG mice. [Figure 24A] Graph showing liver metastases from hCD34+ humanized NCG mice bearing established NCI-H69 tumors treated with either PBS or KC020 (SEQ ID NO: 77) (FIG. 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 (FIG. 24B). [Figure 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) (FIG. 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 (FIG. 24B). [Figure 24C] Graph showing tumor kinetics and in vivo efficacy of KC020 in NCI-H82 / PBMC co-transplant model at E:T ratio of 1:5 in NCG mice. [Diagram 25] Graphs depicting binding of KC020 (SEQ ID NO: 77) subjected to stress conditions for 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 freeze-thaw cycles, and storage at 40° C. for 2 weeks. [Figure 26] Graphs depicting the binding of KC020 (SEQ ID NO: 77) subjected to low pH stress 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 treatment at pH 3.5. [Figure 27] Graph showing survival curve of KC020 treated animals implanted with 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 performed every two weeks. The negative control used in the study was PBS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0232] Detailed Description definition Unless otherwise indicated, the amino acid numbering indicated for the dimerization domains is according to the EU numbering system.

[0233] The use of the terms "a" and "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.

[0234] Unless specifically stated otherwise or apparent from the context, as used herein, the term "or" is understood to be inclusive, covering both "or" and "and."

[0235] The term "and / or" when used herein should be interpreted as an independent disclosure of each of the specified features or components with or without the other.

[0236] 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.

[0237] The term "treatment", for the purposes of this disclosure, refers to both therapeutic treatment and prophylactic or anti-tumor measures. Those in need of treatment include those already with the disorder as well as those predisposed to having the disorder or those in which the disorder is to be prevented.

[0238] The term "about" or "approximately" with respect 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, within + / - 5 percent, within + / - 4 percent, within + / - 3 percent, within + / - 2 percent, or within + / - 1 percent of a given value or range.

[0239] 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.

[0240] 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 allow adjacent modules to move relative to one another.

[0241] As used herein, the term "rigid linker" refers to a peptide that includes at least a portion made up of amino acids that present a rigid structure and maintain the distance between two modules.

[0242] As used herein, the term "helical linker" means a linker composed of amino acid residues that adopt an α-helical conformation.

[0243] As used herein, the term "cleavable linker" refers to a peptide that contains an enzymatic cleavage site 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.

[0244] 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.

[0245] 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.

[0246] The term "multispecific" encompasses "bispecific," "trispecific," "tetraspecific," "pentaspecific," and "hexaspecific" antibodies and the like.

[0247] As used herein, in the context of a binding agent, 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.

[0248] As used herein, a phrase such as "at least two polypeptide chains," e.g., in the phrase "the binding agent comprises at least two polypeptide chains," refers to a number of polypeptide chain species and not to an absolute numerical value.

[0249] Expressions referring to ranges of values ​​in the form "A to B" etc. should be understood herein to include each individual value and any subranges contained within 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 contained within 1 to 10, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0250] 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%.

[0251] Antigen-binding domain (Ab) As disclosed herein, the 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.

[0252] 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.

[0253] The specificity of the binding agents of the disclosure may therefore be conferred by their antigen-binding domain.

[0254] 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.

[0255] In some embodiments, the antigen binding domain is an antigen binding domain capable of binding to DR2 or a cell expressing DR2, for example comprising antigen binding domain 1 (ABD1).

[0256] 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).

[0257] 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).

[0258] 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.

[0259] In some embodiments, the antigen-binding domain binds to at least one antigen that is not selected from among DR2, PD-1, or CD47.

[0260] In some embodiments, a binding agent may comprise more than one antigen-binding domain.

[0261] For example, in some embodiments, a binding agent may comprise two or more antigen-binding domains.

[0262] In some embodiments, the binding agent may comprise three or more antigen-binding domains.

[0263] In some embodiments, the binding agent may comprise four or more antigen-binding domains.

[0264] In some embodiments, the binding agent may comprise five or more antigen-binding domains.

[0265] In some embodiments, the binding agent may comprise six or more antigen-binding domains.

[0266] In some embodiments, the binder may be 1-12, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 4-5, 4-6, 4-7, 4-8, 4 The antibody may comprise up 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.

[0267] 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.

[0268] In some embodiments, the binding agent of the present disclosure may comprise one or more antigen-binding domains, and 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.

[0269] 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 an immune cell. In 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.).

[0270] 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.

[0271] In some embodiments, the protein expressed on the surface of an immune cell is selected from CD47 or CD3.

[0272] In some embodiments, the protein expressed on the surface of an immune cell is CD47.

[0273] In some embodiments, the protein expressed on the surface of an immune cell is CD3.

[0274] The antigen-binding domain may be derived from a natural antibody (of human or animal origin) or a synthetic antibody.

[0275] In some embodiments, the antigen-binding domains of a native antibody are engineered to form a single chain.

[0276] In some embodiments, the antigen-binding domain may be derived from an IgG, such as an IgG1, IgG2, IgG3 or IgG4. In certain embodiments, the antigen-binding domain is derived from a human IgG heavy chain.

[0277] In some embodiments, the antigen-binding domain may be derived from a heavy chain-only antibody (HCAb).

[0278] 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.

[0279] In some embodiments, the antigen-binding domain of a binding agent disclosed herein is a VHH.

[0280] 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.

[0281] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to Kabat CDRs.

[0282] In some embodiments, the complementarity determining regions (CDRs) of a VHH disclosed herein correspond to the IMGT CDRs.

[0283] In certain embodiments, the binding agent of the disclosure may comprise an antigen-binding domain VHH from human or mouse or rat, or a transgenic mouse or rat in which the mouse or rat VHH has been camelized, IgG1, IgG2a, IgG2b, IgG2c or IgG3 or a combination thereof, human VHH, camelized human VHH. The antibody may be obtained by immunizing a mouse or rat or a 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.

[0284] 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.

[0285] 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 vicugna (Lama vicugna).

[0286] 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. NAR The fragment may originate from a shark antibody.

[0287] 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 produced, 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 NAR Antibodies may be humanized by converting non-CDR residues to those of 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 disclosure thus include antigen-binding domains that are humanized.

[0288] In yet another particular 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 camelid-like (i.e. by camelization).

[0289] 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- One of skill in the art will appreciate 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 mutated CH3 domain, a native CH2-CH3 domain, or a mutated CH2-CH3 domain as disclosed herein.

[0290] 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.

[0291] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where 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.

[0292] In some exemplary embodiments, a binding agent of the 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.

[0293] In some exemplary embodiments, a binding agent of the 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.

[0294] In some exemplary embodiments, a binding agent of the 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.

[0295] In some exemplary embodiments, a binding agent of the 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.

[0296] In some exemplary embodiments, a binding agent of the 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.

[0297] In some exemplary embodiments, a binding agent of the 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.

[0298] In some exemplary embodiments, a binding agent of the 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.

[0299] In some exemplary embodiments, a binding agent of the 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.

[0300] In some exemplary embodiments, a binding agent of the 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.

[0301] In some exemplary embodiments, a binding agent of the 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.

[0302] In some exemplary embodiments, a binding agent of the 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.

[0303] In some exemplary embodiments, a binding agent of the 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.

[0304] In some exemplary embodiments, a binding agent of the 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.

[0305] In some exemplary embodiments, a binding agent of the 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.

[0306] In some exemplary embodiments, a binding agent of the 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.

[0307] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0308] 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.

[0309] 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:7 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:7.

[0310] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:7.

[0311] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0312] 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.

[0313] 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.

[0314] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:14.

[0315] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0316] 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.

[0317] 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 CDRs that are identical to the IMGT CDRs of SEQ ID NO:21.

[0318] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:21.

[0319] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0320] 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.

[0321] 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 CDRs that are identical to the IMGT CDRs of SEQ ID NO:28.

[0322] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:28.

[0323] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0324] 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.

[0325] 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.

[0326] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:35.

[0327] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0328] 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.

[0329] 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 CDRs that are identical to the IMGT CDRs of SEQ ID NO:42.

[0330] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:42.

[0331] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[0332] 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.

[0333] 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:49 and CDRs that are identical to the IMGT CDRs of SEQ ID NO:49.

[0334] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:49.

[0335] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having 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.

[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:56 and CDRs identical to the Kabat CDRs of SEQ ID NO:56.

[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:56 and CDRs identical to the IMGT CDRs of SEQ ID NO:56.

[0338] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:56.

[0339] Exemplary embodiments of ABD2 Antigen-binding domain 2 (ABD2) may, for example, be selected from antigen-binding domains that bind to PD-1, some non-limiting exemplary embodiments of which are provided herein.

[0340] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), comprising 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.

[0341] In some exemplary embodiments, the binding agents of the 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.

[0342] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a heavy chain having 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.

[0343] 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.

[0344] 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 CDRs that are identical to the IMGT CDRs of SEQ ID NO:63.

[0345] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:63.

[0346] Exemplary embodiments of ABD3 Antigen binding domain 3 (ABD3) may, for example, be selected from antigen binding domains that bind CD47, some non-limiting exemplary embodiments of which are provided herein.

[0347] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where 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.

[0348] In some exemplary embodiments, the binding agents of the 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.

[0349] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises a heavy chain having 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.

[0350] 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.

[0351] 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 IMGT CDRs of SEQ ID NO:70.

[0352] In some exemplary embodiments, a binding agent of the disclosure may comprise one or more antigen-binding domains, where at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0353] Exemplary embodiments of other antigen-binding domains In some embodiments, the binding agent comprises one or more antigen-binding domains, 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 antigen-binding domain binds to a different antigen.

[0354] 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.

[0355] Thus, in some embodiments, the binding agent may comprise an antigen-binding domain that binds DR2, PD-1, and / or CD3. In other embodiments, the binding agent may comprise an antigen-binding domain that binds DR2 and / or CD3. In yet other embodiments, the binding agent may comprise an antigen-binding domain that binds PD-1 and / or CD3.

[0356] 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, beta 1 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 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, or SIGLEC-7.

[0357] Polypeptide Chain The binding agents of the present disclosure comprise one or more polypeptide chains.

[0358] 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 forming a single chain.

[0359] Polypeptide chains of the disclosure include, for example and without limitation, an antigen binding domain, a linker, and a dimerization domain that promotes assembly of at least two polypeptide chains.

[0360] In some embodiments, the polypeptide chains of the present disclosure may be monospecific.

[0361] In some embodiments, the polypeptide chains of the present disclosure may be multispecific.

[0362] In some embodiments, the polypeptide chains of the present disclosure may be monovalent.

[0363] In some embodiments, the polypeptide chains of the present disclosure may be multivalent.

[0364] In some cases, the polypeptide chain does not comprise a dimerization domain.

[0365] In some cases, the polypeptide chain comprises a dimerization domain.

[0366] In some aspects of the present disclosure, the polypeptide chains may be monospecific.

[0367] Exemplary embodiments of monospecific polypeptide chains include polypeptide chains that comprise one antigen-binding domain. Another exemplary embodiment of monospecific polypeptide chains includes polypeptide chains that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and framework regions. Yet another exemplary embodiment of monospecific polypeptide chains includes polypeptide chains that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and different framework regions. Further exemplary embodiments of monospecific polypeptide chains include polypeptide chains that comprise 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 the same antigen or epitope.

[0368] 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.

[0369] In exemplary configurations, the antigen binding domain or domains may be located N-terminal, C-terminal or on each side of the dimerization domain.

[0370] 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.

[0371] 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.

[0372] 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.

[0373] The polypeptide chain has the formula I in N-terminal to C-terminal fashion: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y may comprise 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 stands for dimerization domain.

[0374] In some embodiments, L b does not have to exist.

[0375] In some embodiments, L c does not have to exist.

[0376] In some embodiments, L b and L c Both do not have to be present.

[0377] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0378] In some embodiments, where m is an integer greater than or equal to 2, [(Ab a )-(L b )] units are the same.

[0379] In other embodiments where m is an integer greater than or equal to 2, the [(Ab a )-(L b )] units are different.

[0380] In other embodiments where m is an integer greater than 2, the [(Ab a )-(L b ) units may include the same and different units.

[0381] In some embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units are the same.

[0382] In other embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units are different.

[0383] In other embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units include same and different units.

[0384] In some embodiments where m is an integer greater than 2, each Ab a are the same or different.

[0385] In some embodiments where n is an integer greater than 2, each Ab d are the same or different.

[0386] In some embodiments, Ab a stands for ABD1.

[0387] In some embodiments, Abd stands for ABD2.

[0388] In some embodiments, Ab d represents ABD3.

[0389] In some embodiments, m is 1 and Ab a stands for ABD1.

[0390] In some embodiments, n is 2 and Ab d One of them represents ABD2.

[0391] In some embodiments, n is 2 and Ab d One of them represents ABD3.

[0392] In some embodiments, n is 2 and Ab d One of them represents ABD2, and the other Ab d represents ABD3.

[0393] In some embodiments, m is 2, 3, 4, 5, or an integer greater than 5.

[0394] In some embodiments, m is 2.

[0395] In other embodiments, m is 3.

[0396] In yet other embodiments, m is 4.

[0397] In a further embodiment, m is 5.

[0398] In other embodiments, m is an integer greater than five.

[0399] In some embodiments, n is 2, 3, 4, 5, or an integer greater than 5.

[0400] In some embodiments, n is 2.

[0401] In other embodiments, n is 3.

[0402] In additional embodiments, n is 4.

[0403] In a further embodiment, n is 5.

[0404] In other embodiments, n is an integer greater than five.

[0405] In embodiments, one or more of the polypeptide chains further comprises a hinge region of an antibody or antigen-binding fragment thereof.

[0406] In some embodiments, the hinge region is N-terminal to the dimerization domain.

[0407] In some embodiments, L b is the hinge region of an antibody or antigen-binding fragment thereof.

[0408] In some embodiments, the hinge region is a native human IgG1 hinge region. In other embodiments, the hinge region is a mutated human IgG1 hinge region.

[0409] In other embodiments, the hinge region is a native human IgG2 hinge region. In other embodiments, the hinge region is a mutated human IgG2 hinge region.

[0410] In other embodiments, the hinge region is a native human IgG3 hinge region. In other embodiments, the hinge region is a mutated human IgG3 hinge region.

[0411] In yet other embodiments, the hinge region is a native human IgG4 hinge region. In other embodiments, the hinge region is a mutated human IgG4 hinge region.

[0412] 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.

[0413] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0414] In some embodiments, the linker L c is a rigid linker.

[0415] In some embodiments, Lc comprises a non-cleavable linker. In other embodiments, Lc consists of a non-cleavable linker.

[0416] In some embodiments, the one or more linkers comprise a flexible linker.

[0417] In some embodiments, the one or more linkers comprise a rigid linker.

[0418] In some embodiments, the flexible linker is a GS linker.

[0419] In some embodiments, the flexible linker comprises one or more GGGGS units.

[0420] In some embodiments, the flexible linker comprises at least 2, 3, 4, 5, or more GGGGS units.

[0421] In some embodiments, the rigid linker comprises multiple PA repeats.

[0422] In some embodiments, the rigid linker is selected from PAPAPKA (SEQ ID NO: 105); APAPAPAPAPKA (SEQ ID NO: 106); APAPAPAPAPAPAPAPAPAPKA (SEQ ID NO: 107); or a combination thereof.

[0423] In some embodiments, the helical linker comprises one or more EAAAK units.

[0424] In some embodiments, the helical linker is selected from AEAAAKEAAAKA (SEQ ID NO: 109); AEAAAKEAAAKEAAAKA (SEQ ID NO: 110); AEAAAKEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 111); or a combination thereof.

[0425] In some embodiments, the dimerization domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:116.

[0426] In some embodiments, the dimerization domain further comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:117.

[0427] In another exemplary embodiment, the polypeptide chain has the formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II) Includes.

[0428] In yet another exemplary embodiment, the polypeptide chain has the formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) Includes.

[0429] In a further exemplary embodiment, the polypeptide chain has Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV) Includes.

[0430] In additional exemplary embodiments, the polypeptide chain has the formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y(Formula V) Includes.

[0431] In a further exemplary embodiment, the polypeptide chain 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) Includes.

[0432] In still further exemplary embodiments, the polypeptide chain 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) Includes.

[0433] In another exemplary embodiment, the polypeptide chain has the formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(Lc1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VIII) Includes.

[0434] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each independently comprises an antigen-binding domain.

[0435] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each independently represents an antigen-binding domain.

[0436] In some embodiments, 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).

[0437] In some embodiments, L b1 comprises one or more linkers and / or hinge regions of an antibody or antigen-binding fragment thereof. In some embodiments, L b1 L b It is.

[0438] In some embodiments, L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers.

[0439] In some embodiments, L b1 , L b2 and / or L b3 One or more of may be absent.

[0440] In some embodiments, L b2 and / or L b3 does not have to exist.

[0441] In some embodiments, L c1 , L c2 , and / or L c3 One or more of may be absent.

[0442] In some embodiments, L c2 and / or L c3 does not have to exist.

[0443] In some embodiments, L c1 is a rigid linker.

[0444] In some embodiments, L c2 is a rigid linker.

[0445] In some embodiments, L c3 is a rigid linker.

[0446] In some embodiments, L c1 , and L c2 is a rigid linker.

[0447] In some embodiments, L c1 , L c2 and L c3 is a rigid linker.

[0448] In some embodiments, L c1 , L c2 and / or L c3 does not have to exist.

[0449] In some embodiments, L c2 , and / or L c3 does not have to exist.

[0450] In some embodiments, L c1 , and / or L c3does not have to exist.

[0451] In some embodiments, L c2 and / or L c3 does not have to exist.

[0452] In some embodiments, L c1 , L c2 , and L c3 does not have to exist.

[0453] The polypeptide chains of the disclosure comprise an antigen-binding domain that is functionally active as a single chain or when part of a binding agent disclosed herein.

[0454] For example, the antigen-binding domain of a polypeptide chain may bind to its target and may be biologically active.

[0455] 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. Additionally, the biological activity of an antigen-binding domain includes its ability to induce signal transduction.

[0456] A polypeptide chain that contains more than one antigen-binding domain is characterized as being multivalent.

[0457] 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.

[0458] 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.

[0459] 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.

[0460] 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.

[0461] 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.

[0462] A polypeptide chain that contains two antigen-binding domains that bind to two non-overlapping epitopes on the same target is characterized as being biparatopic. A polypeptide chain that contains antigen-binding domains that bind to three, four or more epitopes on the same target is characterized as being multiparatopic.

[0463] 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.

[0464] 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.

[0465] For example, the polypeptide chains of the disclosure may include at least one antigen-binding domain that specifically binds to an antigen expressed by a tumor cell or the tumor cell environment (i.e., a tumor-specific antigen-binding domain).

[0466] 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.

[0467] 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.

[0468] In some exemplary embodiments, the antigen-binding domain may bind to dopamine receptor D2 (DR2).

[0469] In some exemplary embodiments, the antigen-binding domain may bind to PD1.

[0470] In some exemplary embodiments, the antigen-binding domain may bind to CD47.

[0471] In exemplary embodiments, the polypeptide chains of the disclosure may comprise at least one tumor-specific antigen-binding domain and at least one immunospecific antigen-binding domain.

[0472] In some embodiments, the tumor-specific antigen-binding domain may be located N-terminal to the dimerization domain.

[0473] In some embodiments, the tumor-specific antigen-binding domain may be located C-terminal to the dimerization domain.

[0474] In some embodiments, the tumor-specific antigen-binding domain may be located both N-terminal and C-terminal to the dimerization domain.

[0475] In some embodiments, multiple immunospecific antigen-binding domains may be located N-terminal to the dimerization domain.

[0476] In some embodiments, the immunospecific antigen-binding domain may be located C-terminal to the dimerization domain.

[0477] In some embodiments, the immunospecific antigen-binding domains may be located both N-terminal and C-terminal to the dimerization domain.

[0478] In exemplary and non-limiting embodiments, a polypeptide chain or binding agent may comprise two immunospecific antigen-binding domains at the C-terminus of 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.

[0479] 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.

[0480] 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.

[0481] In certain embodiments and aspects of the 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.

[0482] 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 natural human heavy chains.

[0483] 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.

[0484] Thus, in some embodiments, the dimerization domain comprises a naturally occurring CH3 domain.

[0485] In an exemplary embodiment, the dimerization domain comprises a native human CH3 domain.

[0486] In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.

[0487] 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).

[0488] In some embodiments, the native CH3 domain is a native IgG2 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG2 CH3 domain.

[0489] In some embodiments, the native CH3 domain is a native IgG3 CH3 domain. In other embodiments, the native CH3 domain is a native human IgG3 CH3 domain.

[0490] 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.

[0491] When the 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 may result in a mixture of homodimers and heterodimers. Different binding agents present in a mixture may be separated by methods known in the art, including, for example, size exclusion chromatography.

[0492] Exemplary heterodimers of the disclosure therefore include those formed by two polypeptide chains having a CH3 domain or a CH2-CH3 domain of a natural antibody and having different sequences or configurations.

[0493] In some embodiments, the polypeptide chains may have a variant dimerization domain that includes, for example, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 amino acid substitutions compared to the native or wild-type sequence.

[0494] 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).

[0495] In exemplary embodiments, the polypeptide chain may have a mutant dimerization domain that has a sequence that is 80%-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 include a mutant dimerization domain that is 85%-99% identical, 90%-99% identical, 95%-99% identical to the sequence of a native IgG1, IgG2, IgG3 or IgG4 constant region or CH2 and / or CH3 domain.

[0496] In some embodiments, the polypeptide chains of the present disclosure may comprise a mutant dimerization domain that comprises an amino acid substitution that favors heterodimer formation. Heterodimers of the present disclosure may thus be formed by polypeptide chains that contain such mutations.

[0497] Thus, in some embodiments, the dimerization domain comprises a mutated constant region of an antibody, such as a mutated human IgG1 constant region, a mutated human IgG2 constant region, a mutated human IgG3 constant region or a mutated 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.

[0498] Thus, in some embodiments, the dimerization domain comprises a mutated CH3 domain. The mutated CH3 domain may have one or more amino acid substitutions, insertions or deletions compared to the native CH3 domain.

[0499] In some embodiments, the dimerization domain comprises a native CH2 and a variant CH3 domain.

[0500] 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.

[0501] In some embodiments, the mutated CH3 domain is a mutated IgG1 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG1 CH3 domain.

[0502] In some embodiments, the mutated CH3 domain is a mutated IgG2 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG2 CH3 domain.

[0503] In some embodiments, the mutated CH3 domain is a mutated IgG3 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG3 CH3 domain.

[0504] In some embodiments, the mutated CH3 domain is a mutated IgG4 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG4 CH3 domain.

[0505] In some embodiments, the mutated CH2 domain is a mutated IgG1 CH2 domain. In other embodiments, the mutated CH2 domain is a mutated human IgG1 CH2 domain.

[0506] In some embodiments, the mutated CH2 domain is a mutated IgG2 CH2 domain. In other embodiments, the mutated CH2 domain is a mutated human IgG2 CH2 domain.

[0507] In some embodiments, the mutated CH2 domain is a mutated IgG3 CH2 domain. In other embodiments, the mutated CH2 domain is a mutated human IgG3 CH2 domain.

[0508] In some embodiments, the mutated CH2 domain is a mutated IgG4 CH2 domain. In other embodiments, the mutated CH2 domain is a mutated human IgG4 CH2 domain.

[0509] In some embodiments, the Fc region may be modified to prevent glycosylation, increase its half-life, 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).

[0510] 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.

[0511] 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.

[0512] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions 356, 357, 370, 399 and / or 439 (according to the EU numbering system).

[0513] In some embodiments, the dimerization domain comprises a mutated CH3 domain comprising one or more mutations at positions corresponding to D399, D / E356 and / or K370 (according to the EU numbering system) compared to the native CH3 domain of human IgG1 or IgG4.

[0514] In other embodiments, the dimerization domain comprises a mutated CH3 domain comprising one or more mutations at positions corresponding to D399, E357 and / or K439 (according to the EU numbering system) compared to the native CH3 domain of human IgG1 or IgG4.

[0515] In some embodiments, the variant CH3 domain further comprises one or more mutations at positions corresponding to Y349, T350, L351, P352, S354, R / Q355, T394 and / or P395 (according to the EU numbering system) compared to a native CH3 domain of human IgG1 or IgG4.

[0516] In an exemplary embodiment, one polypeptide chain of a given binding agent may comprise a mutated CH3 domain, e.g., with amino acid substitutions at positions 357, 399, and 439 (according to the EU numbering system), while another polypeptide chain of the binding agent may comprise a mutated CH3 domain, e.g., with amino acid substitutions at positions 356, 370, and 399.

[0517] In an exemplary embodiment, one polypeptide chain of a given binding agent may comprise a mutated CH3 domain, e.g., with amino acid substitutions at positions 357, 399 and 439 (according to the EU numbering system), while the other polypeptide chain of the binding agent may comprise a mutated CH3 domain, e.g., with amino acid substitutions at positions 356, 370 and 399. One or both polypeptide chains of a given binding agent may optionally further comprise a mutation at a position selected from 349, 350, 351, 352, 354, 355, 394 and / or 395. One polypeptide chain of a given binding agent may thus comprise a first dimerization domain (DD1) having an amino acid sequence as disclosed herein, and the other polypeptide chain of a given binding agent may thus comprise a second dimerization domain (DD2) having an amino acid sequence as disclosed herein.

[0518] In some embodiments, the amino acid at position 356 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 370 may be replaced with a positively charged amino acid. In some embodiments, the amino acid at position 399 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 357 may be replaced with a neutral amino acid. In some embodiments, the amino acid at position 439 may be replaced with a negatively charged amino acid.

[0519] For example, to favor heterodimer formation, one of the polypeptide chains may be mutated by replacing a) aspartic acid (D) or glutamic acid (E) at position 356 with a neutral amino acid, b) lysine (K) at position 370 with a positively charged amino acid, and c) aspartic acid (D) at position 399 with a neutral amino acid, and the other polypeptide chain may be mutated by replacing a) glutamic acid (E) at position 357 with a neutral amino acid, b) aspartic acid (D) at position 399 with a neutral amino acid, and c) lysine (K) at position 439 with a negatively charged amino acid.

[0520] Exemplary embodiments of polypeptide chains of the disclosure may include a dimerization domain that includes a mutated CH3 domain in which the aspartic acid (D) or glutamic acid (E) at position 356 may be changed to glutamine (Q), the lysine (K) at position 370 may be changed to glutamic acid (E), and the aspartic acid (D) at position 399 may be changed to asparagine (N).

[0521] Another exemplary embodiment of a polypeptide chain of the disclosure may include a dimerization domain that includes a mutated CH3 domain in which the glutamic acid (E) at position 357 may be changed to a glutamine (Q), the aspartic acid (D) at position 399 may be changed to an asparagine (N), and the lysine (K) at position 439 may be changed to a glutamic acid (E).

[0522] In some embodiments, the dimerization domain comprises an amino acid substitution (compared to a native CH3 domain): a. An amino acid substitution at position D399; b. an amino acid substitution at position D / E356; c. an amino acid substitution at position E357; d. an amino acid substitution at position K370; e. an amino acid substitution at position K439; f. an amino acid substitution at position Y349; g. an amino acid substitution at position T350; h. an amino acid substitution at position L351; i. an amino acid substitution at position P352; j. an amino acid substitution at position S354; k. an amino acid substitution at position R355; l. an amino acid substitution at position Q355; m. an amino acid substitution at position T394; n. An amino acid substitution at position P395; o. an amino acid substitution at position Y349; and Any combination of two or more of pa~o. The mutant CH3 domain is selected from the group consisting of:

[0523] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and Y349.

[0524] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and T350.

[0525] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and L351.

[0526] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and P352.

[0527] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and S354.

[0528] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and R355.

[0529] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and Q355.

[0530] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370 and T394.

[0531] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, D / E356, K370, Y349 and S354.

[0532] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357 and Y349.

[0533] In some embodiments, the variant CH3 domain comprises amino acid substitutions at positions D399, E357, K439 and T350.

[0534] In some embodiments, the variant CH3 domain comprises amino acid substitutions at positions D399, E357, K439E and L351.

[0535] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357 and P352.

[0536] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357 and S354.

[0537] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357 and R355.

[0538] In some embodiments, the mutated CH3 domain may comprise mutations at positions D399, K439, E357 and Q355.

[0539] In some embodiments, the variant CH3 domain may comprise amino acid substitutions at positions D399, K439, E357 and P395.

[0540] In some embodiments, the variant CH3 domain comprises amino acid substitutions at positions D399, E357, K439, Y349 and S354.

[0541] It should be understood that other amino acid substitutions in the native or variant CH3 domain (at other positions or at the same positions with other amino acid residues) can be made without affecting the ability of the polypeptide chains to form dimers.

[0542] In some embodiments, the amino acid substitution at position Y349 is selected from Y349K, Y349D, or Y349R. More particularly, in some embodiments, the amino acid substitution at position Y349 is Y349K. In other embodiments, the amino acid substitution at position Y349 is Y349D.

[0543] In some embodiments, the amino acid substitution at position S354 is selected from S354K, S354D, S354W or S354M. More particularly, in some embodiments, the amino acid substitution at position S354 is S354K. In other embodiments, the amino acid substitution at position S354 is S354D. In other embodiments, the amino acid substitution at position S354 is S354M.

[0544] In some embodiments, the amino acid substitution at position L351 is L351Y, L351W, L351H, L351R, L351D, L351A, L351T. More particularly, in some embodiments, the amino acid substitution at position L351 is L351Y. In other embodiments, the amino acid substitution at position L351 is L351W. In other embodiments, the amino acid substitution at position L351 is L351R.

[0545] In some embodiments, the amino acid substitution at position T350 is T350L, T350I or T350V. More particularly, in some embodiments, the amino acid substitution at position T350 is T350I. In other embodiments, the amino acid substitution at position T350 is T350V.

[0546] In some embodiments, the amino acid substitution at position P352 is P352Y, P352V, P352R, P352T, P352L, P352G, P352E, P352C, P352K or P352D. More particularly, in some embodiments, the amino acid substitution at position P352 is P352R. In other embodiments, the amino acid substitution at position P352 is P352E.

[0547] In some embodiments, the amino acid substitution at position T394 is T394N.

[0548] In some embodiments, the amino acid substitution at position P395 is P395I. In other embodiments, the amino acid substitution at position P395 is P395G. In other embodiments, the amino acid substitution at position P395 is P395E.

[0549] In some embodiments, the amino acid substitution at position R355 is R355K. In other embodiments, the amino acid substitution at position R355 is R355W.

[0550] In some embodiments, the amino acid substitution at position Q355 is Q355K. In other embodiments, the amino acid substitution at position Q355 is Q355W.

[0551] In some embodiments, the mutated CH3 domain comprises the mutations D399N, D / E356Q and K370E according to EU numbering.

[0552] In some embodiments, the mutated CH3 domain comprises the mutations D399Q, D / E356Q and K370E according to EU numbering.

[0553] In another embodiment, the mutated CH3 domain comprises the mutations D399N, E357Q and K439E according to EU numbering.

[0554] In some embodiments, the mutated CH3 domain may include the mutations D399Q, D / E356Q, K370E, Y349K and S354K.

[0555] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and L351W.

[0556] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and S354M.

[0557] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and T350I.

[0558] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E and T350V.

[0559] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352R.

[0560] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E and P352E.

[0561] In some embodiments, the mutated CH3 domain may comprise the mutations D399Q, D / E356Q and K370E.

[0562] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and L351Y.

[0563] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E, and L351H.

[0564] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E, and R355K.

[0565] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E, and Q355K.

[0566] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and S354K.

[0567] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and T350L.

[0568] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E and T394N.

[0569] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352Y.

[0570] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352V.

[0571] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352T.

[0572] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352L.

[0573] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352G.

[0574] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, D / E356Q, K370E and P352C.

[0575] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E and L351T.

[0576] In some embodiments, the mutated CH3 domain may include the mutations D399N, D / E356Q, K370E and L351A.

[0577] In some embodiments, the mutant CH3 domain comprises the mutations D399Q, E357Q, K439E, Y349D and S354D.

[0578] In some embodiments, the mutant CH3 domain comprises the mutations D399N, E357Q, K439E and L351R.

[0579] In some embodiments, the mutant CH3 domain comprises the mutations D399N, E357Q, K439E and L351Y.

[0580] In some embodiments, the mutant CH3 domain comprises the mutations D399N, E357Q, K439E and T350I.

[0581] In some embodiments, the mutated CH3 domain may comprise the mutations D399N, E357Q, K439E and T350V.

[0582] In some embodiments, the mutated CH3 domain may include the mutations D399Q, K439E, E357Q.

[0583] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, S354K.

[0584] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, S354W.

[0585] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, Y349R.

[0586] In some embodiments, the mutated CH3 domain may include the mutations D399N, K439E, E357Q, T350L.

[0587] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, R355W.

[0588] In some embodiments, the mutated CH3 domain may include the mutations D399N, K439E, E357Q, Q355W.

[0589] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, P395I.

[0590] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, P395G.

[0591] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, P395E.

[0592] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, P352K.

[0593] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, P352D.

[0594] In some embodiments, the mutant CH3 domain may include the mutations D399N, K439E, E357Q, L351D.

[0595] In some embodiments, the binding agent comprises two polypeptide chains; One of the two polypeptide chains comprises a first dimerization domain (DD1) comprising a mutated CH3 domain comprising one or more amino acid substitutions at positions corresponding to D399, D / E356 and / or K370 according to EU numbering; and The other of the two polypeptide chains comprises a second dimerization domain (DD2) comprising a mutated CH3 domain containing one or more amino acid substitutions at positions corresponding to D399, E357 and / or K439 according to EU numbering.

[0596] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q and K370E according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q and K439E according to EU numbering.

[0597] In some embodiments, the first dimerization domain (DD1) and / or the second dimerization domain (DD2) comprise a mutated CH3 domain further comprising amino acid substitutions at positions corresponding to Y349, T350, L351, P352 and / or S354 according to EU numbering.

[0598] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399Q, D / E356Q, K370E, Y349K and S354K according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399Q, E357Q, K439E, Y349D and S354D according to EU numbering.

[0599] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and L351W according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and L351R according to EU numbering.

[0600] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and S354M according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and L351Y according to EU numbering.

[0601] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and T350I according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and T350I according to EU numbering.

[0602] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and T350V according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and T350V according to EU numbering.

[0603] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and P352R according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and L351R according to EU numbering.

[0604] In some embodiments, the first dimerization domain (DD1) comprises a mutated CH3 domain comprising the mutations D399N, D / E356Q, K370E and P352E according to EU numbering, and the second dimerization domain (DD2) comprises a mutated CH3 domain comprising the mutations D399N, E357Q, K439E and L351R according to EU numbering.

[0605] 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.

[0606] Heterodimers can be produced by co-expressing two different polypeptide chains (eg, chain A and chain B).

[0607] In some embodiments, the polypeptide chains may comprise a dimerization domain comprising a wild-type human CH2 and a mutant human CH3 that favors the formation of heterodimers. 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.

[0608] Heterodimers were produced by transfection of a set of polypeptide chains (chain A and chain B as exemplified in Table 1) that specifically contained CH3 mutations.

[0609] [Table 1]

[0610] Depending on the ratio of chain A to chain B, co-expression of two such polypeptide chains may result in the formation of homodimers.

[0611] Thus, in some circumstances, co-expression of polypeptide chain A with polypeptide chain B may result in heterodimers of chain A and chain B, homodimers of chain A, homodimers of chain B, and mixtures thereof. It is also possible that residual monomers of chain A and / or chain B are present. Since each of the monomers, heterodimers and homodimers contains an antigen-binding domain, each component of the mixture may have some level of activity.

[0612] 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.

[0613] In other embodiments, the polypeptide chains disclosed herein may comprise mutant dimerization domains that contain mutations known in the art to favor heterodimer formation.

[0614] For example, the 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.

[0615] Exemplary embodiments of such 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 mutation T366S, L368A, Y407V in the second CH3 domain, or mutations S354C, T366W in the first CH3 domain and mutation S366S, L368A, Y407V in the second CH3 domain). mutations Y349C, T366S, L368A, Y407V), 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), and In-line mutations E236K, D278K), XmAb mutations (1st CH3 domain mutations S364H, F405A, 2nd CH3 domain mutations Y349T, T394F), DuoBody mutations (1st CH3 domain mutation F405L, 2nd CH3 domain mutation K409R), Azymetric mutations (1st CH3 domain mutations T350V, L351Y, S400E, F405A, Y407V, 2nd 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 IgA-derived 45 residues on the CH3 of IgG1 and mutations in the second CH3 domain IgA These include the IgG1-derived 57 residues in CH3), the A107 mutation (mutations K370E and K409W in the first CH3 domain, and mutations E357N, D399V, and F405T in the second CH3 domain).

[0616] Linker (L) The different modules of the polypeptide chains disclosed herein may be associated with each other via linkers.

[0617] In some embodiments, the linker used to join one or more modules of the polypeptide chain is not a cleavable linker.

[0618] In an exemplary embodiment, the linker (Lc) located immediately adjacent to the C-terminus of the dimerization domain does not comprise a cleavable linker.

[0619] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains does not comprise a cleavable linker.

[0620] In other embodiments, the linker used to join one or more modules of the polypeptide chain may comprise a non-cleavable linker.

[0621] In an exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.

[0622] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains is a non-cleavable linker.

[0623] 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.

[0624] In some embodiments, the linker immediately adjacent to the N-terminus of the dimerization domain may preferably comprise the hinge region of an antibody.

[0625] In some embodiments, the hinge region is a naturally occurring hinge region.

[0626] 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.

[0627] In some embodiments, the native hinge region is a native IgG2 hinge region. In other embodiments, the native hinge region is a native human IgG2 hinge region.

[0628] In some embodiments, the native hinge region is a native IgG3 hinge region. In other embodiments, the native hinge region is a native human IgG3 hinge region.

[0629] 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.

[0630] In some embodiments, the hinge region is a mutant hinge region.

[0631] In some embodiments, the mutated hinge region is a mutated IgG1 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG1 hinge region.

[0632] In some embodiments, the mutated hinge region is a mutated IgG2 hinge region. In other embodiments, the mutated hinge region is a mutated human IgG2 hinge region.

[0633] In some embodiments, the mutated hinge region is a mutated IgG3 hinge region. In other embodiments, the mutated hinge region is a mutated human IgG3 hinge region.

[0634] In some embodiments, the mutated hinge region is a mutated IgG4 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG4 hinge region.

[0635] In some embodiments, all modules of the polypeptide chain are linked via a non-cleavable linker.

[0636] 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, 1% or less of the total polypeptide content of a given solution or composition.

[0637] 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.

[0638] Additional exemplary embodiments of non-cleavable linkers further include linkers that upon administration of the binding agent in an individual retain their integrity for at least 1, 2, 3, 4, 5, 6, 12, 24, 48 hours or longer following administration.

[0639] In a further exemplary embodiment, the linker includes both a non-cleavable linker and a cleavable linker.

[0640] In some embodiments, the linker is not cleavable.

[0641] 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.

[0642] 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 a protease, typically an extracellular protease, such as a protease produced by a tumor or an activated immune effector cell, such as ADAMS, ADAMTS, e.g., ADAMS; ADAMS; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, such as BACE or renin; aspartic cathepsins, such as cathepsin D or cathepsin E; caspases, such as 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, such as cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, such as 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;Type II transmembrane serine proteases (TTSPs), 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 include such a linker at a position corresponding to Lc;

[0643] Exemplary embodiments of the linker 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.

[0644] In some embodiments, the hinge region or a portion thereof may be used to link the module to the 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.

[0645] In some cases, the hinge region may have one or more amino acid substitutions, insertions and / or deletions compared to the native hinge region. The mutant hinge region comprises, for example, a sequence (mutated hinge region) that is 80%-99% identical to the sequence of a native IgG1, IgG2, IgG3 or IgG4 hinge region. Exemplary and non-limiting embodiments of mutant hinge regions include 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.

[0646] 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 , (GS) n , (G4S) n , (GGS) n , (GGGS) n , (GGGGS) n , (GGSG) n , (GGGS) n and the like, where n and m can be integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater, such as 15, 20 or 25.

[0647] Specific exemplary and non-limiting embodiments of flexible linkers include those that comprise or consist 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.

[0648] 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).

[0649] The rigid linker of the present disclosure typically comprises 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).

[0650] Specific exemplary and non-limiting embodiments of rigid linkers include those that comprise or consist 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.

[0651] 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.

[0652] Although helical linkers may be characterized as rigid, they are separated into separate 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.

[0653] Specific exemplary and 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.

[0654] SEQ ID NO: 112 is of the formula X(EAAAK) n It should be understood that X and X2 are 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; X3 is absent or, when present, is 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO:112.

[0655] In an exemplary embodiment, the linker (L in Formulas II-VIII) immediately adjacent to the C-terminus of the dimerization domain is c1 (identified as) 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 that comprise or consist 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).

[0656] In an exemplary embodiment, a linker (L in Formulas II-VIII) joining the first two antigen-binding domains located at the C-terminus of the dimerization domain is c2 The linker (identified as N,N-diaminophenyl) 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 that comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 100-110 or SEQ ID NO: 112 (where n is 1).

[0657] The present disclosure also provides linkers having the addition of 1-10 amino acids (and any range or value between 1 and 10, such as 1-5) 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.

[0658] The present disclosure also provides linkers having a deletion of 1, 2, 3, 4 or 5 amino acids (and any value included within 1 and 5) at one or both of the N-terminus or C-terminus of any of SEQ ID NOs:100-112.

[0659] 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.

[0660] 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 sub-ranges within and including such ranges.

[0661] 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.

[0662] Binder Binding agents of the disclosure include, for example, the antigen-binding domains disclosed herein.

[0663] Binding agents of the present disclosure include, for example, the polypeptide chains disclosed herein.

[0664] Binding agents of the present disclosure include, for example, dimers of the polypeptide chains disclosed herein.

[0665] Binding agents of the present disclosure include, for example, multimers of the polypeptide chains disclosed herein.

[0666] Binding agents of the 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.

[0667] In some embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof.

[0668] In some embodiments, the binding agent comprises an antibody-like molecule.

[0669] In some embodiments, the binding agent may be fused to the protein scaffold.

[0670] In some embodiments, the binding agent comprises an immune cell modulating agent.

[0671] Thus, in some embodiments, the binding agents include antibodies and antigen-binding fragments thereof, such as, but not limited to, single domain antibodies from, for example, 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.

[0672] In other embodiments, the binding agents include antigen-binding fragments, such as, but not limited to, Fab, Fab', F(ab')2, complementarity determining regions, and variable regions, including VH, VHH, VL.

[0673] Binding agents also include immune cell modulating agents, 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, and the like.

[0674] 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.

[0675] 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.

[0676] 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 those 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.

[0677] A binding agent of the disclosure can be, for example, a compound represented by Formula I in N-terminal to C-terminal fashion: 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 stands for dimerization domain.

[0678] In some embodiments, L b In some embodiments, L b2 and / or L b3 does not have to exist.

[0679] In some embodiments, L c In some embodiments, L c1 , L c2 , and / or L c3 does not have to exist.

[0680] In some embodiments, L b and L c Both do not have to be present.

[0681] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0682] In some embodiments, the polypeptide chain comprises two or more antigen binding domains.

[0683] In some embodiments, the polypeptide chain comprises three or more antigen-binding domains.

[0684] In some embodiments, the polypeptide chain comprises four or more antigen-binding domains.

[0685] In some embodiments, the polypeptide chain comprises five or more antigen-binding domains.

[0686] In some embodiments, the polypeptide chain comprises six or more antigen-binding domains.

[0687] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen binding domains.

[0688] In some embodiments, the binder may be 1-12, e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-12, 4-5, 4-6, 4-7, 4-8, 4 The antibody may comprise up 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.

[0689] In some embodiments, the binding agent comprises a single polypeptide chain.

[0690] In some embodiments, the binding agent comprises two polypeptide chains.

[0691] In some embodiments, the binding agent comprises three polypeptide chains.

[0692] In some embodiments, the binding agent comprises four polypeptide chains.

[0693] In some embodiments, the binding agent comprises five polypeptide chains.

[0694] In some embodiments, the binding agent comprises six polypeptide chains.

[0695] In some embodiments, the binding agent comprises seven polypeptide chains.

[0696] In some embodiments, the binding agent comprises eight polypeptide chains.

[0697] In some embodiments, the binding agent comprises nine polypeptide chains.

[0698] In some embodiments, the binding agent comprises 10 polypeptide chains.

[0699] In some embodiments, the binding agent comprises more than 10 polypeptide chains.

[0700] In some embodiments, the binding agent has Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0701] 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).

[0702] 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).

[0703] In some embodiments, the binding agent has the formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0704] In some embodiments, the binding agent 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).

[0705] In some embodiments, the binding agent 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).

[0706] In some embodiments, the binding agent 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).

[0707] In some embodiments, the binding agent has the formula III in an N-terminal to C-terminal manner: 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).

[0708] In some embodiments, the binding agent has the formula III in an N-terminal to C-terminal manner: 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).

[0709] In some embodiments, the binding agent has the formula III in an N-terminal to C-terminal manner: 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).

[0710] In some embodiments, the binding agent has the formula III in an N-terminal to C-terminal manner: 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 the other represents Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2), and the other represents Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 3 (ABD3).

[0711] In some embodiments, the binding agent has the formula III in an N-terminal to C-terminal manner: 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 stands for antigen-binding domain 1 (ABD1), and Ab d1stands for antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3).

[0712] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in an N-terminal to C-terminal manner, the amino acid sequence of formula III, Ab a1 is an antigen-binding domain that binds to DR2, and Ab d1 is the antigen-binding domain that binds to PD-1, and Ab d2 is an antigen-binding domain that binds to CD47.

[0713] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in an N-terminal to C-terminal manner, the amino acid sequence of formula III, Ab a1 is an antigen-binding domain that binds to DR2, and Ab d1 is an antigen-binding domain that binds to CD47, and Ab d2 is the antigen-binding domain that binds to PD-1.

[0714] 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 each independently comprise the amino acid sequence of

[0715] 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 each independently comprise the amino acid sequence of

[0716] In some embodiments, the binding agent comprises two identical polypeptide chains.

[0717] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula II (having the same or different amino acid sequences).

[0718] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in formula III (having the same or different amino acid sequences).

[0719] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula IIIa (having the same or different amino acid sequences).

[0720] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula IIIb (having the same or different amino acid sequences).

[0721] 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 (wherein the antigen-binding domains have the same or different amino acid sequences).

[0722] 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.

[0723] 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.

[0724] 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.

[0725] 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.

[0726] In some embodiments, the binding agents of the present disclosure are monospecific or multispecific.

[0727] A monospecific binding agent includes a binding agent that is specific for a single epitope on a given antigen.

[0728] Exemplary embodiments of monospecific binding agents include binding agents that comprise one antigen-binding domain. Another exemplary embodiment of monospecific binding agents includes binding agents that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and framework regions. Yet another exemplary embodiment of the binding agent chain includes binding agents that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and different framework regions. Further exemplary embodiments of monospecific binding agents include binding agents that comprise 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.

[0729] Multispecific binding agents include binding agents specific for more than one epitope (of the same or different antigens) or more than one antigen. 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.

[0730] 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.

[0731] 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 specificity. 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.

[0732] In some embodiments, the binding agents of the present disclosure are monovalent or multivalent.

[0733] 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.

[0734] 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.

[0735] Exemplary embodiments of monospecific binding agents As indicated herein, the binding agents of the present disclosure may be monospecific.

[0736] Exemplary, non-limiting exemplary embodiments of monospecific binding agents include those provided herein.

[0737] For example, a binding agent may comprise only one antigen-binding domain. Alternatively, a binding agent may comprise more than one identical antigen-binding domain.

[0738] Exemplary embodiments of monospecific binding agents comprise an antigen-binding domain disclosed herein comprising ABD1, ABD2, ABD3 or a heavy chain disclosed herein.

[0739] In an exemplary embodiment, the monospecific binding agent may comprise an antigen-binding domain and a dimerization domain disclosed herein.

[0740] Exemplary embodiments of dimerization domains are provided throughout the specification and include, without limitation, a CH3 domain (including native CH3 domains and native human CH3 domains, mutated CH3 domains), and may also include a CH2 domain (native CH2 domain or mutated CH2 domain).

[0741] In some embodiments, the dimerization domain is an Fc region of an antibody or portion thereof.

[0742] In some embodiments, the dimerization domain may also comprise a hinge region of an antibody or portion thereof, hi some embodiments, the hinge region or portion thereof is a human hinge region or portion thereof.

[0743] In some embodiments, the binding agent may comprise ABD1 and a dimerization domain.

[0744] In some embodiments, the binding agent may comprise ABD2 and a dimerization domain.

[0745] In some embodiments, the binding agent may comprise ABD3 and a dimerization domain.

[0746] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7.

[0747] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0748] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21.

[0749] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28.

[0750] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35.

[0751] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42.

[0752] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49.

[0753] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56.

[0754] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63.

[0755] In some exemplary embodiments, a binding agent of the present disclosure may comprise a heavy chain and a dimerization domain having an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0756] In some exemplary embodiments, a binding agent of the present disclosure may comprise 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:71.

[0757] In some exemplary embodiments, a binding agent of the present disclosure may comprise 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:72.

[0758] In some exemplary embodiments, a binding agent of the present disclosure may comprise 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:73.

[0759] In some exemplary embodiments, a binding agent of the present disclosure may comprise 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:74.

[0760] In some exemplary embodiments, a binding agent of the present disclosure may comprise 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:75.

[0761] Other exemplary embodiments of monospecific binding agents are provided herein.

[0762] Exemplary embodiments of multispecific binding agents As indicated herein, the binding agents of the present disclosure may be multispecific.

[0763] Exemplary, non-limiting exemplary embodiments of multispecific binding agents include those provided herein.

[0764] For example, in some embodiments, a binding agent may comprise more than one antigen-binding domain, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, and where at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein.

[0765] In other embodiments, the binding agent may comprise more than one antigen-binding domain, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, where at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and where 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.

[0766] In yet other embodiments, the binding agent may comprise more than one antigen-binding domain, where at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, where at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and where 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.

[0767] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 2 (ABD2).

[0768] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 3 (ABD3).

[0769] In some embodiments, the binding agent comprises at least one antigen binding domain 2 (ABD2) and at least one antigen binding domain 3 (ABD3).

[0770] 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).

[0771] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0772] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 70.

[0773] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0774] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0775] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 70.

[0776] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0777] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0778] 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) and comprises a heavy chain having 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) and comprises a heavy chain having 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) and comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:70.

[0779] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0780] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0781] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0782] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0783] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0784] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0785] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0786] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0787] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0788] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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: 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.

[0789] In some embodiments, the binding agent comprises one or more polypeptide chains, and 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.

[0790] Other exemplary embodiments of multispecific binding agents are provided herein, including those in Table 7.

[0791] In some embodiments, the binding agent of the present disclosure comprises an antigen-binding domain that binds DR2, an antigen-binding domain that binds PD1, and an antigen-binding domain that binds CD47. In some embodiments, the 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 via blocking SIRPα / CD47 interaction. In some embodiments of the present disclosure, the binding agent may exhibit its anti-tumor activity via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0792] variant Variants of the sequences disclosed herein are also encompassed by the disclosure.

[0793] Variants encompassed by the present disclosure include those which 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.

[0794] 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).

[0795] 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.

[0796] One of ordinary skill in the art will recognize that a particular amino acid is more positively charged, neutral, negatively charged, or has 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 about 3-9 (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 4-7 (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 with positively charged side chains include, for example, arginine (R), histidine (H), and lysine (K). Amino acids with negatively charged side chains include, for example, aspartic acid (D) and glutamic acid (E). Amino acids with 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).

[0797] In some embodiments, the isoelectric point of an antibody is modified via amino acid substitution. See, for example, 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.

[0798] [Table 2]

[0799] 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., blastp program, BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gapx dropoff 50, expectation 10.0, word size 3) and activated filters.

[0800] 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.

[0801] The percentage of similarity is an indication 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.

[0802] 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 a portion of an original sequence.

[0803] In some embodiments, polypeptide chains having an amino acid sequence that is 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.

[0804] In some embodiments, the variants may have at least 80% sequence identity with the sequences disclosed herein. In other embodiments, the variants may have at least 85% sequence identity with the sequences disclosed herein. In further embodiments, the variants may have at least 90% sequence identity with the sequences disclosed herein. In further embodiments, the variants may have at least 95% sequence identity with the sequences disclosed herein. In other embodiments, the variants may have at least 99% sequence identity with the sequences disclosed herein.

[0805] 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).

[0806] 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 a hinge region of a native antibody.

[0807] 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.

[0808] 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.

[0809] 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, at least 99% identical to the CH3 domain of a native antibody.

[0810] 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.

[0811] 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.

[0812] DNA segments and vectors encoding one or more modules or entire polypeptide chains are specifically provided.

[0813] The DNA segments and / or vectors may be provided in separate vials and sold as a kit.

[0814] Sets of DNA segments that contain sequences that allow for the directed assembly of modules and cloning vectors incorporating the DNA segments or entire polypeptide chains are specifically contemplated.

[0815] DNA segments and vectors may be provided as part of kits for assembling DNA constructs capable of expressing the polypeptide chains or binding agents disclosed herein.

[0816] The kit may include at least one or more DNA segments or vectors that enable a user to generate a polypeptide chain comprising a mutant dimerization domain having amino acid substitutions at positions 356, 357, 370, 399 and / or 439 (according to the EU numbering system) as disclosed herein.

[0817] 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, altering 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, changing codon preferences, and generating splice variants. Codon-optimized nucleic acids encoding the polypeptide chains described herein are encompassed by the present disclosure.

[0818] 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.

[0819] 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.

[0820] 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. If the 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 the binding agent may be encoded by a single vector or by separate vectors (vector set).

[0821] Polypeptides are often expressed in mammalian cells. For long-term production of recombinant proteins, stable expression systems may be used, in which DNA segments are integrated into the host cell genome by the use of selection markers or maintained in 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 specific 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.

[0822] 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.

[0823] The present disclosure therefore relates to an isolated cell transformed or transfected with a vector, a nucleic acid, a set of vectors or a set of nucleic acids encoding at least one of the polypeptide chains described herein. The present disclosure therefore also relates to an isolated cell capable of expressing the polypeptide chains or binding agents disclosed herein.

[0824] 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.

[0825] The method may also include the step of purifying the binding agent from the cells or cell debris.

[0826] In some embodiments, the production method allows reaching a purity level (of the dimer) of at least 80%, at least 85%, at least 90%, at least 99%.

[0827] In some embodiments, the purity level of the binder is between 80.0% and 99.9%.

[0828] In some embodiments, the purity level of the binder is between 95.0% and 99.9%.

[0829] In some embodiments, the purity level of the binder is at least 90.0+ / -5.0%.

[0830] In some embodiments, the purity level of the binder is at least 95.0+ / -5.0%.

[0831] In some embodiments, the purity level of the binder is equal to or greater than 95%.

[0832] In some embodiments, the purity level of the binder is 97.0+ / -1.0%.

[0833] In some embodiments, the purity level of the binder is equal to or greater than 97%.

[0834] In some embodiments, the purity level of the binder is 99.0+ / -1.0%.

[0835] In some embodiments, the purity level of the binder is equal to or greater than 99%.

[0836] 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.

[0837] Typically, heterodimers are generated by co-transfection 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.

[0838] Heterodimers can also be made by transfecting cells with a single vector encoding both polypeptide chains. The collected supernatants may contain heterodimers or a mixture of monomers, heterodimers and / or homodimers.

[0839] 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.

[0840] The method may also include the step of isolating and / or purifying the binding agent from impurities.

[0841] The methods of the present disclosure therefore result in compositions comprising homodimers, heterodimers or mixtures of monomers, heterodimers and / or homodimers.

[0842] 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.

[0843] 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.

[0844] 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.

[0845] In exemplary embodiments, the polypeptide chains or binding agents of the 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).

[0846] Therapeutic moieties include, but are not limited to, yttrium-90, scandium-47, rhenium-186, iodine-131, iodine-125, and many others recognized by those of skill in the art, such as 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.

[0847] In another exemplary embodiment, the polypeptide chains or binding agents of the present disclosure are conjugated with a detectable moiety, including, for example and 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, for example and without limitation, 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 (for example and without limitation, 125 I, In 111 , Tc 99 , I 131 and positron emitting isotopes for PET scanners), 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 a signal from the detectable moiety to be detected.

[0848] Pharmaceutical Compositions 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 pharma- ceutically acceptable carrier.

[0849] In some embodiments, the pharmaceutical composition comprises a conjugated polypeptide chain or a conjugated binding agent as disclosed herein. In some embodiments, the pharmaceutical composition comprises a polypeptide chain or a binding agent conjugated to a therapeutic moiety. In some embodiments, the pharmaceutical composition comprises a polypeptide chain or a binding agent conjugated to a detectable label.

[0850] In addition to the active ingredient, the pharmaceutical composition may contain pharma- ceutical acceptable carriers, including water, PBS, saline, gelatin, oil, alcohol, and other excipients and auxiliaries that facilitate processing of the active compound into a pharma- ceutical usable preparation. In other cases, such preparations may be sterilized.

[0851] As used herein, a "pharmaceutical composition" refers to a therapeutically effective amount of an agent together with pharma- ceutically 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 dosing 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 of 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 affect 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, pericanceromally, mucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, and intratumorally.

[0852] Furthermore, as used herein, "pharmaceutical acceptable carriers" or "pharmaceutical carriers" are known in the art and include, but are not limited to, 0.01-0.1M or 0.05M phosphate buffer or 0.8% saline. Additionally, such pharmaceutical 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, as well as electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

[0853] For any compound, the therapeutically effective dose may be estimated initially either in cell culture assays or animal models, such as mice, rats, rabbits, dogs, or pigs. Animal models may also be used to determine concentration ranges and routes of administration. Such information may 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 may 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 that is lethal to 50% of the population) statistic may be calculated and compared. 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.

[0854] 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.

[0855] In some embodiments, the pharmaceutical composition is made from a binder having a purity level of 80.0%-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%.

[0856] In some embodiments, the pharmaceutical composition comprises a binding agent that is stable in solution under one or more stress conditions as described herein.

[0857] How to use The polypeptide chains and binding agents of the disclosure may be used for the treatment of a disorder or disease.

[0858] In some embodiments, the polypeptide chains and binding agents may be used to target therapeutics and / or diagnostics to target cells, circulating proteins or tissues.

[0859] In some embodiments, the polypeptide chains and binding agents may be conjugated to a therapeutic moiety and used for therapeutic methods.

[0860] In some embodiments, the polypeptide chains and binding agents may be conjugated to a detectable moiety and used for detection or diagnostic methods.

[0861] In some embodiments, the polypeptide chains and binding agents of the present disclosure may be used to target tumors in vivo.

[0862] In some embodiments, the polypeptide chains and binding agents are used to promote tumor regression and / or reduce tumor volume in vivo.

[0863] The polypeptide chains and binding agents of the present disclosure may therefore be used for the treatment of cancer.

[0864] The methods of the disclosure may include administering to an individual in need thereof the polypeptide chains, binding agents, or mixtures disclosed herein, or a pharmaceutical composition comprising said polypeptide chains, binding agents, or mixtures.

[0865] In some embodiments, the polypeptide chains and binding agents are administered in combination with a chemotherapeutic agent.

[0866] According to the present disclosure, an individual in need may be a human. Further according to the present disclosure, an individual in need may be an animal.

[0867] In some embodiments, the treatment of disorders or diseases caused by or associated with the expression of neoantigens is specifically contemplated.

[0868] In some embodiments, the treatment of disorders or diseases caused by or associated with overexpression of the antigen is specifically contemplated.

[0869] In some embodiments, the disorder or disease may be cancer.

[0870] In other embodiments, the disorder or disease may be an infectious disease.

[0871] In other embodiments, the disorder or disease may be an immune dysregulation.

[0872] In other embodiments, the disorder or disease may be a metabolic dysregulation.

[0873] In some embodiments, the binding agent is administered intravenously.

[0874] In some embodiments, the binding agent is administered by injection.

[0875] The polypeptide chains and binding agents of the present disclosure may be used for detection purposes.

[0876] Detection of a particular target may be performed in vitro by contacting a sample containing or suspected of containing a target with a polypeptide chain or binding agent comprising an antigen-binding domain for such target and quantifying a signal associated with positive or negative binding using a detection device.

[0877] The sample may originate from a mammal (e.g., a human). The sample may be a tissue sample obtained from a mammal or a cell culture supernatant.

[0878] In some embodiments, the sample may be a serum sample, a plasma sample, a blood sample, semen, or ascites fluid obtained from a mammal.

[0879] Detection of a particular target may be performed in vivo by administering to an individual a polypeptide chain or binding agent comprising an antigen-binding domain for such target and quantifying a signal associated with positive or negative binding using a detection device.

[0880] Once the presence of a target is detected in a sample or in an individual, an agent (eg, an antibody, a small molecule, a polypeptide chain, or a binding agent disclosed herein) may be administered to the individual.

[0881] In addition to the embodiments described and provided in this disclosure, the following non-limiting embodiments are specifically contemplated.

[0882] Tables 3, 4, 5 and 6 show VHHs that have biological activity on their own (e.g. as fusions with a dimerization domain) and as part of multimeric, multivalent and / or multispecific binding agents, as exemplified in Table 7. EXAMPLES

[0883] Example 1 Immunization of animals and construction of immune libraries Single domain antibodies were generated by immunizing alpacas (Vicugna pacos) or llamas (llama glama) with human antigens, cDNA plasmids encoding the human antigens, and / or cells overexpressing the human antigens.

[0884] For example, anti-DR2 single domain antibodies were generated by immunizing alpaca (Vicugna pacos) with DR2 antigen including human DR2 protein, human DR2 cDNA plasmid, and human DR2 overexpressing cell line; SH-SY5Y-D2 cells. Anti-PD-1 single domain antibodies were generated by immunizing alpaca (Vicugna pacos) with PD-1 antigen including human PD-1 protein, human PD-1 cDNA plasmid, and human PD-1 overexpressing cell line; CHO-PD1 cells. For anti-CD47, single domain antibodies were generated by immunizing llama (llama glama) with CD47 antigen including human CD47 protein, human CD47 cDNA, and (activated) human PBMCs.

[0885] The sequences of single domain antibodies were revealed from clone picking following phage display panning of the reconstituted immune library against the antigen.

[0886] DNA fragments encoding the VHHs were generally subcloned into constructs for expression of the polypeptide chains (see, for example, Table 6 or Table 7) and for in vitro and in vivo testing.

[0887] Controls were also generated by replacement of one or more VHHs with HEWL-specific VHHs.

[0888] Example 2 Transfection, expression, and purification of binding agents Transfection and expression of polypeptide chains in ExpiCHO or Expi293 cells Protein dimers (e.g., homodimers or heterodimers) were expressed from DNA constructs in 2.5 mL or 400 mL culture volumes using the ExpiCHO™ Expression System (Thermo Fisher, Catalog No. A29133) or the Expi293™ Expression System (Thermo Fisher, Catalog No. A14635).

[0889] Briefly, freshly thawed CHO cells were passaged two or more times and allowed to recover in culture prior to transfection. Cells were then cultured at 4 × 10 6 ~6×10 6 The cells were passaged every 3-4 days until they reached 2 x 10 cells / mL, at which point they were cultured at 2 x 10 in ExpiCHO™ Expression Medium warmed to 37°C. 5 ~3×10 5 The day before transfection, cells were diluted to 3 × 10 6 ~4×10 6 On the day of transfection, cells were diluted to 6 x 10 cells / mL. 6 The DNA was further diluted to 10 cells / mL. 1 μg DNA / mL culture volume was diluted with cold OptiPRO™ medium (100 μL for 2.5 mL culture volume; 16 mL for 400 mL culture volume). ExpiFectamine™ CHO Reagent (8 μL for 2.5 mL culture volume; 1280 μL for 400 mL culture volume) was added to the medium containing DNA and incubated with the ExpiFectamine™ / DNA complexes for 1-5 minutes at room temperature. The DNA complexes were then transferred to the culture with swirling (6×10 6Cells were incubated at 37°C with shaking (INFORS HT shaker, 125 rpm) under 8% CO2 and 80% humidity. 18-22 hours after the start of transfection, ExpiCHO™ Feed (0.6 mL per 2.5 mL culture volume; 96 mL per 400 mL culture volume) and ExpiCHO™ Enhancer (15 μL per 2.5 mL culture volume; 2.4 mL per 400 mL culture volume) were added to the cells. Cells were returned to an INFORS HT incubator set at 37°C with shaking at 125 rpm (25 mm orbit) under 8% CO2 and 80% humidity. Eight days after transfection, the supernatant was clarified by centrifugation at 4000×g for 30 minutes. Supernatants were filter sterilized using Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units, 1000 mL filter units (Thermo Science, Cat. No. 567-0020) and stored at 4° C. or frozen for later analysis.

[0890] Freshly thawed HEK293 cells were passaged two or more times prior to transfection and allowed to recover in culture. The cells were then cultured at 3 × 10 6 ~5×10 6 The cells were passaged every 3-4 days until they reached 3×10 cells / mL, at which point they were cultured at 3×10 in Expi293™ Expression Medium warmed to 37°C. 5 ~5×10 5 The day before transfection, cells were diluted to 2.5 x 10 6 ~3×10 6 On the day of transfection, cells were diluted to 3 x 10 cells / mL. 6The 1 μg DNA / mL culture volume was diluted with OptiMEM™ I Reduced Serum Medium to obtain a final volume of 150 μL for 2.5 mL culture volume and 24 mL for 400 mL culture volume. ExpiFectamine™ 293 Reagent (8 μL for 2.5 mL culture volume; 1.3 mL for 400 mL culture volume) was added to Opti-MEM™ I Reduced Serum Medium (140 μL for 2.5 mL culture volume; 22.5 mL for 400 mL culture volume) and incubated at room temperature for 5 minutes. The diluted ExpiFectamine™ was added to the diluted DNA and incubated at room temperature for 15 minutes. The ExpiFectamine™ / DNA solution was added dropwise (3×10 6 The cells were transferred to a 100% culture (1000 cells / ml) of 1000 mM NaCl. The cells were incubated at 37°C with 8% CO2 and 80% humidity with shaking (INFORS HT shaker, 125 rpm) overnight. 18-22 hours after the start of transfection, ExpiFectamine™ 293 Transfection Enhancer 1 (15 μL per 2.5 mL culture volume; 2.4 mL per 400 mL culture volume) and ExpiFectamine™ 293 Transfection Enhancer 2 (50 μL per 2.5 mL culture volume; 24 mL per 400 mL culture volume) were added to the cells. The cells were returned to an INFORS HT incubator set at 37°C with 8% CO2 and 80% humidity with shaking at 125 rpm (25 mm orbit). Five days after transfection, the supernatant was clarified by centrifugation at 4000×g for 30 minutes. Supernatants were filter sterilized using Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units, 1000 mL filter units (Thermo Science, Cat. No. 567-0020) and stored at 4° C. or frozen for later analysis.

[0891] purification Proteins are purified using 3 mL MabSelect™ SuRe™ resin with a gravity column (GE Healthcare, Catalog No. 17-5438-02) or 40 mL MabSelect™ SuRe™ resin with AKTA PURE (GE Healthcare, Piscataway, NJ) depending on the volume of the supernatant. The resin was incubated with 0.5 M NaOH overnight and equilibrated with Tris-based buffer pH 7.4 (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) before injection. The supernatant was applied to the gravity column or to the 40 mL column at 5 ml / min. The resin column was washed with 3 CV (column volumes) of Tris-based buffer pH 7.4 at a flow rate of 10 mL / min. Proteins were eluted with 3 CV of 0.1 M citric acid pH 3 at 10 mL / min. Fractions identified by proteins from the visual output of the chromatogram (absorbance at 280 nm) were pooled together. The pooled fractions were neutralized with 1 M Tris-HCl pH 9.0 to achieve a pH of approximately 5-6 before being transferred into phosphate buffered saline (PBS) pH 6.0 buffer prepared from PBS 10× pH 7.2 (15 mM potassium dihydrogen phosphate, 1552 mM sodium chloride, 27 mM sodium phosphate dibasic, ThermoFisher, catalog no. 70013073).

[0892] Buffer exchange was performed by sample concentrator for proteins purified from gravity column, or by dialysis or desalting column for proteins purified from AKTA PURE. Proteins purified from gravity column were concentrated by sample concentrator VivaSpin2, 50 kDa MWCO (GE Healthcare, Cat. No. 28932257) by centrifugation at 3500-4000×g at 4°C, then diluted 4-fold with PBS pH6, and repeated until the sample was 200-fold. Dialysis was performed in 4 L of PBS pH6 at 4°C overnight using 7 kDa molecular weight cutoff dialysis tubing (ThermoFisher, Cat. No. 68799). Meanwhile, desalting column was incubated with 0.5 M NaOH overnight and equilibrated with PBS pH6. A volume of 15 mL of the neutralized protein sample was loaded onto a HiPrep 26 / 10 desalting column (GE Healthcare, Cat. No. 17-5087-02) at 0.5 mL / min, and then the protein was eluted with 2 CV of PBS pH 6. The loading and elution steps were repeated until no neutralized protein sample was left from the affinity column elution. Fractions identified by protein from the visual output of the chromatogram (absorbance at 280 nm) were pooled together.

[0893] Samples were filter sterilized using Nalgene™ Rapid-Flow™ Sterile Disposable Filter Units, 150 mL filter units (Thermo Scientific, Cat. No. 565-0010). Final protein samples were quantified using the Pierce™ Bicinchoninic Acid Protein Assay kit (ThermoFisher, Cat. No. 23227) and tested for endotoxin levels using Endosafe® LAL Reagent cartridges (Charles River, Cat. No. PTS2005). Final protein samples were analyzed on SDS-PAGE gels under reducing or non-reducing conditions.

[0894] Production of the binder in CHO cells followed by conventional two-step purification using Protein A affinity chromatography followed by size exclusion chromatography (SEC) resulted in purities (of the dimer) ranging from 95.5% to 99.5%.

[0895] The purity of KC020 was found to be greater than 97% after size exclusion chromatography (SEC) and SDS caliper. The DNA sequences of the top three clones were verified; the charge profile and glycosylation profile of KC020 were found to be similar to that of the monoclonal antibody. The table below shows the KC020 titers after 14 days from three representative clones selected for cell line development.

[0896] [Table 3]

[0897] SDS-PAGE and Western blotting Samples were prepared for SDS-PAGE analysis under reducing or non-reducing conditions in NuPAGE™ LDS Sample Buffer (ThermoFisher Cat. No. NP0007) with or without NuPAGE™ Sample Reducing Agent (ThermoFisher Cat. No. NP0004). Samples under reducing conditions were denatured by heating at 70°C for 10 min. Samples (16 μL) were loaded next to BSA standards on 3-8% Tris-Acetate minigels (1.5 mm, 15 wells). Electrophoresis was performed using an X-Cell SureLock™ minigel apparatus at 125 volts for approximately 1 hour. Gels were stained using GelCode™ stain (ThermoFisher Cat. No. 24594).

[0898] For Western blot analysis, proteins were transferred to nitrocellulose membranes using the iBlot™ system (Thermo Fisher, Cat. No. IB301031) according to the manufacturer's instructions.

[0899] Detection of the His tag was performed with Anti-Penta His-HRP antibody. Briefly, the membrane was blocked by incubation in 20 ml of Qiagen blocking buffer (Qiagen, 1018862) for 1 hour at room temperature with shaking, followed by incubation in 20 ml of Starting Blocking™ T20 (PBS) Blocking (Thermo Fisher, Cat. No. 37528) for 1 hour at room temperature with shaking. The membrane was washed 3 times for 10 minutes with 1X TBS Tween™-20. The membrane was incubated with Anti-Penta His-HRP (Qiagen, Cat. No. 1014992) previously diluted 1:2000 in blocking buffer for 1 hour at room temperature with shaking. The membrane was washed 3 times for 10 minutes with 1X TBS Tween™-20. Signals were visualized using Super Signal™ West Pico PLUS (ThermoFisher, Cat. No. 34080) according to the manufacturer's instructions. Images were recorded using an Azure Biosystem imaging system.

[0900] The results of these experiments indicate that the binding agents (homodimers or heterodimers) of the present disclosure can be efficiently expressed in cells and purified, and that the polypeptide chain formats disclosed herein make it possible to achieve yields of binding agent (homodimers or heterodimers) in the gram / L range (data not shown).

[0901] Example 3 In vitro and in vivo testing of binding agents containing anti-D2 antigen-binding domains cell line OCI-AML3 cells are human-origin acute myeloid leukemia cells that are used as target cells in PBMC-dependent cytotoxicity assays.THP-1 is a monocytic cell line derived from a patient with acute monocytic leukemia.THP-1 cells are used as target cells in PBMC-dependent cytotoxicity assays.

[0902] NCI-H82, NCI-H69 and NCI-H510A are human small cell lung cancer (SCLC) cell lines used in SCLC xenograft tumor models in immunodeficient mice. NCI-H82, NCI-H69 and NCI-H510A overexpress dopamine receptor D2 and are used herein as models for D2 targeting studies in particular.

[0903] NCI-H727 cells are human non-small cell lung cancer (NSCLC) cells used for NSCLC xenograft tumor model in immunodeficient mouse models. D2 has been shown to be upregulated in NCI-H727 cells.

[0904] PANC1 is a human pancreatic cell line used for xenograft tumor modeling in immunodeficient mouse models.

[0905] Manufacture of proteoliposomes To determine the binding specificity of the anti-D2 binders, human D2-presenting proteoliposomes were prepared using the ProteoLiposome BD kit from CellFree Science (CellFree Science, Cat. No. CFS-CPLE-BD) according to the supplemented protocol.

[0906] Briefly, the required volumes of nuclease-free water, 5X transcription buffer, NTP mix, RNase inhibitor, SP6 RNA polymerase and plasmid DNA were mixed in a 1.5 mL tube and incubated at 37°C for 6 hours to perform the transcription reaction. To perform the translation reaction, the mixture of mRNA mix, feeding buffer, WEPRO7240, creatine kinase and Asolectin liposomes was prepared as per the manufacturer's instructions. The translation reaction mixture was added to a slide-A-Lyzer Mini Dialysis device (Thermo Scientific, Cat. No. 69570) and incubated at 15°C for 72 hours to perform the translation reaction.

[0907] The proteoliposomes were purified by washing with PBS and centrifugation at 15000 rpm for 10 min at 4° C. After repeating the washing step three times, the collected proteoliposomes were resuspended in an appropriate amount of PBS and stored at −80° C.

[0908] Binding of anti-D2 binders (e.g., Table 6) to human D2-presenting proteoliposomes (Figure 1, Figure 25, Figure 26) was evaluated by ELISA. Briefly, proteoliposomes or empty liposomes containing target proteins were coated onto 96-well plates. The plates were covered and left at 4°C overnight. The next day, the plates were washed once with PBS and blocked with blocking buffer for 1 h at room temperature. Binders were tested at the indicated concentrations diluted in blocking buffer and incubated at 37°C for 1 h. After incubation, the plates were washed 3 times with washing buffer. The plates were incubated with anti-human Fc-HRP (Sigma-Aldrich, Cat. No. AP113P) diluted 1:5000 for 1 h at room temperature and then washed 3 times with PBS-T washing buffer. Signal was developed using SuperSignal™ ELISA Pico Chemiluminescent Substrate (Thermofisher, Cat. No. 37069). Plates were read on a SpectraMax™ i3x Multi-Mode Microplate Reader (Molecular Devices).

[0909] Representative results of such experiments are presented in Figure 1, which demonstrates that the anti-D2 binding agents tested bind specifically to D2 proteoliposomes (EC50 of 0.1488 nM) with very high affinity.

[0910] GPCR BRET assay The effect of various anti-D2 binders on signaling downstream of D2 was evaluated (Figure 2). Prior to transfection, HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin / streptomycin and 10% fetal bovine serum. Polyethylenimine (PEI) transfection reagent was used for all transfections. HEK293 cells were co-transfected with hDR2 and one of the biosensors. The total amount of DNA transfected was kept constant at 1 μg per mL of cell culture to be transfected. The PEI:DNA ratio (μg:μg) was fixed at 3:1. DNA and PEI were first diluted separately in 150 mM NaCl. The volume of diluent in each tube corresponds to 5% of the volume of the cell culture to be transfected. After dilution of DNA and PEI, the PEI-containing solution was added to the DNA solution, and the DNA / PEI mixture was immediately vortexed for 5 seconds. The DNA / PEI mixture was incubated at room temperature for at least 20 min to allow the formation of DNA / PEI complexes. During the incubation, the HEK293 cells were detached, counted, and resuspended in culture medium. At the end of the incubation period, the DNA / PEI mixture was added to the cells. The cells were finally distributed at a density of 35,000 cells in 96-well plates.

[0911] 48 hours after transfection, BRET experiments were performed to detect downstream signaling of D2 in agonist test mode. Culture medium was aspirated and replaced with 50 μl of Hank's Balanced Salt Solution buffer from a 96-well plate. For assays performed in HEK239 cells, 10 μl of 10 μM e-Coelenterazine Prolume Purple was added to each well at a final concentration of 1 μM. Dopamine was added to each well as a positive control using an HP D300 digital dispenser. Dopamine was assayed with each biosensor at 16 concentrations. Antibodies were added manually to each well (1 μM final concentration per well). Anti-D2 binders were assayed in duplicate with each biosensor at 8 concentrations. Cells were incubated with dopamine for 10 minutes at room temperature and with antibodies for 60 minutes at room temperature. BRET signals were collected using an integration time of 0.4 seconds on a Synergy NEO plate reader.

[0912] The EC50 of various anti-D2 binders in GPCR BRET assays transfected with various G protein sensors is presented in Figure 2. Dopamine was used as a positive control to demonstrate GPCR response with G proteins including Gαz, Gαi2, GαoA, and Barr2. Unexpectedly, all anti-D2 binders including KC011, KC012, KC013, and KC014 demonstrated specific GPCR signaling through Gαz. The results indicate that anti-D2 binders bind to unique epitopes on D2 and initiate highly specific signaling pathways.

[0913] RNAseq and qRT-PCR validation The mechanism of action of KC013 was investigated in vitro in NCI-H69 cells through the use of next-generation sequencing technology to evaluate the global transcriptional profile after treatment. Suspension NCI-H69 cells were harvested and 5 × 10 6The cells were resuspended at a concentration of 1.0 μM cells / mL. Ten million cells per 2 mL of complete medium were transferred to wells of a 6-well plate. KC013 and negative control KC018 (in which the VHH portion was replaced with the anti-4HEM VHH shown in SEQ ID NO: 114) were added to the appropriate wells at a final concentration of 1.0 μM. The cells were returned to a 37° C., 5% CO2 incubator and treated for 3 hours. After the incubation period, the cells were homogenized using QiaShredder columns and total RNA was isolated from the disrupted cell lysates using the RNeasy RNA Isolation Kit as per the manufacturer's protocol. A total of 500 ng of total RNA was snap frozen and shipped to Genome Quebec for analysis.

[0914] RNA integrity was assessed using an Agilent bioanalyzer. Libraries were prepared using the NEB mRNA stranded NEBNext Ultra II kit (NEB, Cat. No. E7645S) incorporating an mRNA enrichment step followed by an Illumina library QC assessment. Samples were sequenced on a NovaSeq 6000 PE100 instrument (Illumina) at 25 million reads / sample. Reads were mapped to the ENSEMBL GRCh38 (Homo sapiens) release 86 human reference genome sequence and annotation. This resulted in count data for 58,051 genes. Genes with less than 1 count per million (CPM) in at least three samples (i.e., minimum group size) were filtered out, leaving 14,983 genes for downstream analysis.

[0915] Gene lists were constructed by using a maximum false discovery rate (FDR) of 0.05 for KC013 treatment compared to KC018 negative control. Enrichment analysis was performed using Enrichr (Icahn School of Medicine, Mount Sinai; http: / / amp.pharm.mssm.edu / Enrichr / ). Over-representation analysis was performed using the PANTHER database (University of Southern California; http: / / pantherdb.org) focusing on Gene Ontology (GO) biological pathways and protein classes using Fischer's exact test with FDR multiple testing correction. The gene list used consisted of 191 upregulated and 634 downregulated genes, for a combined total of 825 significantly altered genes (FDR<0.05) (Figure 3). Pathway analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen) (Figure 4A&B). Heatmaps were constructed using Log2-transformed transcript read counts along with complete linkage clustering and Pearson distance measurement (FDR<0.05, 825 genes).

[0916] Pathway analysis of the RNA-Seq data revealed downregulation of several cancer-related pathways, including CREB, Wnt, and mTOR signaling pathways, as well as activation of p53 through multiple signaling pathways, compared to negative control (KC018)-treated cells, including NCI-H69 and NCI-H82 SCLC.

[0917] We validated the pathway analysis of RNA-Seq data by quantitative reverse transcriptase PCR (qRT-PCR) using three different cell lines, including NCI-H69 (Figure 5A), NCI-H82 (Figure 5B), and HEK-D2 (Figure 5C). Briefly, suspension cells were grown in complete medium in T175 flasks. Cells were plated at 5 × 10 per well in triplicate 6-well plates. 6 Cells / mL were seeded and treated in triplicate with 1.0 μM KC013 or 1.0 μM KC018 for 3 and 6 h. After the indicated incubation period, replicate samples were scraped off and transferred to 15 mL Falcon tubes for each time point. Cells were washed twice with 1x PBS and cell pellets were flash frozen in liquid nitrogen and stored at -80°C for downstream analysis. Total RNA was extracted using the Quick-RNA Miniprep Plus kit (Cedarlane, Cat. No. R1057) according to the manufacturer's instructions. The amount of RNA recovered for each sample was quantified by measuring absorbance at 260 and 280 nm using a NanoPhotometer. 1 μg of RNA was used for reverse transcription using iScript™ Reverse Transcription Supermix according to the manufacturer's instructions. The resulting cDNA was then diluted 1:20 using ddH2O containing 40 ng / mL glycogen.

[0918] Gene expression analysis was performed using SsoAdvanced Universal IT SYBR Green in an iQTM5 Real-Time PCR System using the relative standard curve method. The final reaction volume was 10 μl per well of a 96-well plate containing 1x SYBR green mix, 200 nM final primer concentration, 2 μl cDNA and ddH2O. Raw qRT-PCR data for target genes was collected as the number of cycles required for a fluorescent signal to cross background level (i.e., threshold). These values ​​are known as cycle threshold or Ct values. To determine the relative gene expression levels of target genes after treatment with KC013 vs. KC018, the geometric mean of the multiple internal control gene method was used, followed by the ΔΔCt calculation method described below. This approach is described in detail in Vandesompele J. et al., Genome Biology 2002 and Hellemans et al., Genome Biology, 2007. Final data was presented as fold change in gene expression normalized to a selected common endogenous reference gene compared to the control binder. Data were presented as the average of three biological replicates performed in three technical replicates. Results were representative data from assays performed at least three times in triplicate.

[0919] The changes in gene expression in NCI-H69 cells upon treatment with KC013 are shown by a heatmap (Figure 3), with color and intensity variations representing changes in individual gene expression. The results of this experiment indicate that a total of 825 significantly altered genes (FDR<0.05) were observed after treatment with KC013, with 634 genes being downregulated.

[0920] Figure 4 depicts the downregulated signaling pathways and protein classes from PANTHER analysis of RNAseq results. The top downregulated signaling pathways include the Wnt pathway, the gonadotropin releasing hormone (GnRH) pathway, and the cadherin signaling pathway (Figure 4A). The most downregulated protein classes by PANTHER analysis are gene-specific transcription regulators (Figure 4B). The Wnt pathway and the cadherin signaling pathway have been suggested to play important roles in tumor cell survival, proliferation, tumorigenesis, and progression. Aberrant Wnt signaling pathways have been shown to play a key role in the initiation of many cancer cells by regulating cancer stem cells (CSCs). The downregulation of these pathways and transcription regulators suggests an antitumor mechanism of KC013.

[0921] Consistent with the RNAseq profiles, distinct signaling components in the Wnt, mTOR, and cAMP pathways were downregulated in NCI-H69 (Figure 5A), NCI-H82 (Figure 5B), and HEK-D2 (Figure 5C) cells upon treatment with KC013.

[0922] Single-dose PK in CB-17 Fox Chase SCID mice CB-17 Fox Chase SCID mice (6-8 weeks old, female) purchased from University Health Network (UHN, Toronto, ON) were used for PK studies. Mice were housed in a pathogen-free environment in the UHN animal facility. Animal studies were performed in accordance with the Canadian Council on Animal Care (CCAC) guidelines and animal use protocols approved by the UHN Animal Care Committee. Briefly, mice were divided into two experimental groups (6 mice / group), and mice in each group received a single dose of 30 mg / kg trastuzumab (UHN pharmacy) or KC013 by either intravenous (Figure 6A) or intraperitoneal (Figure 6B) injection. For each study, blood samples were collected at 0.083 hours (for PK iv studies only), 1 hour (for PK ip studies only), 3 hours, 6 hours, 1 day, 3 days, 7 days, 10 days, 15 days, 21 days, and 28 days after injection. Blood samples were centrifuged and serum samples were collected and stored at -80°C until the time of analysis. Trastuzumab and KC013 concentrations in mouse serum were measured by quantitative enzyme-linked immunosorbent assay (ELISA) as described below. Pharmacokinetic data analysis was performed by non-compartmental methods using Phoenix WinNonlin software (Certara).

[0923] Antibodies in mouse serum were quantified by quantitative ELISA (Figure 6). Briefly, mouse anti-human IgG antibody (Bio-rad, Cat. No. MCA878G, using a dilution of 1 / 200) was used to coat 96-well plates overnight at 4°C. The next day, the plates were washed three times and blocked with 2% BSA for 1 h at room temperature. Meanwhile, standards (STD, Bio-rad, Cat. No. MCA6092), quality control (QC), and each serum sample were diluted in wash buffer containing 1% mouse serum. 100 μl of STD, QC, and diluted serum samples were added to the 96-well plate and incubated for 2 h at room temperature. After three washes, the plate was incubated with secondary antibody (goat anti-human Fc antibody, 1 / 5000 dilution, Cedarlane, Cat. No. 109-035-098) for 1 h at room temperature. After washing the plate three times, 100 μl of detection substrate was added to each well. When the highest STD reading reached 0.9-1.1 at 650 nm wavelength, 50 μl of stop solution was added to each well to stop the reaction. The plate was read at 450 nm wavelength for antibody measurement.

[0924] The results of these experiments indicate that IV injection of KC013 (FIG. 6A) showed a T1 / 2 of 214 hours, while IP injection (FIG. 6B) showed a T1 / 2 of 159 hours. The Cmax achieved for KC013 is similar to that of the fully human antibody trastuzumab. This data demonstrated the long half-life of the anti-D2 binder in SCID mice.

[0925] Tumor prevention model in CB-17 Fox Chase SCID mice CB-17 Fox Chase SCID mice (6-8 weeks old, female) were purchased from either Charles River Laboratories (St. Constant, QC) or UHN (Toronto, ON). Animal care and research guidelines were the same as those described above.

[0926] The efficacy of anti-D2 binders was evaluated in various tumor prevention models, NCI-H510A (Figure 7C, Figure 8), NCI-H727 (Figure 7A, Figure 8), PANC1 (Figure 8), and NCI-H69 (Figure 7B, Figure 8). For each model, tumor cells (8 million cells / mouse for NCI-H510A, 6 million / mouse for NCI-H727, 5 million / mouse for PANC1, or 3 million / mouse for NCI-H69) in DPBS were injected subcutaneously (sc) into the right flank of mice. One day after cell inoculation, mice inoculated with each cell line were randomized into two experimental groups (10 or 5 mice / group), and mice in each group received 16 mg / kg ip. of either KC018 (negative control) or KC013 twice a week for a total of 8 weeks. Tumor volumes were measured using calipers and mice were weighed once or twice weekly. Tumor volume was calculated using the formula: 1 / 2(length x width) 2 For calculation of percentage of tumor growth inhibition (TGI), KC013 treated groups were compared with their respective negative controls. TGI was calculated by the following formula:

[0927]

number

[0928] TV day z represents the tumor volume of an individual animal on the specified study day (day z), and TV day x represents the tumor volume of an individual animal on the staging day (day x).

[0929] Statistical tests were performed by Student's t test (two-tailed).

[0930] As shown in FIG. 7, KC013 demonstrated 49% tumor growth inhibition in the NCI-H727 tumor preventive model (FIG. 7A), 46% tumor growth inhibition in the NCI-H69 tumor preventive model (FIG. 7B), and 77% tumor growth inhibition in the NCI-H510A tumor preventive model (FIG. 7C) compared to the isotype control.

[0931] As shown in Figure 8, KC013, KC012, and KC014 demonstrated universal tumor growth inhibition in all of the tumor prevention SCLC tumor models, with KC013 providing the best in vivo efficacy over other anti-D2 binders. Since all of these anti-D2 binders initiated unique Gαz signals in the BRET assay, the in vivo efficacy achieved in the SCLC tumor models may be related to the unique Gαz signals in tumor cells. The in vivo efficacy is also explained by the downregulated signaling pathways that are important for tumor progression.

[0932] Example 4 In vitro and in vivo testing of binding agents containing anti-PD1 and anti-CD47 antigen-binding domains ELISA binding assay Binding of binders containing anti-PD-1 and CD47 antigen-binding domains to recombinant human or cynomolgus PD-1 (Cedarlane, Catalog No. HPLC-10377-H08H-100) or recombinant human or cynomolgus CD47 (Cedarlane, Catalog Nos. 12283-H08H-200 and 90869-C08H) was assessed by ELISA (Figure 9A, Figure 9B, Figure 9C, Figure 9D, Figure 22, Figure 23). Pembrolizumab was used as a positive control in the PD1 binding assay. A monoclonal anti-CD47 antibody (clone B6H12) was used as a positive control in the CD47 binding assay (Biolegend, Catalog No. 401401).

[0933] Briefly, recombinant proteins were coated onto 96-well plates. Plates were covered and placed at 4°C overnight. The following day, plates were washed once with PBS and blocked with blocking buffer for 1 h at room temperature. Binders were tested at the indicated concentrations, diluted in blocking buffer and incubated at 37°C for 1 h. After incubation, plates were washed 3 times with wash buffer. Plates were incubated with anti-human Fc-HRP (Sigma-Aldrich, Cat. No. AP113P) diluted 1:5000 for 1 h at room temperature and then washed 3 times with PBS-T wash buffer. Signal was developed using SuperSignal™ ELISA Pico Chemiluminescent Substrate (Thermofisher, Cat. No. 37069). Plates were read on a SpectraMax™ i3x Multi-Mode Microplate Reader (Molecular Devices).

[0934] As shown in Figure 9A, the anti-PD1 binder KC009 efficiently binds to human, cynomolgus monkey, and rat recombinant PD1 proteins. The cross-species binding activity of KC009 implies the feasibility of using cynomolgus monkeys and rats as toxicity models for KC009.

[0935] The binding of KC036 was compared to pembrolizumab. Both KC036 and pembrolizumab were diluted 1 / 5 starting at 500 nM. As shown in Figure 9B, KC036 binds similarly to pembrolizumab by ELISA. The negative control KC036 did not bind to PD-1 recombinant protein.

[0936] The binding of KC015 was compared to the anti-CD47 antibody B6H12 (ThermoFisher, Cat. No. 14-0479-82). Both KC015 and B6H12 antibodies were diluted 1 / 5 starting at 50 nM. As shown in Figure 9C, KC015 showed higher binding affinity than clone B6H12 by ELISA. The negative control KC016 did not bind to the CD47 recombinant protein.

[0937] As shown in Figures 9C and 9D, the anti-CD47 binding agents bound efficiently to human and cynomolgus CD47 recombinant proteins, while the negative control showed no binding to both CD47 recombinant proteins.

[0938] Blood cell binding of binding agents by flow cytometry To evaluate the binding activity of KC015 on various blood cell subtypes, fresh blood was collected from two healthy donors in Becton Dickinson Vacutainers. For white cell preparation, 10 mL of 1X red blood cell lysis buffer (eBiosceince, 00-4300-54) was added to each 1 mL of fresh blood and incubated at RT for 15 min. The cell suspension was then centrifuged at 300 g for 5 min at room temperature, followed by one additional wash with PBMC medium (RPMI + 10% heat-inactivated FBS). White blood cell counts were performed before staining with binders.

[0939] For preparation and staining of red blood cells (RBCs), fresh blood was transferred to a 15 mL conical bottom polypropylene tube and mixed with PBS wash buffer (PBS + 2% heat inactivated FBS). The tube was centrifuged at 800 g for 10 min at room temperature with suspension. Red blood cells were collected from the bottom of the tube, resuspended in PBS wash buffer (PBS + 2% heat inactivated FBS), and cell number was counted before staining with binder.

[0940] For platelet cell preparation, fresh blood was transferred to a 15 mL conical bottom polypropylene tube and mixed with PBS wash buffer (PBS + 2% heat inactivated FBS). The tube was centrifuged at 800 g for 10 min at room temperature with interruption. Platelet rich plasma was collected from the top of the tube (2 / 3 of the plasma volume). The platelet rich plasma was transferred to 9 mL PBS wash buffer and centrifuged at 1250 g for 15 min at room temperature with interruption. Platelet cell number was counted before staining with binder.

[0941] Fc receptor blocking antibodies were added to the cell suspension at a dilution of 1 / 100 and incubated on ice for 10 min. Primary binders were added to the cell suspension at serial antibody concentrations and incubated on ice for 30 min. Cells were washed once with PBS wash buffer before staining with secondary detection antibodies. T cells were also stained with CD3 marker antibody (Biolegend, 557705). Monocytes were stained with CD14 antibody (Biolegend, 301814). Granulocyte populations were gated based on FSC and SSC. Red blood cells were stained with CD235ab antibody (Biolegend, 306620). Platelets were stained with CD41 antibody (Biolegend, 303704). All cells were stained with 7-AAD (BD, 559925) with a dilution of 1 / 100 before analysis on a FACSCantoII cytometer.

[0942] As shown in FIG. 9E, KC015 binds efficiently to human T cells, but less efficiently to RBCs and platelets.

[0943] FACS binding assay of cells Binding of the binders to the cells was assessed by flow cytometry. To determine the binding specificity of binders containing the anti-PD-1 antigen-binding domain, CHO parental and CHO-PD-1 cell lines were used in binding assays (Figure 10). Briefly, 1x10 5The cells were incubated with Fc receptor blocking antibody (1 / 100 dilution, Cedarlane, Cat. No. 422302) for 10 min at room temperature. A binder containing anti-PD-1 antigen-binding domain was added to FACS staining buffer at 20 μg / mL and incubated with the cells for 30 min on ice. PD-1 antibody (clone EH12.2H7, Biolegend) was used as a positive control. After washing three times with FACS staining buffer, APC-conjugated secondary antibody recognizing human Fc region (Cedarlane, Cat. No. 409306) was mixed with the cells. After staining for 30 min, the cells were washed three times and then resuspended in 100 μl of 7-amino-actinomycin D (7-AAD) solution (1 / 500 dilution, BD Bioscience, Cat. No. 559925). The cells were analyzed by BD FACSCanto™ II Flow cytometer (BD Bioscience). Data was analyzed using FlowJo via gating on single and live cells.

[0944] As shown in Figure 10, KC036 bound only to CHO-PD1 cells and did not bind to non-PD1 expressing cells (parental CHO cells), demonstrating the high specificity of the anti-PD-1 module KC009.

[0945] PBMC-mediated cytotoxicity assay PBMC-mediated cytotoxicity was evaluated by flow cytometry. Briefly, tumor cells (THP-1 or OCI-AML3) were stained with CellTrace violet for 10 min at 37°C. Cells were washed and mixed with human PBMCs (Cedarlane, Cat. No. 70025.1) at a 5:1 ratio. Anti-CD3 antibody or CD33 bispecific T cell engager (BiTE, G&P Bioscience, Cat. No. FLC2032) was added to activate T cells. Binders were added at the indicated concentrations to the corresponding wells to determine the effect of anti-PD-1 modules on cytotoxicity. After 48 or 72 h of incubation, cells were washed three times and then resuspended in 100 μl of 7-AAD solution (1 / 100 dilution, BD Bioscience, Cat. No. 559925). Cells were analyzed using a BD FACSCanto™ II Flow cytometer (BD Biosciences). Data was analyzed using FlowJo with gating on single and live cells only.

[0946] As shown in FIG. 11, KC036 (SEQ ID NO: 94) demonstrated checkpoint inhibition (CPI) activity by increasing T-cell dependent cytotoxicity to levels similar to the positive controls pembrolizumab and nivolumab.

[0947] PD-1 / PD-L1 blockade bioassay The blocking activity of binders containing the anti-PD-1 antigen-binding domain was assessed using a PD-1 / PD-L1 blocking assay in a luminescent NFAT-RE reporter system (Promega, Cat. No. J1255) and compared to a positive control. PD-L1 aAPC / CHO-K1 (target) cells were thawed and plated at the recommended density in a 96-well plate and allowed to adhere to the plate overnight. The next day, binders were diluted to 350 nM in assay buffer (Ham's F12 medium with 10% low IgG FBS) and eight 2.5x serial dilutions were performed. The media from the 96-well plate was decanted and 40 μl of diluted binder and 40 μl of Jurkat (effector) cells were added to the plate. The plate was incubated at 37°C for 6 hours. Bio-Glo luciferase assay buffer and substrate were combined and 80 μl of the solution was transferred to each well. The plate was incubated at room temperature for 5 minutes and luminescence was measured using a plate reader.

[0948] The results of these experiments showed that KC036 achieved similar in vitro CPI activity as pembrolizumab, suggesting that the binder can induce antitumor activity by restoring T cell function in "exhausted" T cells (Figure 12).

[0949] The anti-PD1 antigen-binding domain of KC036 is replaced with other anti-PD-1 antigen-binding domains listed in Table 4 in a manner similar to that described herein.

[0950] Example 5 In vitro and in vivo testing of binding agents containing anti-CD47 antigen-binding domains Binding agents comprising anti-D2 and anti-CD47 antigen binding domains (KC040) or anti-D2, anti-PD-1 and anti-CD47 antigen binding domains (SEQ ID NO: 70) were tested for binding in HEK293T or HEK293T-CD47KO cells (CD47 knockout) (Figure 13) or primary human T cells (PBMCs, StemCell) (Figure 14).

[0951] As shown in FIG. 13, binders containing the anti-CD47 module bind to parental HEK293T cells and not to CD47 knockout cells (HEK293T-CD47KO).

[0952] As shown in FIG. 14, binding agents containing an anti-CD47 module bind to primary human T cells.

[0953] T cell activation assay The ability of binders containing anti-CD47 modules to induce polyclonal T cell activation was performed in the presence (Figure 16) or absence (Figure 15A) of concanavalin A (ConA, Cedarlane, 14951-250). Briefly, PBMCs were incubated with the indicated concentrations of binders for 24 hours in the presence or absence of 10 μg / mL ConA. Plates were spun down and cells were incubated with Fc receptor blocking antibodies (Biolegend, 1 / 200 dilution) for 10 minutes on ice. Plates were spun down again and cells were stained with APC-conjugated anti-CD3 (Cedarlane, Cat. No. 300312) and FITC-conjugated anti-CD69 antibodies (Biolegend, Cat. No. 310904, 1 / 500 dilution) for 30 minutes on ice. Cells were washed three times and then resuspended in 100 μl of 7-AAD solution (BD Bioscience, 1 / 100 dilution). Cells were analyzed using a BD FACSCanto™ II Flow cytometer (BD Bioscience). Data was analyzed using FlowJo with gating on single and live cells only.

[0954] PBMC-mediated cytotoxicity assay PBMC-mediated cytotoxicity was evaluated by flow cytometry (Figure 15B). Briefly, tumor cells (THP-1 or OCI-AML3) were stained with CellTrace violet for 10 min at 37°C. Cells were washed and mixed with PBMCs at a ratio of 5:1. Anti-CD3 antibody or CD33 bispecific T cell engager (BiTE, G&P Bioscience, Cat. No. FLC2032) was added to activate T cells. Binders were added at the indicated concentrations to the corresponding wells to determine the effect of CD47 on cytotoxicity. After 48 or 72 h of incubation, cells were washed three times and then resuspended in 100 μl of 7-AAD solution (1 / 100 dilution, BD Bioscience, Cat. No. 559925). Cells were analyzed by a BD FACSCanto™ II Flow cytometer (BD Bioscience). Data was analyzed using FlowJo with gating on single cells and live cells only.

[0955] As shown in Figure 15, the binders containing the anti-CD47 module did not activate T cells (Figure 15A) or mediate T cell-dependent cytotoxicity against THP-1 tumor cells (Figure 15B). Although the binders showed binding to HEK293T and primary T cells, they did not activate T cells in vitro, which may result in better safety for immune cell redirection without activating T cells.

[0956] As shown in Figure 16, concanavalin A-mediated T cell activation is not affected by binders containing the anti-CD47 module. Concanavalin A, a superantigen, leads to T cell activation through the TCR-MHC complex. These results suggest that the anti-CD47 module did not interfere with T cell activation induced through the TCR-MHC complex.

[0957] CD47 / SIRPα blockade bioassay The CD47 / SIRPα blocking assay in a luminescent reporter system was used to evaluate the blocking activity of binders containing anti-CD47 antigen-binding domains and compared with B6H12 antibody (Promega, Cat. No. CS316013) as a positive control. CD47 CHO-K1 target cells were thawed and resuspended in Ham's F12 medium containing 10% FBS. The cell suspension was dispensed with 100 μL of CD47 CHO-K1 cells per well of a 96-well plate and incubated overnight at 37° C., 5% CO2. The next day, 1.5× serial dilutions of the test molecules were prepared in assay buffer (RMPI-1640 medium containing 2% FBS). The cell medium was removed and 50 μL of serially diluted antibodies were added to the corresponding wells. SIRPα effector cells were thawed and resuspended in assay buffer. The cell suspension was dispensed with 75 μL of SIRPα cell suspension per well. Plates were incubated for 4 hours at 37°C with 5% CO2. At the end of the 4-hour incubation, 75 μL of Bio-Glo-NL Luciferase Assay Reagent was added per well, and luminescence was measured after 5–10 minutes using a Spectra Max ix3 Plate Reader.

[0958] As shown in Figure 17, KC015 (SEQ ID NO: 75) exhibited checkpoint inhibition (CPI) activity by blocking the interaction between CD47 and SIRPα. The negative control (KC016: SEQ ID NO: 76) did not exhibit any CPI activity.

[0959] Example 6 In vitro and in vivo testing of binding agents containing anti-DR2, PD-1 and CD47 antigen-binding domains Established tumor model in NCG mice To determine the antitumor efficacy of multivalent and multispecific binding agents, an established tumor model in humanized NCG mice was used in the study (Figure 18, Figure 22). Briefly, NCG mice (5-6 weeks old, female) were purchased from Charles River Laboratories (St. Constant, QC). Tumor cells (8 million cells / mouse for NCI-H82) in DPBS were injected subcutaneously (sc) into the right flank of the mice. When tumors reached 100 mm 3 After the mice had grown to 100 μg / kg, they were randomly divided into experimental groups (10 or 9 mice / group). Human PBMCs (10 million cells) were injected intravenously (iv). The next day, mice in each group were given binder (28 mg / kg) treatment intraperitoneally (ip) twice a week for a total of 8 doses. Tumor volume was measured using calipers and mice were weighed twice a week. Tumor volume and tumor growth inhibition were calculated as described above ("Tumor prevention tumor model in CB-17 Fox Chase SCID mice" in the Methods section). Statistical tests were performed by Student's t-test (two-tailed).

[0960] As shown in Figure 18, the binding agent comprising anti-D2, anti-PD-1 and anti-CD47 antigen-binding domains exhibited a TGI of 52%. The results implied that the trispecific binding agent could inhibit tumor growth in vivo. The binding agent comprising the anti-CD47 antigen-binding domain disclosed herein is expected to provide better safety than other activating antibodies for immune cell redirection.

[0961] In vitro T cell recruitment assay To test whether a binding agent targeting a tumor antigen (D2) and T cells could recruit T cells and bridge T cells to tumor cells, T cells were co-cultured with NCI-H69 cell clusters in the presence of the binding agent.

[0962] Briefly, the level of T cell recruitment to NCI-H69 SCLC cell clusters was quantified for various binders containing anti-D2, anti-PD1 and / or anti-CD47 modules, as well as variations of the module combinations (Figure 19). Suspension NCI-H69 cells were harvested, transferred to 12-well plates and returned to a 37°C, 5% CO2 incubator for 72 hours to allow for the growth of larger cell clusters. After 72 hours, 5.0 x 10 5 PBMCs and binders (final concentration of 25 μg / mL) were added to the NCI-H69 cells. The plates were returned to the 37° C., 5% CO2 incubator for 24 hours to allow recruitment of T cells into the NCI-H69 cell clusters.

[0963] After 24 hours, the media from each well was transferred to a microcentrifuge tube and each tube was briefly centrifuged using a benchtop mini centrifuge for a 5 second pulse spin. The supernatant was separated and the pellet resuspended in 150 μL of flow buffer (PBS with 5 mM EDTA). The pellet and supernatant fractions were evaluated using bright field microscopy to ensure efficient separation of large cell clusters. The resuspended pellet from the microcentrifuge tube was transferred to a 96-well plate. The plate was centrifuged at 1200 RPM to pellet the cells, the plate was inverted to remove the supernatant, and Human TruStain FcX Block (BD Bioscience, Cat. No. 559925) was added at 2 μL per 100 μL of flow buffer per well and incubated on ice for 10 minutes. The plates were centrifuged at 1200 RPM to pellet the cells, and APC-conjugated anti-human CD3 (Cedarlane, Cat. No. 300312) and FITC-conjugated anti-human CD69 antibodies (Cedarlane, Cat. No. 310904) were added at 0.5 μL each per 100 μL flow buffer per well, including appropriate isotype controls in separate wells. After addition of the fluorescent conjugated antibodies, the plates were covered and incubated on ice for 30 minutes. After the incubation period, the plates were centrifuged at 1200 RPM to pellet the cells, and washed with 150 μl flow buffer. The plates were centrifuged at 1200 RPM to pellet the cells, and resuspended in 7-AAD solution containing 1.0 μL 7-AAD per 100 μL flow buffer per well.

[0964] Cell populations were analyzed by flow cytometry. Raw reads were collected on a BD FACSCanto II instrument. A total of 100,000 events were collected for data analysis. Total live cell populations were assessed by gating on 7-AAD negative cells (PerCP-Cy5.5). Total T cell populations were assessed by gating on CD3 positive cells (APC) and activated T cell populations were assessed by gating on dual CD3 (APC) and CD69 (FITC) positive cells. Mean fluorescence intensity (MFI) of CD3 (APC) and CD69 (FITC) were quantified and compared between treatment groups. All samples described in this report were tested in triplicate. Results represent the mean + / - standard deviation.

[0965] As shown in FIG. 19, the binding agents were found to effectively recruit T cells to NCI-H69 cell clusters in vitro.

[0966] Tumor prevention model in CB-17 Fox Chase SCID mice Binders containing the anti-D2 module were tested in a tumor prevention model as described in Example 3.

[0967] Briefly, binders (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) were tested in a similar tumor prevention model in NCG mice inoculated with NCI-H82 cells, except that human PBMCs and NCI-H82 cells were co-transplanted at a ratio of 1:5 (Figures 20A-B and Figure 24C).

[0968] As shown in Figure 20B, binders containing KC001, KC009 and KC010 (SEQ ID NO: 77) showed the best efficacy of 74% compared to other D2 modules with KC009 and KC010 (Figure 20A & B). The superiority of KC001 efficacy data was also consistent with the D2 efficacy data as VHH-hinge-CH2-CH3 (Figure 20), in which KC001 (KC013; SEQ ID NO: 73) showed the best efficacy in several tumor models including NCI-H69, NCI-H510A, and NCI-H727.

[0969] In vivo efficacy of KC015 in a PBMC pre-transplant model The antitumor activity of KC015 was evaluated in NCG mice using a PBMC-pre-implanted NCI-H82 xenograft tumor model (Figure 21A). The antitumor activity of KC020 was also studied in PBMC-pre-implanted NCI-H69, RKO, and SCLC-21H xenograft tumor models (Figure 21B). Briefly, NCG mice (5-6 weeks old, female) were purchased from Charles River Laboratories (St. Constant, QC). Ten million PBMCs were implanted into NCG mice 3 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. Formula: 1 / 2(length x width) 2 ) was used to calculate tumor volumes. For calculation of tumor growth inhibition (TGI) percentage, treatment groups were compared to their respective negative controls. TGI was calculated as described herein. Statistical testing was performed by Student's t-test (two-tailed).

[0970] At the end of the study, the animals were sacrificed and the tumors and livers were collected for metastasis analysis.

[0971] The results of these experiments showed that KC015 (SEQ ID NO: 75) inhibited NCI-H82 tumor growth in a PBMC-preimplanted model. KC020 (SEQ ID NO: 77) inhibited the growth of NCI-H69, RKO and SCLC-21H xenograft tumors in a human PBMC-preimplanted model of cancer.

[0972] In vivo efficacy of KC015 in established tumor models in SCID mice The antitumor efficacy of the KC015 binder was evaluated in an established MDA-MB-231 tumor model in SCID mice (Figure 21C). Tumor cells (5 million cells / mouse) in PBS were injected subcutaneously (sc) into the right flank of SCID mice (5-6 weeks old, female; Charles River Laboratories, St. Constant, QC). When tumors reached 100 mm 3 After the mice had grown to 100 mg / kg, they were randomly divided into experimental groups (9 mice / group). The next day, mice in each group were given a binder (8 mg / kg) treatment intraperitoneally (ip) once a week for a total of 8 doses. Tumor volumes were measured using calipers and mice were weighed twice a week. Tumor volumes and tumor growth inhibition were calculated as above. Statistical tests were performed by Student's t-test (two-tailed).

[0973] As shown in FIG. 21C, an established MDA-MB-231 tumor model did not grow upon treatment with KC015, indicating potent antitumor activity in this tumor model.

[0974] In vivo efficacy of IgG4 versus IgG1 versions of binders IgG4 is commonly used for therapeutic antibodies to avoid ADCC effects and cytokine release storms that can lead to T cell killing in this case. To determine the antitumor efficacy of binders containing the IgG4 CH3 domain, an established tumor model in humanized NCG mice was used as described above.

[0975] As shown in FIG. 22, no significant differences were observed between the IgG4 and IgG1 versions of the binder (compare KC021 (SEQ ID NO: 78) and KC027: SEQ ID NO: 85).

[0976] Micro-PET / CT imaging of binder biodistribution in a tumor-bearing mouse model MicroPET / CT imaging of the radiolabeling of the binders and their biodistribution was performed (Figure 23). Preparations of the binders were first buffer exchanged with 0.1 M sodium carbonate (pH 9.0) using an Amicon® Ultra-4 Centrifugal Filter (MWCO 30 kDa, UFC803008). The binders were then modified with desferrioxamine (DFO) by reaction with a 14-fold molar excess of p-isothiocyanatobenzyldesferrioxamine (p-NCS-Bz-DFO, Macrocyclics). Briefly, 20-40 μL of a 20 mM (15 μg / μL) solution of p-NCS-Bz-DFO in anhydrous DMSO was reacted with 4-7 mg of binder (3-4 mg / mL in 0.1 M NaHCO3 buffer, pH 9.0) for 30 min at 37 °C with mixing at 300 rpm on an Excella E24 mixer (New Brunswick Scientific). The reaction was divided into small aliquots to avoid cross-linking and aggregation of the antibody. After the 30 min reaction, excess p-NCS-Bz-DFO was removed by ultrafiltration, which was repeated four times on Amicon Ultra-4 centrifugal filter devices (Millipore, 30 kDa cutoff) using excess 0.25 M Na acetate buffer, pH 5.5, with centrifugation at 5000 × g for 5 min. The purity and homogeneity of the DFO binders were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4-20% Tris-HCl gels (Bio-Rad) under reducing (2-mercaptoethanol) and nonreducing conditions.

[0977] After DFO conjugation, the binder was purified by the following protocol: 89 Radiolabeled with Zr as oxalate 89Zr was purchased from Washington University (St. Louis, MO) and the solution was neutralized by incubation with 2 M NaCO3 (pH 10.0) for 3 min. Then, 0.25 M Na acetate buffer (pH 7.0) containing 32 mM gentisic acid (5 mg / mL) and 0.5 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES buffer) was added, followed by the addition of binder (KC020; SEQ ID NO: 77) (1-5.0 mg / mL) containing D2, PD-1 and CD47 specific antigen binding domains. Radiolabeling was performed at room temperature for 60-90 min with a specific activity of 0.28 mCi / mg. Radiochemical purity was measured by instant thin layer silica gel chromatography (ITLC-SG) developed in 22 mM citric acid in 20 mM Na2CO3 (pH 5.0).

[0978] After radiolabeling, [ 89 The [Zr]-DFO binder was injected into mice bearing two different types of xenografts (NCI-H69 and NCI-H82). Mice were anesthetized using isoflurane (Fresenius Kabi Canada Ltd.) anesthesia (induction at 5% and maintenance at 1.5-2%), followed by [ 89 An iv injection of [Zr]-DFO binder was administered, giving 200 μL, 2.86±0.2 MBq, and 30 mg / Kg. Animals were imaged using a Mediso nanoScan® SPECT / CT / PET 82S system at 24, 96, and 144 hours after injection.

[0979] As shown in Figures 23A and 23B, the radiolabeled binding agent was found to exhibit accumulation in tumors in both NCI-H69 and NCI-H82 xenograft models, suggesting recruitment of the binding agent via tumor antigens.

[0980] Example 7 In vivo testing of binders in the hCD34+ humanized NCG mouse model hCD34+ humanized NCG mice were purchased from Charles River Laboratories. HSC engraftment levels were assessed by flow cytometry one day before tumor implantation. Mice were randomized into two groups, and NCI-H69 cells were implanted subcutaneously (sc) into the right flank of each animal (8 million cells per animal). Tumors grew to an average of 100 mm 3 Once tumor volumes reached 100-200 mg / kg, negative control and test articles were administered twice weekly by IP injection. Tumor volumes, tumor growth inhibition (TGI) percentages and statistical analyses were performed as described in Example 3.

[0981] At the end of the study, the animals were sacrificed and the tumors and livers were collected for metastasis analysis.

[0982] The results of these experiments, presented in Figures 24A-B, showed that KC020 (SEQ ID NO: 77) reduced metastasis of NCI-H69 cells to the liver in a hCD34+ humanized NCG mouse model. The results in Figure 24C also showed the efficacy of KC020 (SEQ ID NO: 77) in a NCI-H82 / PBMC co-transplant model.

[0983] Example 8 Testing of binders in other solid tumors To determine the anti-tumor efficacy of anti-D2 binders including KC013, KC011, KC012, and KC014 in NCI-H510A and NCI-H727 sc preventative xenograft models of human small cell lung cancer, female SCID mice were purchased from Charle River Laboratories and used for the study at 4-6 weeks of age. For NCI-H510a cells, cells were implanted subcutaneously (sc) in the right flank of each animal at 8 million cells per mouse. For NCI-H727 cells, cells were implanted subcutaneously (sc) in the right flank of each animal at 1 million and 6 million cells per mouse. The following day, vehicle control and test articles were administered once a week by ip injection. Tumor volume, tumor growth inhibition (TGI) percentage, and statistical analysis are performed as described in Example 3.

[0984] To evaluate the efficacy of anti-D2 binders in the PANC1 sc prophylactic xenograft model of human pancreatic cancer, female SCID mice were purchased from Charles River Laboratories and used for the study at 4-6 weeks of age. Cells were implanted subcutaneously (sc) into the right flank of each animal at 5 million cells each. Treatment began one day after cell implantation. Negative control antibodies and test articles were administered once a week by ip injection. Tumor volumes, tumor growth inhibition (TGI) percentages and statistical analysis were performed as described in Example 3.

[0985] Example 9 stability studies A solution of purified binder at a concentration of 20 mg / mL (+ / -0.5) was prepared and divided into two batches. One batch was stored at -80°C (not stressed condition) and the other batch was subjected to various stress conditions including 3 days of agitation (25°C), 5 cycles of freezing and thawing, 2 weeks of storage at 40°C, or low pH (3.5) treatment for 4 and 48 hours. Both batches were tested for binding to D2-containing proteoliposomes (Figures 25A and 26A), recombinant PD-1 (Cedarlane, Catalog No. 10377-H08H-50) (Figures 25B and 26B) or recombinant CD47 (Cedarlane, Catalog No. 12283-H08H-200) (Figures 25C and 26C) as described in Examples 3 and 4.

[0986] The results presented in Figures 25 and 26 indicate that the binding of KC020 to its target is not affected by the stress conditions tested.

[0987] Additionally, the biophysical characteristics of the solutions (solubility and aggregation) did not change significantly under these conditions.

[0988] Similar results were obtained with several other binders (data not shown).

[0989] Example 10 Testing of binding agents in the NCI-H82 tail vein metastasis model of SCLC NCG mice (5-6 weeks old, female) were purchased from Charles River Laboratories (St. Constant, QC). NCG mice were inoculated with 50,000 NCI-H82 cells via tail vein injection. Mice were given KC020 treatment by intraperitoneal injection 24 hours prior to tumor cell injection. KC020 was administered every other week for a total of eight doses. Animals were monitored for weight loss and general health for the entire duration of the study. Overall, KC020 treatment significantly prolonged survival in mice implanted iv with NCI-H82 SCLC compared to the vehicle-treated negative control group (Figure 27).

[0990] For in vivo bioluminescence imaging studies, a total of 20 NCG mice were randomly assigned into four groups, including PBS, KC020 3mg / kg, KC020 10mg / kg, and KC020 30mg / kg groups. On day 0, all mice received one treatment of KC020 or PBS. For the KC020 3mg / kg group, mice received one treatment of 10mg / kg human IgG one day prior to KC020 treatment. NCI-H82-luciferase cells were inoculated into NCG mice via tail vein injection at 100,000 cells per mouse. All mice were treated twice weekly with either PBS or KC020 for a total of eight doses. Bioluminescence imaging was performed once weekly 3 weeks after tumor cell inoculation. Prior to imaging, mice were dosed via intraperitoneal injection with 15 mg / mL firefly luciferase substrate in DPBS at a dose volume of 200 μL. The results of this experiment show that KC020 treatment prevented tumor development at all doses tested, including the lowest dose of 3 mg / kg.

[0991] The results presented herein indicate, inter alia, that the binding agents of the present disclosure bind to each of their targets, exhibit in vitro CPI activity, demonstrate in vivo tumor suppression efficacy in both preventative and established xenograft models of human cancer, prevent metastasis development, and prolong mouse survival in a tail vein model of human cancer. Additionally, the binding agents of the present disclosure are manufactured in very high yields in productive cell lines and exhibit favorable stability under accelerated stability testing.

[0992] The embodiments and examples described herein are illustrative and are not meant to limit the scope of the claims. We intend that variations of the above-described embodiments, including alternatives, modifications, and equivalents, are covered by the claims. The cited list of this application is incorporated herein by reference.

[0993] References All patents, patent applications and publications referenced throughout this application are hereby incorporated by reference.

[0994] Angal, S. et al., A Single Amino Acid Substitution Abolishes the Heterogeneity of Chimeric Mouse / human IgG4 Antibody, Mol Immunol 30, 105-108, 1993 Chen X et al., Fusion Protein Linkers: Properties, Design and Functionality, Adv Drug Deliv Rev. 2013; 65(10):1357-1369. Conrath, KE et al., Camel Single-Domain Antibodies as Modular Building Units in Bispecific and Bivalent Antibody Constructs. J. Biol. Chem. 2001, 276, 7346-7350. Coppieters, K. et al., Formatted Anti-Tumor Necrosis Factor alpha VHH Proteins Derived from Camelids Show Superior Potency and Targeting to Inflamed Joints in a Murine Model of Collagen-induced Arthritis. Arthritis Rheum. 2006, 54, 1856-1866. Detallea L, et al., Generation and Characterisation of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus infection. Antimicrobial Agents and Chemotherapy 60(1) 2015 De Vlieger et al., Single-domain Antibodies and their Formatting to Combat Viral Infections, Antibodies 8(1), 1-22, 2019 Duggan S. Caplacizumab: First Global Approval. Drugs (2018) 78:1639-1642 Fleischmann R, et al., A Novel Individualized Treatment Approach in Open-Label Extension Study of Ozoralizumab (ATN-103) in Subjects with Rheumatoid Arthritis on a Background of Methotrexate. Abstract 1311, ACR / ARHP Annual Meeting (2012). Fridy PC, et al., A Robust Pipeline for Rapid Production of Versatile Nanobody Repertoires. Nat Methods. 2014 December; 11(12): 1253-1260. Godar M et al. Therapeutic Bispecific Antibody Formats: a Patent Applications Review (1994-2017) Expert Opinion on Therapeutic patents, 2018; 28(3):251-276 Ha, J-H et al. Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins, Front Immunol, 2016; 7:394. Hamers-Casterman C, et al., Naturally-occurring Antibodies Devoid of Light-chains. Nature 1993, 363:446-448. Hmila, I. et al., VHH, Bivalent Domains and Chimeric Heavy Chain-only Antibodies with High Neutralizing Efficacy for Scorpion Toxin AahI’. Mol. Immunol. 2008, 45, 3847-3856. Hultberg, A et al., Llama-derived Single Domain Antibodies to Build Multivalent, Super Potent and Broadened Neutralizing Anti-viral Molecules. PLoS One 2011, doi: 10.1371 / journal.pone.0017665. Jahnichen S, et al, CXCR4 Nanobodies (VHH-based single variable domains) Potently Inhibit Chemotaxis and HIV-1 Replication and Mobilize Stem Cells. Proc Natl Acad Sci USA. (2010) 107: 20565-20570. Kovalenko OV et al. Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis, J Biol Chem. 2013, 288:17408-17419 Muyldermans,S and Smider, 2016. Distinct Antibody Species: Structural Differences Creating Therapeutic Opportunities. Current Opinion in Immunology 2016, 40:7-13 Muyldermans, S et al., 1994. Sequence and Structure of VH Domain From Naturally Occurring Camel Heavy Chain Immunoglobulins Lacking Light Chains. Protein Eng. 7: 1129-1135. Saunders K.O., Conceptual Approach to Modulating Antibody Effector Functions and Circuation Half-Life, Front. Immunol. 10:1296, 2019. Schoen P, et al., Anti-RANKL Nanobody(Registered Trademark) ALX-0141 Shows Sustained Biomarker Inhibition in a Phase I Study in Healthy Postmenopausal Women. DOI: 10.1530 / boneabs.1.PP135 (2013) Simmons, D.P et al., Dimerisation Strategies for Shark IgNAR Single Domain Antibody Fragments. J. Immunol. Methods 2006, 315, 171-184. Svecova D, et al., A Randomized, Double-Blind, Placebo-Controlled Phase 1 Study of Multiple Ascending Doses of Subcutaneous M1095, an Anti-Interleukin-17A / F Nanobody(Registered Trademark), in Moderate-to-Severe Psoriasis. J Am Acad Dermatol. 2019 Mar 26. Tatusova, T. A, et al. (1999), BLAST 2 Sequences, a New Tool for Comparing Protein and Nucleotide Sequences, FEMS Microbiol Lett. 174:247-250) Van Roy M, et al., The Preclinical Pharmacology of the High Affinity Anti-IL-6R Nanobody(Registered Trademark) ALX-0061 Supports its Clinical Development in Rheumatoid Arthritis. Arthritis Research & Therapy (2015) 17:135 Vincke C. et al. General Strategy to Humanize a Camelid Single-Domain Antibody and Identification of a Universal Humanized Nanobody Scaffold, J.Biol Chem. 2009, 284(5):3273-3284 Vu, KB, et al., 1997. Comparison of Llama VH Sequences from Conventional and Heavy Chain Antibodies. Mol. Immunol. 34: 1121-1131.

[0995] Match list

[0996]

Table 4-1

[0997]

Table 4-2

[0998]

Table 4-3

[0999]

Table 5

[1000]

Table 6

[1001]

Table 7-1

[1002]

Table 7-2

[1003] [Table 8-1]

[1004] [Table 8-2]

[1005] [Table 8-3]

[1006] [Table 8-4]

[1007] [Table 8-5]

[1008] [Table 8-6]

[1009] [Table 8-7]

[1010] [Table 8-8] [Table 8-9]

[1011] SEQ ID NO: 98 (native human IgG1 hinge region) EPKSCDKTHTCPPCP SEQ ID NO: 99 (Linker-HL1) EPKIPQPQPKPQPQPQPGGSGSAEAAAKAPKAP SEQ ID NO:100 (Flexible Linker-FL2) GGGGSGGGGS SEQ ID NO:101 (Flexible Linker-FL18) GGGGSGGGGSGGGGS SEQ ID NO: 102 (Flexible Linker-FL4) GGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO: 103 (Flexible Linker-FL5) GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SEQ ID NO:104 (flexible linker) (GGGGS) n (n is an integer selected from 1 to 10) SEQ ID NO: 105 (Rigid Linker - RL5) PAPAPKA SEQ ID NO: 106 (Rigid Linker - RL7) APAPAPAPAPKA SEQ ID NO: 107 (Rigid Linker - RL12) APAPAPAPAPAPAPAPAPAPKA SEQ ID NO: 108 (rigid linker) (X(PAPAP)) n KA (n is an integer selected from 1 to 10, X is present or absent, and is A) SEQ ID NO: 109 (Helical Linker-RL1) AEAAAKEAAAKA SEQ ID NO:110 (Helical Linker-RL2) AEAAAKEAAAKEAAAKA SEQ ID NO:111 (Helical Linker - RL4) AEAAAKEAAAKEAAAKEAAAKEAAAKA SEQ ID NO: 112 (helical linker X(EAAAK) n Y (n is an integer selected from 1 to 10, more preferably 2 to 5, and X and Y are independently present or absent, and preferably A). SEQ ID NO: 113 Anti-HEWL VHH (KC017)

[1012] [ka]

[1013] (X is E or Q) SEQ ID NO: 114 Anti-4HEM VHH (VHH part of KC018)

[1014] [ka]

[1015] (X is E or Q) SEQ ID NO: 115 Anti-CD3 VHH (KC019)

[1016] [ka]

[1017] (X is E or D) SEQ ID NO: 116 Native human IgG1 CH3

[1018] [ka]

[1019] SEQ ID NO: 117 Native human IgG1 CH2

[1020] [ka]

[1021] SEQ ID NO: 118 Mutant human IgG1 hinge region EPKSSDKTHTCPPCP SEQ ID NO: 119 Mutated human IgG1 hinge region EPKSSDKTHTSPPSP SEQ ID NO: 120 Mutated human IgG1 hinge region DKTHTCPPC SEQ ID NO: 121 Native human IgG2 hinge region ERKCCVECPPCP SEQ ID NO: 122 Mutated human IgG2 hinge region ERKSSVECPPCP SEQ ID NO: 123 Mutated human IgG2 hinge region ERKSSVESPPCP SEQ ID NO: 124 Mutated human IgG2 hinge region ERKSSVESPPSP SEQ ID NO: 125 Native human IgG3 hinge region ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP SEQ ID NO: 126 Mutated human IgG3 hinge region EPKSSDTPPPCPRCP SEQ ID NO: 127 Mutated human IgG3 hinge region EPKSSDTPPPSPRCP SEQ ID NO: 128 Mutant human IgG3 hinge region EPKSSDTPPPSPRSP SEQ ID NO: 129 Native human IgG4 hinge region ESKYGPPCPSCP SEQ ID NO: 130 Mutated human IgG4 hinge region ESKYGPPCPPCP SEQ ID NO: 131 Mutant human IgG4 hinge region ESKYGPPSPSCP SEQ ID NO: 132 Mutant human IgG4 hinge region ESKYGPPSPSSP SEQ ID NO: 133 Signal peptide MEWSWVFLFFLSVTTGVHS SEQ ID NO: 134 Epitope tag HHHHHH SEQ ID NO: 135 Native human IgG1 constant region

[1022] [ka]

[1023] SEQ ID NO: 136 Mutated CH3 domain (Chain A- Mutations D399N; K370E; E356Q)

[1024] [ka]

[1025] SEQ ID NO: 137 Mutant CH3 domain (Chain B - Mutations D399N; K439E; E357Q)

[1026] [ka]

[1027] SEQ ID NO: 138 Mutant human IgG1 constant region (Chain A- Mutations D399N; K370E; E356Q)

[1028] [ka]

[1029] SEQ ID NO: 139 Mutant human IgG1 constant region (chain B - mutations D399N; K439E; E357Q)

[1030] [ka]

[1031] SEQ ID NO: 140 Mutated human IgG4 constant region

[1032] [ka]

Claims

1. A binding agent comprising one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2, and optionally at least one antigen-binding domain capable of binding to an immunomodulator, at least one antigen-binding domain capable of binding to an immune cell, and / or at least one antigen-binding domain capable of binding to a protein expressed on the surface of an immune cell, wherein 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. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7; r. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:14; s. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:21; t. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:28; u. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 35; v. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:42; w. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:49; or x. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:56 A binder comprising:

2. The antigen-binding domain capable of binding to an immunomodulatory factor is an antigen-binding domain 2 (ABD2) capable of binding to PD-1, 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; or c. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

63.

2. The binder of claim 1 , comprising:

3. The antigen-binding domain capable of binding to a protein expressed on the surface of an immune cell is antigen-binding domain 3 (ABD3) capable of binding to CD47, 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. a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 3. The binder according to claim 1 or 2, comprising:

4. The binding agent of claim 1 , wherein the immunomodulator is an immune checkpoint protein.

5. The binding agent of claim 4, wherein the immune checkpoint protein is PD-1.

6. The binding agent of claim 1 or 5, wherein the protein expressed on the surface of an immune cell is CD47 or CD3.

7. A binder comprising three or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises the amino acid sequence set forth in SEQ ID NO:7, at least one of which is antigen-binding domain 2 (ABD2) capable of binding to PD-1 and comprises the amino acid sequence set forth in SEQ ID NO:63, and at least one of which is antigen-binding domain 3 (ABD3) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO:70; b. a binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO: 70; c. a binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO: 70; d. a binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO:70; e. a binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO: 70; f. a binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO:70; g. A binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO:70; or h. A binding agent comprising three or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) capable of binding to dopamine receptor D2 and comprises 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) capable of binding to PD-1 and comprises 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) capable of binding to CD47 and comprises the amino acid sequence set forth in SEQ ID NO:

70. A binder selected from

8. The binding agent described in claim 1, comprising two polypeptide chains, each of which comprises, from N-terminus to C-terminus, a) the amino acid sequence shown 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, b) a hinge, and c) a CH2-CH3 domain.

9. A binding agent comprising one or more antigen-binding domains, at least one of the antigen-binding domains is an antigen-binding domain 2 (ABD2) capable of binding to PD-1; 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; or c. a heavy chain variable region having an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:63 Including, Binder.

10. A binding agent comprising one or more antigen-binding domains, at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) capable of binding to CD47; 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. A heavy chain variable region having an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:

70. Including, Binder.

11. 11. The binding agent of any one of claims 1 to 10, wherein the binding agent comprises at least two polypeptide chains that can assemble to form a dimer, and each polypeptide chain is the same or different and comprises one or more antigen-binding domains.

12. Each of the polypeptide chains independently has the formula I in N-terminal to C-terminal fashion: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y 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; The binder according to claim 11.

13. The binder of claim 12, wherein m is an integer of 2, 3, 4, 5 or greater than 5; and / or n is an integer of 3, 4, 5 or greater than 5.

14. Each of the polypeptide chains independently comprises, in N-terminal to C-terminal fashion: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (form II); X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV); X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V) X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (form VI); X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (form VII); 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 (form VIII) any one of the amino acid sequences; Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each of which represents an antigen-binding domain, wherein at least one of said antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3); L b1 comprises one or more linkers and / or hinge regions of an antibody or antigen-binding fragment thereof; L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers; and DD represents the dimerization domain; The binder according to claim 11.

15. Each of the polypeptide chains independently comprises, in an N-terminal to C-terminal manner: a. The amino acid sequence of formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) [In the formula, 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 which represents antigen-binding domain 2 (ABD2) and Ab a1 , Ab d1 or Ab d2 one of which represents antigen-binding domain 3 (ABD3)]; b. Amino acid sequence of formula IIIa: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa); or c. Amino acid sequence of formula IIIb: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb) The binder of claim 11 , comprising:

16. The binding agent of claim 12, wherein the dimerization domain comprises a constant region of an antibody or portion thereof, a CH3 domain or a CH2-CH3 domain, the CH3 domain being a native CH3 domain or a mutated CH3 domain, and the CH2 domain being a native CH2 domain or a mutated CH2 domain.

17. the variant CH3 domain comprises: a. Mutations D399N, D / E356Q, K370E; b. Mutations D399N, K439E, E357Q; c. Mutations D399Q, D / E356Q, K370E, Y349K and S354K; d. Mutations D399N, D / E356Q, K370E and L351W; e. Mutations D399N, D / E356Q, K370E and S354M; f. Mutations D399N, D / E356Q, K370E and T350I; g. Mutations D399N, D / E356Q, K370E and T350V; h. Mutations D399N, D / E356Q, K370E and P352R; i. Mutations D399N, D / E356Q, K370E and P352E; j. Mutations D399Q, D / E356Q and K370E; k. Mutations D399N, D / E356Q, K370E and L351Y; l. Mutations D399N, D / E356Q, K370E, and L351H; m. mutations D399N, D / E356Q, K370E, and R355K; n. Mutations D399N, D / E356Q, K370E, and Q355K; o. Mutations D399N, D / E356Q, K370E and S354K; p.mutations D399N, D / E356Q, K370E and T350L; q. Mutations D399N, D / E356Q, K370E and T394N; r. Mutations D399N, D / E356Q, K370E and P352Y; s. mutations D399N, D / E356Q, K370E and P352V; t. mutations D399N, D / E356Q, K370E and P352T; u. Mutations D399N, D / E356Q, K370E and P352L; v. Mutations D399N, D / E356Q, K370E and P352G; w. mutations D399N, D / E356Q, K370E and P352C; x. Mutations D399N, D / E356Q, K370E and L351T; y. mutations D399N, D / E356Q, K370E and L351A; z. Mutations D399Q, E357Q, K439E, Y349D and S354D; aa. mutations D399N, E357Q, K439E and L351R; bb. Mutations In D399N, E357Q, K439E and L351Y; cc. mutations D399N, E357Q, K439E and T350I; dd. mutations D399N, E357Q, K439E and T350V; ee. mutations D399Q, K439E, and E357Q; ff. mutations D399N, K439E, E357Q, and S354K; gg. mutations D399N, K439E, E357Q, and S354W; hh. mutations D399N, K439E, E357Q, and Y349R; ii. Mutations D399N, K439E, E357Q, and T350L; jj. mutations D399N, K439E, E357Q, and R355W; kk. mutations D399N, K439E, E357Q, and Q355W; ll. Mutations D399N, K439E, E357Q, and P395I; mm. mutations D399N, K439E, E357Q, and P395G; nn. mutations D399N, K439E, E357Q, and P395E; oo. mutations D399N, K439E, E357Q, and P352K; pp. mutations D399N, K439E, E357Q, and P352D; or qq. Mutations D399N, K439E, E357Q, and L351D The binding agent of claim 16, comprising a mutation selected from:

18. 18. The binding agent of any one of claims 12 to 17, wherein each of the linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues and is independently a flexible linker, a helical linker, a rigid linker, or a hinge region of an antibody or antigen-binding fragment thereof.

19. 19. The binding agent of any one of claims 1 to 18, wherein one or more antigen-binding domains are humanized.

20. 20. The binding agent of any one of claims 1 to 19, which is an antibody or antigen-binding fragment thereof, an antibody-like molecule, or a protein scaffold or comprises an immune cell modulator.

21. The binding agent of claim 1, wherein the binding agent comprises one or more polypeptide chains, and at least one of the polypeptide chains comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 77-83, 85-88 or 71-74.

22. a. a binding agent comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:77; b. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:78; c. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:79; d. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:80; e. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:81; f. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:82; g. A binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:83; h. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:85; i. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:86; j. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:87; k. A binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:88; l. A binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:71; m. a binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:72; n. A binding agent comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO: 73; or A binder comprising two polypeptide chains, each having the amino acid sequence set forth in SEQ ID NO:

74. A binder selected from:

23. 23. The binding agent of any one of claims 1 to 22, conjugated to a therapeutic moiety, to a detectable moiety, to a protein allowing for extended half-life, or to a nanoparticle.

24. 24. A composition or pharmaceutical composition comprising the binding agent according to any one of claims 1 to 23.

25. 24. A nucleic acid or set of nucleic acids encoding a binding agent according to any one of claims 1 to 23.

26. A vector comprising the nucleic acid of claim 25.

27. 27. A cell expressing a binding agent according to any one of claims 1 to 23 or comprising a nucleic acid according to claim 25 or a vector according to claim 26.

28. 24. A kit comprising the binding agent of any one of claims 1 to 23.

29. A binding agent according to any one of claims 1 to 23, or a composition or pharmaceutical composition according to claim 24, for use as a medicament.

30. The binding agent, composition or pharmaceutical composition of claim 29 for use in the treatment of cancer, a solid tumor, an advanced metastatic solid cancer, a blood cancer, lung cancer, myeloma, prostate cancer, breast cancer, rectal cancer, pancreatic cancer, glioblastoma, an infectious disease or an immune dysregulation disorder, optionally wherein the lung cancer is small cell lung cancer, non-small cell lung cancer or metastatic lung cancer, and optionally wherein the metastatic lung cancer is metastatic small cell lung cancer or metastatic non-small cell lung cancer.

31. 26. A method for producing a binding agent according to any one of claims 1 to 22, comprising the step of transforming a cell with one or more vectors comprising the nucleic acid or set of nucleic acids according to claim 25.

32. Use of a binding agent according to any one of claims 1 to 23, or a composition or pharmaceutical composition according to claim 24, in the manufacture of a medicament for treating cancer.

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