Binding agents targeting tumor cells expressing CD36

Abstract

The present disclosure relates generally to binding agents capable of targeting tumor cells and / or immune cells and their use for the treatment of cancer. Binding agents of the present disclosure comprise one or more antigen binding domains capable of binding single domain antibodies of cluster of differentiation 36 (CD36), programmed cell death protein 1 (PD-1), and / or cluster of differentiation 47 (CD47). The binding agents of the present disclosure are monospecific or multispecific, and may be in monomeric or multimeric form.

Description

Technical Field

[0001] The present disclosure generally relates to binding agents capable of targeting tumor cells and / or immune cells and their use for treating cancer. The binding agents of the present disclosure comprise one or more antigen-binding domains of single-domain antibodies capable of binding cluster of differentiation 36 (CD36), programmed cell death protein 1 (PD-1), and / or cluster of differentiation 47 (CD47). The binding agents of the present disclosure are monospecific or multispecific and can be in monomeric or multimeric form.

[0002] Background

[0003] Camelids and cartilaginous fish naturally produce antibodies consisting of functional homodimeric heavy-chain only antibodies (HCAb) (Hamers-Casterman et al., 1993; Muyldermans and Smider, 2016). The heavy chain of HCAb lacks the first constant domain (CH1) and differs from conventional antibodies only in a few amino acid substitutions that usually involve 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). The antigen-binding fragment of HCAb is called VHH or The molecular weight of VHH is approximately 15 kDa, making it suitable for applications that require enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic applications, it is generally necessary to increase the half-life of VHH to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al., Antibodies 8(1), 1-22, 2019). Although methods such as PEGylation, N-glycosylation, HSA or other carrier protein fusions have been utilized to increase the half-life of VHH, such constructs may introduce immunogenicity or have limited effects.

[0004] VHH has been utilized as a building block to manufacture bispecific and multispecific antibodies. In some studies, bivalent constructs have shown higher avidity or affinity than the monovalent form (Conrath et al., 2001; Coppieters et al., 2006; Hmila et al., 2008; Simmons et al., 2006 and Hultberg et al., 2011, et al., 2010; Fridy et al., 2014).

[0005] Currently, many VHH-based therapeutic agents are in advanced research stages or have received US FDA approval. These include the bivalent monospecific antibody Caplacizumab against the antigen vWF, which has been approved for thrombotic thrombocytopenic purpura (Duggan, 2018). The trivalent nanobody complex ALX-0171 against RSV is in the advanced development stage for respiratory syncytial virus infection (Detallea et al., 2015). ALX-0061 is a monovalent antibody against the antigen IL-6R but conjugated to an HSA nanobody to extend its half-life, and it is in the clinical development stage 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). The anti-RANKL ALX-0141 is a bivalent antibody against the antigen RANKL and conjugated to HSA to extend its half-life (Schoen et al., 2013). Ozoralizumab is a bivalent nanobody against the antigen TNF and conjugated to HSA to extend its half-life (Fleischmann et al., 2012).

[0006] CD36 is a transmembrane protein also known as fatty acid translocase (FAT), FAT / CD36, SCARB3, GP88, gpIV, and gpIIIb, which plays a major role in lipid metabolism as well as other cellular functions such as inflammation, immune recognition, and apoptosis (Wang, J et al., Theranostic 9(17), p. 4893-4908, 2019, the full text of which is incorporated herein by reference). CD36 serves as a receptor for various ligands including apoptotic cells, thrombospondin-1 (TSP-1), and fatty acids (FA). CD36 promotes tumor metastasis and treatment resistance by facilitating lipid uptake and FA oxidation and inhibits angiogenesis by binding to TSP-1 (Chen, Y-J et al., Journal of Cancer 12(16), p. 4762-4773, 2021, the full text of which is incorporated herein by reference). CD36 is involved in tumor cell metabolism, anti-angiogenesis, metastasis, treatment resistance, and tumor immune editing.

[0007] The amino acid sequence and structure of human CD36 were first disclosed in Armesilla, A et al. (The Journal of Biological Chemistry, 269(29), p. 18895-18991, 1994; GenBank Accession No. CAA83662.1, Uniprot Accession No. P16671-1), the full text of which is incorporated herein by reference).

[0008] The present applicant has developed novel binding agents for treating cancer that target tumor cells and / or the immune system.

[0009] Overview

[0010] The present disclosure generally relates to binding agents that comprise one or more antigen-binding domains and are capable of targeting tumor cells and / or immune cells, among other things.

[0011] In some embodiments, the binding agents of the present disclosure bind to the cluster of differentiation 36 (CD36) protein or a portion thereof.

[0012] In some embodiments, the binding agent is a single-domain antibody (sdAb) that binds to the CD36 protein (such as human CD36) or a portion thereof.

[0013] In certain cases, the anti-CD36 single-domain antibodies of the present disclosure can block the binding between CD36 and its ligands.

[0014] In certain cases, the anti-CD36 single-domain antibodies of the present disclosure can regulate lipid metabolism.

[0015] In other cases, the anti-CD36 single-domain antibodies of the present disclosure can inhibit fatty acid oxidation.

[0016] In other cases, the anti-CD36 single-domain antibodies of the present disclosure can inhibit fatty acid uptake by tumors.

[0017] In other cases, the anti-CD36 single-domain antibodies of the present disclosure can inhibit the growth of tumor cells or can be used to inhibit the growth of tumor cells.

[0018] In other cases, the anti-CD36 single-domain antibodies of the present disclosure can inhibit tumor metastasis or can be used to inhibit tumor metastasis.

[0019] In other cases, the anti-CD36 single-domain antibodies of the present disclosure can be used to reduce the resistance of tumor cells to chemotherapeutic drugs and / or immune checkpoint inhibitor therapies.

[0020] The binding function of the sdAb is conferred by a single polypeptide chain (i.e., the variable domain of the heavy chain). This unique property provides formatting flexibility. For example, multiple sdAbs or sdAb antigen-binding domains can be fused into a single polypeptide chain, conferring multivalency and / or multispecificity. In addition, multiple polypeptide chains can be assembled to increase the diversity or affinity of the interaction with CD36. Furthermore, the sdAb antigen-binding domain can be fused to any type of polypeptide chain, including, for example but not limited to, fusion with the heavy and / or light chains of a natural antibody or antigen-binding fragments thereof, fusion with a protein scaffold, fusion with an immune cell modulator, etc.

[0021] Accordingly, the present disclosure is not only directed to the single-domain antibodies disclosed herein, but more generally to binding agents that include one or more antigen-binding domains of the single-domain antibodies disclosed herein.

[0022] According to the present disclosure, the binding agent binds to CD36 and also binds to at least one other antigen. For example, the binding agent can bind to CD36 and an immune checkpoint protein. In another example, the binding agent can bind to CD36 and a protein expressed on the surface of an immune cell.

[0023] Thus, exemplary embodiments of the binding agents of the present disclosure include binding agents that bind to CD36, an immune checkpoint protein, and a protein expressed on the surface of an immune cell.

[0024] In certain cases, the binding agent is capable of binding to CD36 and programmed cell death protein 1 (PD-1). Accordingly, in some embodiments, the binding agent includes at least one antigen-binding domain that binds to CD36 and at least one antigen-binding domain that binds to PD-1.

[0025] In other cases, the binding agent is capable of binding to CD36 and cluster of differentiation 47 (CD47). Accordingly, in some embodiments, the binding agent includes at least one antigen-binding domain capable of binding to CD36 and at least one antigen-binding domain capable of binding to CD47.

[0026] Accordingly, the present disclosure provides binding agents that are capable of binding to the CD36 protein or a portion thereof, the programmed cell death protein 1 (PD-1) protein or a portion thereof, and the cluster of differentiation 47 (CD47) protein or a portion thereof. In some embodiments, the binding agent can bind to cells that express the CD36 protein or a portion thereof, the PD-1 protein or a portion thereof, and / or the CD47 protein or a portion thereof.

[0027] In certain aspects, the binding agents of the present disclosure can bind to the human CD36 protein, the human PD-1 protein, and / or the human CD47 protein and / or cells that express the human CD36 protein, human PD-1, and / or human CD47.

[0028] The binding agents of the present disclosure include one or more antigen-binding domains. The binding agent can, for example, include at least one antigen-binding domain that can bind to the CD36 protein or a portion thereof or a cell that expresses the CD36 protein or a portion thereof, at least one antigen-binding domain that can bind to the PD-1 protein or a portion thereof or a cell that expresses the PD-1 protein or a portion thereof, and / or at least one antigen-binding domain that can bind to the CD47 protein or a portion thereof or a cell that expresses the CD47 protein or a portion thereof.

[0029] Thus, the binder of the present disclosure comprises at least one antigen-binding domain capable of binding to human CD36 protein or a portion thereof, at least one antigen-binding domain capable of binding to human PD-1 protein or a portion thereof, and / or at least one antigen-binding domain capable of binding to human CD47 protein or a portion thereof. According to the present disclosure, the binder may further comprise other antigen-binding domains.

[0030] In some embodiments, the antigen-binding domain is from an antibody capable of specifically binding to CD36 protein or a portion thereof. Thus, the antigen-binding domain may be derived from a single-domain antibody capable of specifically binding to CD36 protein or a portion thereof.

[0031] In some embodiments, the antigen-binding domain is from an antibody capable of specifically binding to PD-1 protein or a portion thereof. Thus, the antigen-binding domain may be derived from a single-domain antibody capable of specifically binding to PD-1 protein or a portion thereof.

[0032] In some embodiments, the antigen-binding domain is from an antibody capable of specifically binding to CD47 protein or a portion thereof. Thus, the antigen-binding domain may be derived from a single-domain antibody capable of specifically binding to CD47 protein or a portion thereof.

[0033] The binder of the present disclosure may be capable of reducing tumor growth and / or causing tumor regression. In some embodiments, the binder has anti-tumor activity. In some embodiments, the binder is capable of recruiting or redirecting immune cells.

[0034] In some cases, the binder of the present disclosure may modulate lipid metabolism.

[0035] In other cases, the binder of the present disclosure may inhibit fatty acid oxidation.

[0036] In other cases, the binder of the present disclosure may inhibit the uptake of fatty acids by tumors.

[0037] In other cases, the binder of the present disclosure may inhibit the growth of tumor cells or may be used to inhibit the growth of tumor cells.

[0038] In other cases, the binder of the present disclosure may inhibit tumor metastasis or may be used to inhibit tumor metastasis.

[0039] In other cases, the binder of the present disclosure may be used to reduce the resistance of tumor cells to chemotherapy drugs and / or immune checkpoint inhibitor therapy.

[0040] In some embodiments, the binder comprises an antigen-binding domain of a single-domain antibody having in vitro and / or in vivo activity. Thus, the binder comprises an antigen-binding domain of one or more single-domain antibodies capable of specifically binding to and inhibiting CD36. In some embodiments, the binder comprises multiple antigen-binding domains, and some of the antigen-binding domains may be from the same or different single-domain antibodies capable of specifically binding to and inhibiting CD36.

[0041] In one exemplary embodiment, the antigen-binding domain is from one or more single-domain antibodies capable of specifically binding to CD36 and having anti-tumor activity.

[0042] In another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies capable of inhibiting tumor cell growth.

[0043] In another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies capable of inhibiting tumor cell metastasis.

[0044] In another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies capable of regulating tumor lipid metabolism.

[0045] In another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies capable of inhibiting fatty acid oxidation.

[0046] In another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies capable of inhibiting fatty acid uptake by tumors.

[0047] In other exemplary embodiments, the antigen-binding domain is from one or more anti-CD36 single-domain antibodies that do not significantly cause platelet activation.

[0048] The binder of the present disclosure may further comprise an antigen-binding domain of one or more antibodies capable of specifically binding to an immune checkpoint protein.

[0049] For example, the binder of the present disclosure may comprise an antigen-binding domain of one or more single-domain antibodies capable of specifically binding to and inhibiting PD-1. In some embodiments, the binder comprises multiple antigen-binding domains, and some of the antigen-binding domains may be from the same or different single-domain antibodies capable of specifically binding to and inhibiting PD-1.

[0050] In some embodiments, the antigen-binding domain is from an anti-PD-1 single-domain antibody that is capable of blocking the interaction between PD-1 and PD-L1. PD-1 / PD-L1 blocking assays are known to those of skill in the art and include, for example, Promega, catalog number J1255.

[0051] In some embodiments, the antigen-binding domain is from an anti-PD-1 single-domain antibody that is capable of blocking the interaction between PD-L1 and / or PD-L2.

[0052] In some embodiments, the antigen-binding domain is from an anti-PD-1 single-domain antibody that is capable of restoring T cell effector function.

[0053] In some embodiments, the binder comprises one or more antigen-binding domains of an antibody that is capable of specifically binding to an immune cell surface protein.

[0054] For example, the antigen-binding domain is from a single-domain antibody that is capable of specifically binding to and inhibiting CD47. In some embodiments, the binder comprises multiple antigen-binding domains, and some of the antigen-binding domains may be from the same or different single-domain antibodies that are capable of specifically binding to and inhibiting CD47.

[0055] In some embodiments, the antigen-binding domain is from an anti-CD47 single-domain antibody that is capable of blocking the interaction between CD47 and SIRPα. CD47 / SIRPα blocking assays are known to those of skill in the art and include, for example, Promega, catalog number CS316013. In some embodiments, the antigen-binding domain is from an anti-CD47 single-domain antibody that is capable of blocking the interaction between CD47 and thrombospondin 1 (TSP-1).

[0056] In some embodiments, the antigen-binding domain is from an anti-CD47 single-domain antibody that is capable of recruiting T cells. In some embodiments, the antigen-binding domain is from an anti-CD47 single-domain antibody that is capable of enhancing macrophage function.

[0057] In some embodiments, the antigen-binding domain that is capable of binding to the CD36 protein or a portion thereof or to a cell that expresses the CD36 protein or a portion thereof is antigen-binding domain 1 (ABD1) as described herein.

[0058] Thus, in certain embodiments, the binder can comprise at least one antigen-binding domain 1 (ABD1). According to the present disclosure, ABD1 comprises at least the CDRH3 amino acid sequence of the anti-CD36 single-domain antibody disclosed herein. Also according to the present disclosure, ABD1 comprises the CDRH3 amino acid sequence of the anti-CD36 single-domain antibody disclosed herein and at least the CDRH1 and / or CDRH2 amino acid sequences. In certain embodiments, ABD1 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the anti-CD36 single-domain antibody disclosed herein. In certain embodiments, the CDRH1, CDRH2, and CDRH3 amino acid sequences are from a single anti-CD36 single-domain antibody. In other embodiments, ABD1 comprises the variable region of the anti-CD36 single-domain antibody disclosed herein.

[0059] In certain embodiments, the binder can comprise at least one antigen-binding domain 1 (ABD1) and at least one other antigen-binding domain. For example, the binder can comprise one or more antigen-binding domains 1 (ABD1) and one or more antigen-binding domains of one or more antibodies.

[0060] In certain cases, the binder can comprise at least one antigen-binding domain 1 (ABD1) and can also bind to immune cells. Thus, in certain embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain that binds to immune cells.

[0061] In certain embodiments, the antigen-binding domain that can bind to PD-1 or cells expressing PD-1 is the antigen-binding domain 2 (ABD2) described herein. In certain embodiments, the antigen-binding domain that can bind to PD-1 or cells expressing PD-1 is not limited to the antigen-binding domain 2 (ABD2).

[0062] Thus, in certain embodiments, the binder can comprise at least one antigen-binding domain 2 (ABD2). According to the present disclosure, ABD2 comprises at least the CDRH3 amino acid sequence of the anti-PD-1 single-domain antibody disclosed herein. Also according to the present disclosure, ABD2 comprises the CDRH3 amino acid sequence of the anti-PD-1 single-domain antibody disclosed herein and at least the CDRH1 and / or CDRH2 amino acid sequences. In certain embodiments, ABD2 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the anti-PD-1 single-domain antibody disclosed herein. In certain embodiments, the CDRH1, CDRH2, and CDRH3 amino acid sequences are from a single anti-PD-1 single-domain antibody. In other embodiments, ABD2 comprises the variable region of the anti-PD-1 single-domain antibody disclosed herein.

[0063] In certain embodiments, the binder can comprise at least one antigen-binding domain 2 (ABD2) and at least one other antigen-binding domain.

[0064] In certain embodiments, the antigen-binding domain that can bind CD47 or cells expressing CD47 is antigen-binding domain 3 (ABD3) as described herein. In certain embodiments, the antigen-binding domain that can bind CD47 or cells expressing CD47 is not limited to ABD3.

[0065] Thus, in certain embodiments, the binder can comprise at least one antigen-binding domain 3 (ABD3). According to the present disclosure, ABD3 comprises at least the CDRH3 amino acid sequence of the anti-CD47 single-domain antibody disclosed herein. Also according to the present disclosure, ABD3 comprises the CDRH3 amino acid sequence of the anti-CD47 single-domain antibody disclosed herein and at least the CDRH1 and / or CDRH2 amino acid sequences. In certain embodiments, ABD3 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the anti-CD47 single-domain antibody disclosed herein. In certain embodiments, the CDRH1, CDRH2, and CDRH3 amino acid sequences are from a single anti-CD47 single-domain antibody. In other embodiments, ABD3 comprises the variable region of the anti-CD47 single-domain antibody disclosed herein.

[0066] In certain embodiments, the binder can comprise at least one antigen-binding domain 3 (ABD3) and at least one other antigen-binding domain.

[0067] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1).

[0068] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2).

[0069] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3).

[0070] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is selected from:

[0071] antigen-binding domain 1 (ABD1),

[0072] antigen-binding domain 2 (ABD2), and / or,

[0073] Antigen-binding domain 3 (ABD3).

[0074] In certain embodiments, the binder can comprise an antigen-binding domain that targets an epitope other than the epitopes recognized by ABD1, ABD2, and ABD3. Thus, the binder can comprise an antigen-binding domain having the same or different specificity as ABD1, ABD2, and / or ABD3.

[0075] In certain embodiments, the binder can comprise multiple antigen-binding domains.

[0076] For example, in certain embodiments, the binder can 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.

[0077] In certain embodiments, the binder can comprise one to twelve antigen-binding domains.

[0078] In certain embodiments, the binders of the present disclosure can be monospecific.

[0079] In certain embodiments, the binders of the present disclosure can be multispecific.

[0080] In certain embodiments, the binders of the present disclosure can be monovalent.

[0081] In certain embodiments, the binders of the present disclosure can be multivalent.

[0082] In certain embodiments, the binders of the present disclosure can be in monomeric form.

[0083] In certain embodiments, the binders of the present disclosure can be in dimeric or higher-order forms, such as trimers, tetramers, pentamers, etc. (e.g., multimers).

[0084] In some cases, the antigen-binding domain of the binder is derived from a heavy-chain antibody. In some cases, the heavy-chain antibody can be obtained by immunizing camelids or transgenic animals.

[0085] The binders of the present disclosure are substantially polypeptide in nature. In certain embodiments, the binders of the present disclosure are substantially composed of one, two, or more than two polypeptide chains. However, the binder can include non-polypeptide moieties, such as small molecules, sugars, radiolabels, etc.

[0086] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from N-terminus to C-terminus, an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD-1, and an antigen-binding domain that binds CD47.

[0087] In certain embodiments, the antigen-binding domains are located on one or more polypeptide chains.

[0088] In certain embodiments, the antigen-binding domains are located on the same polypeptide chain.

[0089] In certain embodiments, the binder comprises a single polypeptide chain.

[0090] In certain embodiments, the binder comprises two polypeptide chains. Thus, the binder can be in the form of a dimer.

[0091] In some embodiments, the binder comprises more than two polypeptide chains, such as 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 binder can be in the form of a multimer. For example, a binder comprising three polypeptide chains is referred to herein as a trimer, a binder comprising four polypeptide chains is referred to herein as a tetramer, and so on.

[0092] In some embodiments, the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises one or more antigen-binding domains.

[0093] It should be understood herein that the polypeptide chains of the present disclosure can alternatively be expressed in tandem.

[0094] In some embodiments, two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen-binding domain.

[0095] In some embodiments, two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen-binding domain.

[0096] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 1 (ABD1).

[0097] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 2 (ABD2).

[0098] In some embodiments, each polypeptide chain comprises the same antigen-binding domain 3 (ABD3).

[0099] In some exemplary embodiments, the binder comprises at least two polypeptide chains assembled to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1) and the same antigen-binding domain 2 (ABD2).

[0100] In other exemplary embodiments, the binder comprises at least two polypeptide chains assembled to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1) and the same antigen-binding domain 3 (ABD3).

[0101] In further exemplary embodiments, the binder comprises at least two polypeptide chains assembled to form a dimer, and each polypeptide chain comprises the same antigen-binding domain 1 (ABD1), the same antigen-binding domain 2 (ABD2), and the same antigen-binding domain 3 (ABD3).

[0102] In some cases, antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and / or antigen-binding domain 3 (ABD3) may occupy the same position on each of the two polypeptide chains.

[0103] In some cases, antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and / or antigen-binding domain 3 (ABD3) may occupy different positions on each of the two polypeptide chains.

[0104] In some embodiments, each polypeptide chain of the binder is the same.

[0105] In some embodiments, each polypeptide chain of the binder is different.

[0106] In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.

[0107] In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.

[0108] In some embodiments, the antigen-binding domains are separated by an amino acid sequence.

[0109] In some embodiments, the amino acid sequence is a linker.

[0110] The binders of the present disclosure include, for example, the antigen-binding domains disclosed herein, the single polypeptide chains disclosed herein, the dimers of the polypeptide chains disclosed herein, or the multimers of the polypeptide chains disclosed herein.

[0111] Thus, the binders of the present disclosure can be in the form of antibodies and their antigen-binding fragments, antibody-like molecules (Fc-, CH3-fusions, etc.), fusions with protein scaffolds, immunomodulators, and the like.

[0112] Advantageously, the binder of the present disclosure may have the form disclosed in PCT / CA2020 / 051753, filed on December 18, 2020 (published on June 24, 2021, publication number WO2021 / 119832A1, the entire content of which is 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, etc., or Formula I, Formula II, Formula III, IIIa and IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII disclosed herein.

[0113] In some embodiments, the binder consists of polypeptide chains, and the polypeptide chains contain one or more antigen-binding domains and a dimerization domain that allows at least two polypeptide chains to form a dimer.

[0114] In some embodiments, the binder of the present disclosure may contain one or more polypeptide chains, and each polypeptide chain independently contains the amino acid sequence of Formula I in the form from the N-terminus to the C-terminus:

[0115] X - [(Ab a ) - (L b )] m - (DD) - [(L c ) - (Ab d )] n - Y

[0116] where m is 0, 1, 2, or an integer greater than 2;

[0117] where n is 0, 1, 2, or an integer greater than 2;

[0118] where m and n are not both 0;

[0119] where Ab a and Ab d each represent an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1);

[0120] where X or Y independently exists or is absent and contains an amino acid sequence;

[0121] where L b and L c each independently contain one or more linkers; and

[0122] where DD represents a dimerization domain.

[0123] In some embodiments, when m is 1, 2, or an integer greater than 2 and / or when n is 1, 2, or an integer greater than 2, Ab aor Ab d at least one of which is antigen - binding domain 2 (ABD2).

[0124] In some embodiments, when m is 1, 2, or an integer greater than 2 and / or when n is 1, 2, or an integer greater than 2, Ab a or Ab d at least one of which is antigen - binding domain 3 (ABD3).

[0125] In some embodiments, when m is 1, 2, or an integer greater than 2 and / or when n is 1, 2, or an integer greater than 2, Ab a or Ab d at least one of which is antigen - binding domain 2 (ABD2) and / or antigen - binding domain 3 (ABD3).

[0126] In some embodiments, the polypeptide chain comprises 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, and so on.

[0127] In some embodiments, the polypeptide chain comprises one to twelve antigen - binding domains.

[0128] In some embodiments, the binder comprises one polypeptide chain.

[0129] In some embodiments, the binder comprises two polypeptide chains, three polypeptide chains, four polypeptide chains, five polypeptide chains, six polypeptide chains, seven polypeptide chains, eight polypeptide chains, nine polypeptide chains, ten polypeptide chains or more than ten polypeptide chains.

[0130] In some embodiments, the polypeptide chains can be covalently linked.

[0131] In some embodiments, the polypeptide chains can be non - covalently linked.

[0132] In some embodiments, the polypeptide chains can associate through electrostatic interactions.

[0133] In some embodiments, when m is 2 or an integer greater than 2, [(Ab a )-(L b )] units are the same or different.

[0134] In some embodiments, when n is 2 or an integer greater than 2, [(L c )-(Ab d )] units are the same or different.

[0135] In some embodiments, when m is 2 or an integer greater than 2, each Ab aare the same or different.

[0136] In some embodiments, when n is an integer of 2 or greater than 2, each Ab d are the same or different.

[0137] In some embodiments, Ab a represents ABD1, ABD2, or ABD3.

[0138] In some embodiments, Ab d represents ABD1, ABD2, or ABD3.

[0139] In some embodiments, Ab d represents ABD1, ABD2, or ABD3.

[0140] In an embodiment, one or more polypeptide chains further comprise the hinge region of an antibody or an antigen-binding fragment thereof. For example, in some embodiments, L b is the hinge region of an antibody or an antigen-binding fragment thereof.

[0141] In some embodiments, the hinge region is the native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0142] In some embodiments, the hinge region is a mutant hinge region having at least 70% identity to the native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0143] In some embodiments, one or more linkers each independently have a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0144] In some embodiments, one or more linkers each independently are a flexible linker, a helical linker, or a rigid linker.

[0145] 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.

[0146] In some embodiments, the rigid linker comprises multiple PA repeat sequences as described herein.

[0147] In some embodiments, the helical linker comprises one or more EAAAK units as described herein.

[0148] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula II: X-(Ab a1 )-(L b1 )-(DD)-(Lc1 )-(Ab d1 )-Y (Formula II).

[0149] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0150] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0151] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises Formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0152] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises 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).

[0153] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises 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).

[0154] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains comprises 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).

[0155] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 each independently comprises an antigen-binding domain.

[0156] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 each independently represents an antigen-binding domain.

[0157] In some embodiments, at least one of Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 or Ab d3 is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0158] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1, Ab d2 or Ab d3 at least one of which is not antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0159] In some embodiments, L b1 comprises one or more linkers and / or the hinge region of an antibody or an antigen-binding fragment thereof. In some embodiments, the hinge region is a native hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.

[0160] In some embodiments, L b2 , L b3 , L c1 , L c2 and L c3 each independently comprises one or more linkers as disclosed herein.

[0161] In some embodiments, the polypeptide chain comprises an antigen-binding domain ABD1 as disclosed herein and an antigen-binding domain that binds to immune cells.

[0162] In some embodiments, the polypeptide chain comprises an antigen-binding domain ABD2 as disclosed herein and an antigen-binding domain that binds to tumor cells.

[0163] In some embodiments, the polypeptide chain comprises an antigen-binding domain ABD3 as disclosed herein and an antigen-binding domain that binds to tumor cells.

[0164] In some embodiments, the polypeptide chain comprises an antigen-binding domain ABD3 as disclosed herein and an antigen-binding domain that binds to immune cells.

[0165] In certain embodiments, the polypeptide chain comprises at least one antigen-binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0166] In certain embodiments, the polypeptide chain comprises at least two antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0167] In certain embodiments, the polypeptide chain comprises at least three antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0168] In certain embodiments, the dimerization domain comprises an immunoglobulin dimerization domain. Other dimerization domains known to those skilled in the art are contemplated herein, including leucine zippers and the like.

[0169] In certain embodiments, the dimerization domain comprises the dimerization domain of IgG, IgM, IgA, IgD or IgE (IgG, IgM, IgA, IgD or IgE from human or animal).

[0170] In certain embodiments, the dimerization domain comprises the CH3 domain of an antibody. The dimerization domain may also comprise the CH2 domain of an antibody.

[0171] In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.

[0172] In some exemplary embodiments, the dimerization domain comprises a mutant CH3 domain.

[0173] In certain embodiments, the dimerization domain comprises a native CH2 and a native CH3 domain.

[0174] In certain embodiments, the dimerization domain comprises a native CH2 and a mutant CH3 domain.

[0175] In certain embodiments, the dimerization domain comprises a mutant CH2 and a mutant CH3 domain.

[0176] In certain embodiments, the dimerization domain comprises a mutant CH2 and a native CH3 domain.

[0177] In certain embodiments, the dimerization domain does not contain the CH1 domain.

[0178] In certain embodiments, the dimerization domain does not contain the CH4 domain.

[0179] In certain embodiments, the dimerization domain comprises the Fc region of an antibody or a portion thereof.

[0180] In certain embodiments, the polypeptide chain comprises at least two or at least three antigen-binding domains and a dimerization domain such that two polypeptide chains can assemble to form a multivalent and / or multispecific binding agent.

[0181] In an embodiment, the antigen-binding domain comprises or consists of the antigen-binding domain of a single-domain antibody (sdAb).

[0182] In an embodiment, the antigen-binding domain comprises a heavy-chain variable region (VH or VHH).

[0183] In certain embodiments, the VHH is derived from human, mouse, rat, etc.

[0184] In certain embodiments, the VHH is from a transgenic mouse or rat capable of expressing a camelid-humanized mouse or rat VHH, a VHH from another species (e.g., human, etc.), or a camelid-humanized VHH from another species (e.g., camelid-humanized human VHH, etc.).

[0185] In certain embodiments, the binder does not contain a variable light chain region (VL or VLL).

[0186] In embodiments, the binder contains a variable light chain region (VL or VLL).

[0187] In embodiments, the antigen-binding domain is a single-chain variable fragment (ScFv).

[0188] In embodiments, the antigen-binding domain is from V NAR fragment.

[0189] In other embodiments, the antigen-binding domain of the polypeptide chain comprises any combination of antigen-binding domains of a single-domain antibody (sdAb), a variable heavy chain region (VH or VHH), a variable light chain region (VL or VLL), a single-chain variable fragment (ScFv), and / or V NAR fragment.

[0190] In certain embodiments, the sdAb or VHH is from a camelid antibody.

[0191] In embodiments, the camelid antibody is from a dromedary, a camel, a llama, an alpaca, etc.

[0192] In other embodiments, the sdAb or VHH is from a chondrichthyan antibody.

[0193] In certain embodiments, the chondrichthyan antibody is a shark antibody.

[0194] In certain embodiments, each antigen-binding domain specifically binds to a different epitope.

[0195] In other embodiments, each antigen-binding domain specifically binds to a different antigen.

[0196] In still other embodiments, each antigen-binding domain specifically binds to a different protein.

[0197] It should be understood herein that a given antigen-binding domain can bind to epitopes present in different proteins. Thus, in certain embodiments, the antigen-binding domain or the binder comprising the antigen-binding domain can bind to more than one protein. In certain embodiments, the antigen-binding domain or the binder can have an affinity for more than one protein.

[0198] In certain embodiments, the binder is an antibody or an antigen-binding fragment thereof.

[0199] In other embodiments, the binder comprises an antibody or an antigen-binding fragment thereof.

[0200] In other embodiments, the binder comprises an antigen-binding domain of an antibody or an antigen-binding fragment thereof.

[0201] In certain embodiments, the binder is a single-domain antibody.

[0202] In other embodiments, the binder comprises a single-domain antibody.

[0203] In other embodiments, the binder comprises an antigen-binding domain of a single-domain antibody.

[0204] In certain embodiments, the binder is a single-chain antibody (e.g., scFv).

[0205] In other embodiments, the binder comprises a single-chain antibody (e.g., scFv).

[0206] In certain embodiments, the binder comprises at least two polypeptide chains capable of forming a dimer.

[0207] In certain embodiments, the two polypeptide chains are the same.

[0208] In certain embodiments, the two polypeptide chains are different.

[0209] In certain embodiments, the polypeptide chains are the same and the binder is a homodimer.

[0210] In certain embodiments, the polypeptide chains are different and the binder is a heterodimer.

[0211] In certain embodiments, the binder is multispecific.

[0212] In certain embodiments, the binder is bispecific, trispecific or tetravalent.

[0213] In certain embodiments, the binder comprises one or more multispecific polypeptide chains.

[0214] In certain embodiments, the binder comprises one or more bispecific, trispecific or tetravalent polypeptide chains.

[0215] In certain embodiments, one or more polypeptide chains have the same valence and specificity.

[0216] In certain embodiments, one or more polypeptide chains have different valence and specificity.

[0217] In certain embodiments, one or more polypeptide chains are each an antibody heavy chain.

[0218] In certain embodiments, the binder is an antibody or an antigen-binding fragment thereof.

[0219] In certain embodiments, the binder is a bispecific antibody.

[0220] In certain embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

[0221] In certain embodiments, one or more of the antigen-binding domains are humanized.

[0222] In certain embodiments, one or more of the antigen-binding domains are partially humanized.

[0223] In certain embodiments, the antigen-binding domain comprises one or more human framework regions.

[0224] In certain embodiments, X or Y is 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.

[0225] In certain embodiments, the polypeptide chain is conjugated to a therapeutic moiety.

[0226] In certain embodiments, the polypeptide chain is conjugated to a detectable moiety.

[0227] In certain embodiments, the polypeptide chain is conjugated to a protein that allows for an extended half-life.

[0228] In certain embodiments, the polypeptide chain is attached to a nanoparticle.

[0229] Other aspects and embodiments of the present disclosure relate to compositions comprising at least one binder disclosed herein.

[0230] In certain embodiments, the composition comprises monomers, dimers, and mixtures thereof.

[0231] In certain embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present in the composition in dimer form.

[0232] In certain embodiments, more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present in the composition in homodimer form.

[0233] In certain embodiments, more 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.

[0234] Other aspects and embodiments of the present disclosure relate to pharmaceutical compositions comprising a binder disclosed herein and a pharmaceutically acceptable carrier.

[0235] Other aspects and embodiments of the present disclosure relate to nucleic acids or a set of nucleic acids encoding polypeptide chains and / or binders.

[0236] The nucleic acid can be in the form of a DNA segment disclosed herein.

[0237] 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.

[0238] Further aspects and embodiments of the present disclosure relate to cells expressing a polypeptide chain or binder disclosed herein.

[0239] Additional aspects and embodiments of the present disclosure relate to cells comprising a nucleic acid or vector disclosed herein.

[0240] Further aspects and embodiments of the present disclosure relate to kits comprising a binder disclosed herein.

[0241] Further aspects and embodiments of the present disclosure relate to kits comprising a nucleic acid disclosed herein.

[0242] Further aspects and embodiments of the present disclosure relate to kits comprising a vector disclosed herein.

[0243] Further aspects and embodiments of the present disclosure relate to kits comprising a cell disclosed herein.

[0244] In additional aspects and embodiments, the present disclosure relates to methods of treating or preventing a disease or disorder associated with CD36, CD36 expression, CD36 overexpression or CD36 upregulation.

[0245] Exemplary embodiments of the disease or disorder include cancer, metabolic diseases such as atherosclerosis, diabetes, non-alcoholic fatty liver disease, thrombosis or inflammation.

[0246] In additional aspects and embodiments, the present disclosure relates to a method of treating cancer, the method comprising administering to a subject in need thereof a binder disclosed herein.

[0247] In further aspects and embodiments, the present disclosure relates to a method of treating cancer, the method comprising administering to a subject in need thereof a composition disclosed herein.

[0248] In further aspects and embodiments, the present disclosure relates to a method of treating cancer, the method comprising administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0249] In certain embodiments, the cancer is metastatic.

[0250] In certain embodiments, the cancer is leukemia, including but not limited to acute myeloid leukemia.

[0251] In certain embodiments, the cancer is a solid tumor.

[0252] In certain embodiments, the cancer is selected from endometrial cancer, breast cancer (e.g., triple negative breast cancer), liver cancer (e.g., hepatocellular carcinoma), lung cancer, prostate cancer, kidney cancer (e.g., renal clear cell carcinoma), ovarian cancer, cervical cancer, pancreatic cancer, gastric cancer, bladder cancer (e.g., urothelial carcinoma of the bladder), brain cancer, oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, colon cancer, and colorectal cancer.

[0253] In certain embodiments, the cancer originates from adipose (fatty) tissue.

[0254] In certain embodiments, the cancer is associated with increased lipid metabolism. For example, the subject's tumor may exhibit increased fatty acid uptake and / or increased fatty acid oxidation.

[0255] For example, the binder can be used to reduce and / or prevent tumor growth in a subject in need thereof. In other examples, the binder can be used to reduce and / or prevent tumor metastasis. In other examples, the binder can be used to reduce resistance to chemotherapy and / or immune checkpoint inhibition therapy.

[0256] Accordingly, the binder can be administered to a subject having cancer selected from leukemia (e.g., acute myeloid leukemia), endometrial cancer, breast cancer (e.g., triple negative breast cancer), liver cancer (e.g., hepatocellular carcinoma), lung cancer, prostate cancer, kidney cancer (e.g., renal clear cell carcinoma), ovarian cancer, cervical cancer, pancreatic cancer, gastric cancer, bladder cancer (e.g., urothelial carcinoma of the bladder), oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, and colorectal cancer.

[0257] In certain embodiments, the binder is administered to a subject who has failed prior treatment with chemotherapy (chemotherapeutic agents) and / or immune checkpoints.

[0258] In certain embodiments, the binding agent is administered to a subject with recurrent leukemia. In certain embodiments, the binding agent is administered to a subject with recurrent acute myeloid leukemia. In certain embodiments, the binding agent is administered to a cancerous subject having increased lipid metabolism such as, but not limited to, increased fatty acid uptake and / or increased fatty acid oxidation.

[0259] In certain embodiments, the binding agent is administered to a cancerous subject to reduce and / or prevent tumor growth.

[0260] In certain embodiments, the binding agent is administered to a cancerous subject to reduce and / or prevent tumor metastasis.

[0261] In certain embodiments, the binding agent is administered to a cancerous subject to reduce resistance to chemotherapy and / or immune checkpoint inhibition therapy.

[0262] In other aspects and embodiments, the present disclosure relates to a method of making the binding agents disclosed herein, the method comprising transforming cells with one or more vectors comprising the nucleic acids disclosed herein.

[0263] In certain embodiments, the method further comprises separating and / or purifying the binding agent from impurities.

[0264] In other embodiments, the method further comprises separating and / or purifying the heterodimer from monomers and / or homodimers.

[0265] In other embodiments, the method further comprises separating and / or purifying the homodimer from monomers and / or heterodimers.

[0266] In certain embodiments, the method further comprises conjugating the binding agent to a therapeutic moiety, a detectable moiety, or a protein that permits an extended half-life or conjugating to a nanoparticle.

[0267] The further scope, applicability, and advantages of the present disclosure will become apparent from the following non-limiting detailed description given herein. However, it should be understood that while the detailed description indicates exemplary embodiments of the present disclosure, it is given by way of example only in conjunction with the accompanying drawings.

[0268] Brief Description of the Drawings

[0269] Figure 1 : The bar graph shows the binding of the CD36 antibody to human CD36 protein by ELISA at a concentration of 10 ug / ml.

[0270] Figure 2: The bar graph shows the binding of the CD36 antibody to OCI-AML3 cells (CD36+) and OCI-AML4 cells (CD36-).

[0271] Figure 3 : The figure illustrates the inhibitory effect of the CD36 antibody on fatty acid oxidation in OCI-AML3 cells at a concentration of 350 nM.

[0272] Figure 4 : The figure shows the effect of the anti-CD36 antibody on platelet activation at a concentration of 50 ug / ml.

[0273] Figure 5 A-5B: The schematic diagram shows the configurations of the monospecific single-domain antibody in Table 6 ( Figure 5 A) and the multivalent, multispecific binder in Table 7 ( Figure 5 B).

[0274] Figure 6 : The figure shows the tumor regression of the anti-CD36 KisoBody KF061 on OCI-AML3 xenograft tumors in the I / O model.

[0275] Figures 7A - 7B : The alignment of the anti-CD36 VHH in Table 3 with the IMGT CDR (A) and Kabat CDR (B) is highlighted.

[0276] Figure 8 A-8D: The alignment of the selected anti-CD36 VHH in Table 3.

[0277] Figure 9 : The alignment of the selected anti-PD-1 VHH in Table 4.

[0278] Detailed Description

[0279] Definitions

[0280] Unless otherwise specified, the amino acid numbering of the dimerization domain is according to the EU numbering system.

[0281] In the context of describing the embodiments (especially in the context of the claims), unless otherwise specified herein or the context clearly dictates the contrary, the use of the terms "a" and "an" and "the" and similar references shall be construed to include both the singular and the plural.

[0282] Unless specifically stated or clearly indicated from the context, the term "or" as used herein is understood to be inclusive, covering both "or" and "and".

[0283] The term "and / or" as used herein shall be regarded as a specific disclosure of each designated feature or component, whether or not in combination with other features or components.

[0284] Unless otherwise specified, the terms "comprising", "having", "including", and "containing" shall be construed as open-ended terms (i.e., meaning "including but not limited to"). The term "consisting of" shall be construed as closed-ended.

[0285] In the present disclosure, the term "treatment" refers to therapeutic treatment and prophylactic or oncology preventive measures. Subjects in need of treatment include those who already have cancer and those who are susceptible to cancer or in need of managing cancer.

[0286] The term "CD36" as used herein refers to the CD36 protein or a polypeptide comprising the CD36 amino acid sequence. The term "CD36" includes the human CD36 protein, the CD36 antigen, and any protein having at least 80% identity with the human CD36 protein.

[0287] The term "anti-CD36 antibody" refers to any form of antibody that binds to the CD36 protein, the CD36 antigen, or a portion thereof, including but not limited to "anti-CD36 single-domain antibody".

[0288] The term "PD-1" as used herein refers to the PD-1 protein or a polypeptide comprising the PD-1 amino acid sequence. The term "PD-1" includes the human PD-1 protein, the PD-1 antigen, and any protein having at least 80% identity with the human PD-1 protein.

[0289] The term "anti-PD-1 antibody" refers to any form of antibody that binds to the PD-1 protein, the PD-1 antigen, or a portion thereof, including but not limited to "anti-PD-1 single-domain antibody".

[0290] The term "CD47" as used herein refers to the CD47 protein or a polypeptide comprising the CD47 amino acid sequence. The term "CD47" includes the human CD47 protein, the CD47 antigen, and any protein having at least 80% identity with the human CD47 protein.

[0291] The term "anti-CD47 antibody" refers to any form of antibody that binds to the CD47 protein, the CD47 antigen, or a portion thereof, including but not limited to "anti-CD47 single-domain antibody".

[0292] The term "antibody" is used in the broadest sense and encompasses various antibody forms and structures, including any immunoglobulin, monoclonal antibody, polyclonal antibody, bivalent antibody, monovalent antibody, bispecific antibody, multispecific (polyspecific) antibody, conventional antibody, single-domain antibody, heavy-chain-only antibody, nanobody, full-length antibody, humanized antibody, chimeric antibody, an antibody that binds to a specific antigen, and any antigen-binding fragment that exhibits the desired antigen-binding activity. An antibody can be naturally occurring (native) or the result of recombinant technology.

[0293] As used herein, the term "subject in need thereof" refers to a subject having, suspected of having, a disease or disorder associated with CD36, CD36 expression, CD36 overexpression, or CD36 upregulation, and who may benefit from treatment with an agent that targets CD36. "Subject in need thereof" includes subjects having cancer or suspected of having cancer.

[0294] As used herein, the expressions "single-domain antibody" and "heavy-chain-only antibody" can be used interchangeably. The term "single-domain antibody" includes naturally occurring single-domain antibodies and single-domain antibodies and variants thereof in which the naturally occurring constant region has been replaced by another dimerization domain (such as the constant region of a human immunoglobulin (such as IgG)), such as humanized or chimeric single-domain antibodies.

[0295] "Naturally occurring single-domain antibodies" include antibodies produced by camelids (camelid antibodies) or sharks. "Naturally occurring single-domain antibodies" also include antibodies produced by transgenic animals that have been modified to express heavy-chain-only antibodies. Exemplary embodiments of transgenic animals are provided in the application with international application number PCT / CA2021 / 050951, filed on July 21, 2021, and published on January 20, 2022, with publication number WO2022 / 011457, the entire content of which is incorporated herein by reference. Although naturally occurring single-domain antibodies generally do not include light chains, the present disclosure does not exclude single-domain antibodies that contain light chains. As used herein, the terms "naturally occurring antibody" or "naturally occurring single-domain antibody" refer to an antibody or single-domain antibody isolated and / or purified from an animal, typically obtained after immunization. The term "naturally occurring" is not intended to cover products that exist in nature without human or mechanical intervention.

[0296] The term "antigen-binding domain" refers to the part of an antibody that participates in antigen binding and includes, for example, one or more CDRs, one or more framework regions (FRs), or the entire variable region. In the context of a single-domain antibody, the term "antigen-binding domain" refers to the part of the single-domain antibody that participates in antigen binding and includes, for example, one or more of CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more framework regions FR1, FR2, FR3, FR4, or the entire variable region (VH or VHH). In the context of a native antibody, the term "antigen-binding domain" refers to the part of the native antibody that participates in antigen binding and includes, for example, one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light-chain or heavy-chain framework regions FR1, FR2, FR3, FR4, or one or both entire variable regions (heavy-chain variable region (VH) and / or light-chain variable region (VL)).

[0297] The term "antigen-binding fragment" as used in the expression "antibody or its antigen-binding fragment" or "single-domain antibody or its antigen-binding fragment" refers to an antibody or single-domain antibody fragment that contains an antigen-binding domain and may or may not contain other parts of the antibody or single-domain antibody, such as amino acid residues of the hinge region, amino acid residues of the constant region, and a part of the Fc region. Regardless of the structure, the antigen-binding fragment binds to the same antigen as the full antibody (such as a single-domain antibody).

[0298] The term "binding agent" refers to a molecule that contains a polypeptide part (such as a polypeptide chain) and is capable of specifically binding to an antigen, or contains a domain capable of specifically binding to an antigen, such as an antigen-binding domain.

[0299] The term "humanized" as used herein means that the antigen-binding domain contains human antibody amino acid residues. A "humanized antigen-binding domain" typically has CDRs derived from a non-human animal antibody, FR amino acid residues derived from a human antibody, and, when applicable, a constant region derived from a human antibody. However, in some cases, a "humanized antigen-binding domain" may also contain CDR amino acid residues derived from a human antibody. The term "humanized" encompasses "fully humanized" and "partially humanized".

[0300] As used herein, the expression "fully humanized" means that the amino acid sequences of all framework regions of a given antigen-binding domain are identical to the amino acid sequences of a human antibody such as a germline human antibody variable region. Thus, the term "fully humanized antigen-binding domain" encompasses antigen-binding domains having CDRs derived from a non-human animal antibody, all FR amino acid residues derived from a human antibody, and, where applicable, a constant region derived from a human antibody. In some cases, a "fully humanized antigen-binding domain" may also contain CDR amino acid residues derived from a human antibody.

[0301] As used herein, the expression "partially humanized" means that the amino acid sequence of one or more or all framework regions of a given antigen-binding domain is not identical to the amino acid sequence of a human antibody such as a germline human antibody variable region. The expression "partially humanized" also applies to antigen-binding domains containing human framework regions that contain back mutations reintroducing one or more of the original non-human amino acid residues into the human framework. Thus, the term "partially humanized antigen-binding domain" encompasses antigen-binding domains having CDRs derived from a non-human animal antibody, some FR amino acid residues derived from a human antibody, and, where applicable, a constant region derived from a human antibody. In some cases, a "partially humanized antigen-binding domain" may also contain CDR amino acid residues derived from a human antibody.

[0302] As used herein, the term "specifically binds" or "binds specifically to" refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. Specific binding can be characterized by binding affinity. A K D value ≤ 10 -6 M (e.g., ≤ 5x10 -7 M, ≤ 2x10 -7 M, ≤ 10 -7 M, ≤ 5x10 -8 M, ≤ 2x10 -8 M, ≤ 10 -8 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M or ≤ 10 -9 M and any ≤ 10 -6 value) can indicate specific binding between a binder (e.g., an antibody such as a single domain antibody) and CD36 (e.g., human CD36).

[0303] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between a binder or its antigen-binding domain and an antigen. Affinity is, for example, represented by K DThe value represents the ratio of the dissociation rate to the association rate (k off / k on or k ass / k diss ) when the binding between an antigen and a binding agent (such as an antibody, such as a single-domain antibody) reaches equilibrium. The affinity can be determined by using any known conventional method, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). Antibodies typically have a K D value ≤ 10 -6 M (e.g., ≤ 5x10 -7 M, ≤ 2x10 - 7 M, ≤ 10 -7 M, ≤ 5x10 -8 M, ≤ 2x10 -8 M, ≤ 10 -8 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M or ≤ 10 -9 M and any value ≤ 10 -6 ). Preferably, the antibody has a K D value in the nanomolar range or lower (e.g., ≤ 9x10 -9 M, ≤ 8x10 -9 M, ≤ 7x10 -9 M, ≤ 6x10 -9 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M, ≤ 1x10 -9 M or lower, e.g., ≤ 1x10 -10 M, ≤ 1x10 -11 M, ≤ 1x10 -12 M). More preferably, the antibody has a K D value in the picomolar range or lower (e.g., ≤ 9x10 -12 M, ≤ 8x10 -12 M, ≤ 7x10 -12 M, ≤ 6x10 -12 M, ≤ 5x10 -12 M, ≤ 4x10 -12 M, ≤ 3x10 - 12 M, ≤ 2x10 -12 M, ≤ 1x10 -12 M or lower).

[0304] As used herein, the terms "competing for binding", "compete(s) with", "competing with" refer to the ability of a first antibody or an antigen-binding fragment thereof (e.g., a single-domain antibody or an antigen-binding fragment thereof) to inhibit the binding interaction of CD36 with a second antibody to the same CD36, the binding interaction of PD-1 with a second anti-PD-1 antibody, and the binding interaction of CD47 with a second anti-CD47 antibody (e.g., a single-domain antibody or an antigen-binding fragment thereof) to any detectable extent.

[0305] As used herein, the term "epitope" refers to the specific atoms or group of amino acid residues on an antigen to which an antibody (e.g., a single-domain antibody) binds. If two antibodies exhibit competitive binding to an antigen, they may bind to the same or closely related epitopes. Epitopes can be linear or conformational (i.e., including amino acid residues that are spaced apart). For example, an antibody or antigen-binding fragment is considered to bind to the same / closely related epitope as a reference antibody if it blocks the binding of the reference antibody (e.g., a single-domain antibody) to the antigen by at least 85%, or at least 90%, or at least 95%. For example, a monoclonal, chimeric, human, or humanized antibody or an antigen-binding fragment thereof can compete with the single-domain antibodies of the present disclosure for binding to human DR2.

[0306] As used herein, the term "sequence identity" refers to the degree of identity between two nucleic acid or two amino acid sequences when compared at their best alignment and with appropriate mutations (such as substitutions, insertions, or deletions). The sequence identity can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. In certain embodiments, framework regions are generally not considered part of the antigen-binding domain. However, certain amino acid residues in one or more framework regions may contribute to antigen binding.

[0307] The term "functional activity" with respect to an antigen-binding domain means that the antigen-binding domain is capable of binding its target, and optionally the antigen-binding domain has one or more biological activities.

[0308] As used herein, the term "flexible linker" refers to a peptide that includes at least a portion composed of flexible amino acid residues that enable adjacent modules to move relative to each other.

[0309] As used herein, the term "rigid linker" refers to a peptide that includes at least a portion composed of amino acids that exhibit a rigid structure and maintain the distance between two modules.

[0310] As used herein, the term "helical linker" refers to a linker composed of amino acid residues that adopt an α-helical conformation.

[0311] As used herein, the term "cleavable linker" refers to a peptide that includes a cleavage site sensitive to proteases selected from ADAMS, ADAMTS, aspartic proteases, caspases, cathepsin cysteine proteases, cysteine proteases, metalloproteases, serine proteases, coagulation factor proteases, type II transmembrane serine proteases (TTSP), and combinations thereof.

[0312] As used herein, the term "monospecific" with respect to a binder refers to a binder that binds to a single antigen or epitope. Thus, a monospecific binder may have one antigen-binding domain or multiple binding domains (identical or different) with the same specificity for a given antigen or epitope.

[0313] As used herein, the term "multivalent" with respect to a binder refers to a binder that binds to multiple antigens or epitopes.

[0314] The term "multivalent" includes "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", etc.

[0315] As used herein, the term "multispecific" with respect to a binder refers to a binder that binds to at least two different antigens or epitopes.

[0316] The term "multispecific" includes "bispecific", "trispecific", "tetraspecific", "pentaspecific", "hexaspecific", etc.

[0317] As used herein, the term "bispecific" in the context of a binder refers to a binder that can bind to two different antigens or proteins or two epitopes of the same antigen or protein.

[0318] As used herein, expressions such as "at least two polypeptide chains" as in "a binder comprises at least two polypeptide chains" refer to the quantity of polypeptide chain substances rather than an absolute numerical value.

[0319] It should be understood herein that an expression in the form of a range value such as "from A to B" includes each individual value and any sub-range included within that range and includes such a range. For example, the expression "from 1 to 10" includes sub-ranges such as, but not limited to, "from 2 to 10", "from 2 to 9", "from 3 to 6", "from 5 to 7", etc., and any single value included in and including between 1 and 10, namely 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0320] As used herein, it should be understood that the term "at least" is intended to include the value and higher values thereof in terms of a given value. 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%.

[0321] The term "about" or "approximately" in terms of a given value means that variations of the value are taken into account. In some embodiments, the term "about" or "approximately" generally should mean a range within + / - 10%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, or + / - 1% of the given value or range.

[0322] The term "codon-optimized" refers to a sequence for which the codons have been changed to another codon that encodes the same amino acid but is preferred or performs better in a given organism (increasing expression, minimizing secondary structure in RNA, etc.). A "codon-optimized" sequence can be obtained by using publicly available software or through service providers including GenScript (OptimumGene TM , U.S. Patent No. 8,326,547).

[0323] The term "single-chain form" in terms of an antigen-binding domain means that the domain necessary for antigen binding is located on a single polypeptide chain.

[0324] As used herein, any of the terms "KF023", "KF024", "KF025", "KF026", "KF027", "KF028", "KF029", "KF030", "KF031", "KF032", "KF033", "KF034", "KF035", "KF036", "KF037", "KF038", "KF039", "KF040", "KF041", "KF042", "KF043", "KF044", "KF045", "KF046", "KF047", "KF048", "KF049", "KF050", "KF051", "KF052", "KF053", "KF054", "KF055", "KF056", "KF057", "KF058", "KF059", or "KF061" refers to a binder comprising two chains, each chain having the corresponding amino acid sequence set forth in Table 6 or Table 7 or described herein.

[0325] Antigen-binding domain (Ab)

[0326] As disclosed herein, a binder may comprise one or more antigen-binding domains.

[0327] For example, the binders of the present disclosure comprise one or more antigen-binding domains capable of binding CD36. The binders may also comprise one or more antigen-binding domains that bind one or more other antigens.

[0328] In certain cases, the antigen-binding domain may bind, for example, an immune checkpoint protein.

[0329] In other cases, the antigen-binding domain may bind, for example, a protein expressed on the surface of an immune cell.

[0330] The antigen-binding domains of the present disclosure may be selected based on their ability to bind a specific target. The antigen-binding domains may also be selected based on their in vivo and / or in vitro functional properties or biological effects, including, for example, their ability to modulate cellular processes such as gene expression, signal transduction, cell growth, cell viability, and the like.

[0331] For example, the binders of the present disclosure comprise one or more antigen-binding domains, each of which independently comprises one or more complementarity-determining regions (CDRs) of an antibody.

[0332] Thus, the specificity of the binders of the present disclosure may be conferred by their antigen-binding domains.

[0333] The binder of the present disclosure may comprise an antigen-binding domain capable of binding CD36 or a cell expressing CD36, an antigen-binding domain capable of binding PD-1 or a cell expressing PD-1, and / or an antigen-binding domain capable of binding CD47 or a cell expressing CD47.

[0334] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding CD36 or a cell expressing CD36, and includes, for example, antigen-binding domain 1 (ABD1). In some embodiments, the cells expressing CD36 include tumor cells.

[0335] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding PD-1 or a cell expressing PD-1, and includes, for example, antigen-binding domain 2 (ABD2). In some embodiments, the cells expressing PD-1 include immune cells.

[0336] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding CD47 or a cell expressing CD47, and includes, for example, antigen-binding domain 3 (ABD3). In some embodiments, the cells expressing CD47 include immune cells or tumor cells.

[0337] 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 epitopes or antigens.

[0338] In some embodiments, at least one of the antigens bound by the antigen-binding domain is not CD36, PD-1, or CD47.

[0339] In some embodiments, the binder may comprise more than one antigen-binding domain.

[0340] For example, in some embodiments, the binder may comprise two or more antigen-binding domains.

[0341] In certain embodiments, the binder may comprise three or more antigen-binding domains.

[0342] In certain embodiments, the binder may comprise four or more antigen-binding domains.

[0343] In certain embodiments, the binder may comprise five or more antigen-binding domains.

[0344] In certain embodiments, the binder may comprise six or more antigen-binding domains.

[0345] In certain embodiments, the binder can comprise from one to twelve antigen-binding domains, such as one to two, one to three, one to four, one to five, one to six, one to seven, one to eight, one to nine, one to ten, one to eleven, one to twelve, two to three, two to four, two to five, two to six, two to seven, two to eight, two to nine, two to ten, two to eleven, two to twelve, three to four, three to five, three to six, three to seven, three to eight, three to nine, three to ten, three to eleven, three to twelve, four to five, four to six, four to seven, four to eight, four to nine, four to ten, four to eleven, four to twelve, five to six, five to seven, five to eight, five to nine, five to ten, five to eleven, five to twelve, six to seven, six to eight, six to nine, six to ten, six to eleven, six to twelve, seven to eight, seven to nine, seven to ten, seven to eleven, seven to twelve, eight to nine, eight to ten, eight to eleven, eight to twelve, nine to ten, nine to eleven, nine to twelve, ten to eleven, ten to twelve, or eleven to twelve.

[0346] In certain embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains and at least one of the antigen-binding domains is capable of binding to a tumor cell.

[0347] In certain embodiments, the binder is capable of binding to a tumor cell. In other embodiments, the binder is capable of binding to a protein expressed on the surface of a tumor cell.

[0348] In certain embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, and at least one of the antigen-binding domains is capable of binding to an immunomodulator. In certain embodiments, at least one of the antigen-binding domains is capable of binding to an immune checkpoint protein. In certain embodiments, the immune checkpoint protein is PD-1.

[0349] In certain embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, and at least one of the antigen-binding domains is capable of binding to an immune cell. In certain embodiments, at least one of the antigen-binding domains is capable of binding to a protein expressed on the surface of an immune cell (such as a T cell, NK cell, monocyte, macrophage, etc.).

[0350] In certain embodiments, the binder is capable of binding to an immune cell. In other embodiments, the binder is capable of binding to a protein expressed on the surface of an immune cell.

[0351] In certain embodiments, the binder of the present disclosure can be multivalent and can comprise at least one antigen-binding domain that binds to tumor cells and at least one antigen-binding domain that binds to immune cells.

[0352] In certain embodiments, the protein expressed on the surface of immune cells is selected from CD47 or CD3.

[0353] In certain embodiments, the protein expression on the surface of immune cells is CD47.

[0354] In certain embodiments, the protein expression on the surface of immune cells is CD3.

[0355] The antigen-binding domain can be derived from natural antibodies (from human or animal sources) or from synthetic antibodies.

[0356] In certain embodiments, the antigen-binding domain of the natural antibody is engineered to form a single chain.

[0357] In certain embodiments, the antigen-binding domain can be obtained from IgG such as IgG1, IgG2, IgG3, or IgG4. In a specific embodiment, the antigen-binding domain is derived from the human IgG heavy chain.

[0358] In certain embodiments, the antigen-binding domain can be obtained from a heavy chain only antibody (HCAb).

[0359] Exemplary embodiments of the antigen-binding domain include, for example but not limited to, single domain antibodies (sdAb), 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.

[0360] In certain embodiments, the antigen-binding domain of the binder disclosed herein is VHH.

[0361] In certain embodiments, the antigen-binding domain of the binder disclosed herein is a VHH comprising the complementarity determining regions of the VHH disclosed herein.

[0362] In certain embodiments, the complementarity determining regions (CDR) of the VHH disclosed herein correspond to Kabat CDR.

[0363] In certain embodiments, the complementarity determining regions (CDR) of the VHH disclosed herein correspond to IMGT CDR.

[0364] In a specific embodiment, the binder of the present disclosure may comprise an antigen-binding domain VHH derived from a human, mouse or rat or from a transgenic mouse or rat in which the mouse or rat VHH has been camelized, a human VHH, a camelized human VHH, IgG1, IgG2a, IgG2b, IgG2c or IgG3 or a combination thereof. The antibody can be obtained by immunizing a mouse or rat or transgenic mouse or rat lacking any of its heavy chains, a functional CH1 domain of IgG1, IgG2a, IgG2b, IgG2c or IgG3 or a combination thereof, or a combination of the above VHHs, with an antigen of interest.

[0365] In a specific embodiment, the polypeptide chain of the present disclosure may comprise an antigen-binding domain of a camelid antibody such as IgG2 or IgG3 VHH. The camelid antibody can be obtained by immunizing a dromedary, bactrian camel, llama or alpaca with an antigen of interest.

[0366] In certain embodiments, the camelid antibody may be derived from so-called Old World camelids such as Camelus bactrianus, Camelus dromaderus or from New World camelids such as Lama pacos, Lama glama and Lama vicugna.

[0367] In another specific embodiment, the polypeptide chain of the present disclosure may comprise an antigen-binding domain of a chondrichthyan antibody such as IgNAR V NAR fragment. The V NAR fragment may be derived from a shark antibody.

[0368] If desired, the antigen-binding domain of a non-human antibody can be humanized. For example, the framework regions of a non-human VH, VHH or HCAb can 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 text of which is incorporated herein by reference. Humanized camelid antibodies can be obtained, for example, by grafting the CDRs onto a common humanized nanobody scaffold (e.g., h-NbBcII10 FGLA ) disclosed in Vincke C. et al. The V NAR antibody can be humanized by converting non-CDR residues to residues of the human germline Vκ1 sequence DPK9, as discussed in Kovalenko OV et al. (J Biol Chem. 2013, 288: 17408-17419), the entire text of which is incorporated herein by reference. Thus, the polypeptide chain of the present disclosure comprises a humanized antigen-binding domain.

[0369] In another specific embodiment, the antigen-binding domain can comprise a human VH (modified or unmodified). The human VH can be obtained, for example, from a synthetic human VH library. Modified human VH includes those in which some amino acid residues have been modified to make them more camel-like (i.e., by camelization).

[0370] Those skilled in the art will understand that the antigen-binding domain can be incorporated into an antibody, antigen-binding fragment, or antibody-like molecule, including but not limited to single-domain antibodies (sdAb), VHH, conventional antibodies or their antigen-binding fragments, bispecific antibodies, single-chain Fv-CH3 (scFv-CH3) fusions, tandem-scFv-CH3 (TaFv-CH3) fusions, diabody-CH3 (Db-CH3) fusions, tandem Db-CH3 (TaDb-CH3) fusions, single-chain Db-CH3 fusions (scDb-CH3), Fab-CH3 fusions, single-chain Fab-CH3 fusions, Fab-scFv-CH3 fusions, bispecific affinity retargeting (DART)-CH3 fusions, Fab-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, bispecific affinity retargeting (DART)-Fc fusions, Fab-DART-Fc fusions, etc. These antibody forms can have the native CH3, mutant CH3 domains, native CH2-CH3 domains, or mutant CH2-CH3 domains disclosed herein.

[0371] Exemplary embodiments of ABD1

[0372] The antigen-binding domain 1 (ABD1) can be selected, for example, from antigen-binding domains that bind CD36, some non-limiting exemplary embodiments of which are provided herein.

[0373] In certain cases, ABD1 is capable of binding human CD36 or a portion thereof or a cell expressing human CD36 or a portion thereof.

[0374] In some exemplary embodiments, the binder of the present disclosure can comprise 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 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.

[0375] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 4, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 5, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 6.

[0376] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 8, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 9, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 10.

[0377] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 11, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 12, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 13.

[0378] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0379] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0380] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0381] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0382] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0383] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0384] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0385] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0386] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0387] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0388] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[0389] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises 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.

[0390] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 71, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 72, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 73.

[0391] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 74, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 75, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 76.

[0392] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 78, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 79, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 80.

[0393] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 81, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 82, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 83.

[0394] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 85, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 86, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 87.

[0395] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 88, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 89, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 90.

[0396] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 92, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 93, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 94.

[0397] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 95, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 96, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 97.

[0398] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 99, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 100, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 101.

[0399] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 102, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 103, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 104.

[0400] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 106, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 107, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 108.

[0401] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 109, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 110, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 111.

[0402] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 113, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 114, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 15.

[0403] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 116, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 117, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 118.

[0404] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 120, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 121, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 122.

[0405] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 123, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 124, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 125.

[0406] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 127, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 128, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 129.

[0407] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 130, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 131, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 132.

[0408] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 134, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 135, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 136.

[0409] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 137, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 138, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 139.

[0410] In some embodiments, the binder of the present disclosure can have a combination of CDRH1, CDRH2, and CDRH3 of ABD1, wherein any one of CDRH1, CDRH2, or CDRH3 has one, two, three, four, or five amino acid substitutions. In some embodiments, the total number of amino acid substitutions in any one of CDRH1, CDRH2, or CDRH3 is ten amino acid substitutions or less (a total of nine, eight, seven, six, five, four, three, two, or one). In some embodiments, the total number of amino acid substitutions in CDRH3 is three or less (one, two, or three).

[0411] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 380.

[0412] QVQLQESGGGLX 2a QPGGSLRLSCAASGX 2b X 2c X 2d SX 2e YAMSWYRQX 2f PGKERELVA LITX 2g X 2h GGX 2i TDYADSVKGRFTIX 2j RDDAEKTVSLQMNSLKPEDTAVYYCSAWRAX 2k L X 2l X 2m PYWGQGTQVTVSS(SEQID NO:380)

[0413] wherein X 2a to X 2m is any (e.g., natural) amino acid residue, or wherein X 2a is A or V, wherein X 2b is L or F, wherein X 2c is A, T, or S, wherein X 2dis F or V, where X 2e is T or S, where X 2f is A or P, where X 2g is S or T, where X 2h is A or T, where X 2i is I or T, where X 2j is A or S, where X 2k is S or T, where X 2l is F, Y or L and / or where X 2m is E or K.

[0414] In some embodiments, the CDR1, CDR2, and / or CDR3 of SEQ ID NO: 380 can independently correspond to the CDR1, CDR2, and / or CDR3 of any one of SEQ ID NO: 7, 28, 98, or 119 and combinations thereof.

[0415] In some embodiments, the framework regions of SEQ ID NO: 380 can be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 380 can contain amino acid residues of human antibodies (e.g., human germline antibody sequences or their consensus sequences).

[0416] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 381.

[0417] QVQLQESGGGLVQPGX 3a SLRLSCX 3b ASGX 3c X 3d X 3e X 3f FTFSX 3g YAMSWYRQX 3h PGK ERELVAAX3 i X 3j X 3k X 3l GX 3m X 3n X 3o X 3p YX 3q DSVKGRFX 3r ISRDX 3s AKNTVYLX 3t MNSLKPE DTAX 3u YYCNAVTLQGWX 3vEYDYWGQGTQVTVSS (SEQ ID NO:381)

[0418] Wherein any one of X 3a to X 3v is any (e.g., natural) amino acid residue, or wherein X 3a is G or E, wherein X 3b is A or T, wherein X 3c is absent or is F, wherein X 3d is absent or is T, wherein X 3e is absent or is S, wherein X 3f is absent or is G, wherein X 3g is S or N, wherein X 3h is A or T, wherein X 3i is M or I, wherein X 3j is Q, P or T, wherein X 3k is S, H or N, wherein X 3l is T, A or D, wherein X 3m is D or G, wherein X 3n is S or T, wherein X 3o is T or I, wherein X 3p is Y or H, wherein X 3q is A or V, wherein X 3r is S or T, wherein X 3s is D or N, wherein X 3t is Q or L, wherein X 3u is V or A and / or wherein X 3v is S or R.

[0419] In some embodiments, X 3c , X 3d , X 3e and X 3f are absent.

[0420] In some embodiments, the CDR1, CDR2 and / or CDR3 of SEQ ID NO:381 can independently correspond to the CDR1, CDR2 and / or CDR3 of any one of SEQ ID NO:14, 21, 35 or 49 and combinations thereof.

[0421] In some embodiments, the framework region of SEQ ID NO:381 can be modified to include amino acid substitutions, additions and / or deletions. In some embodiments, the framework region of SEQ ID NO:381 can comprise the amino acid residues of a human antibody (e.g., a human germline antibody sequence or its consensus sequence).

[0422] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 382.

[0423] QVQLQESGGGLVQPGGSLRLSCAASGFRFSX 4a SIMX 4b WYRQAPX 4c KERELVASISSD SX 4d RTNYAESVKGRFTISRDNEQX 4e X 4f X 4g FLEMNX 4h LKPVDTAVYFCTRPX 4i IRGRDWG QGTQVTVSS(SEQ ID NO:382)

[0424] wherein X 4a to X 4i is any (e.g., natural) amino acid residue, or wherein X 4a is S or T, wherein X 4b is S or T, wherein X 4c is E or G, wherein X 4d is V or R, wherein X 4e is R or S, wherein X 4f is M or T, wherein X 4g is V or L, wherein X 4h is S or N, wherein X 4i is S or N.

[0425] In some embodiments, the CDR1, CDR2, and / or CDR3 of SEQ ID NO: 382 can independently correspond to the CDR1, CDR2, and / or CDR3 of any one of SEQ ID NO: 56 or 84 and combinations thereof.

[0426] In some embodiments, the framework region of SEQ ID NO: 382 can be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework region of SEQ ID NO: 382 can comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus sequence thereof).

[0427] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 383.

[0428] QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYPMGWFRQAPGKEREFVAAIDWSG DITYYADSVKGRFAISRDNAKNMVYLQMNSLKPEDTAVYYCNAGPLDSWERNDFPSW X 5a QGTQVTVSS(SEQ ID NO:383)

[0429] wherein X 5a is any (e.g., natural) amino acid residue, or wherein X 5a is G or D.

[0430] In some embodiments, the CDR1, CDR2, and / or CDR3 of SEQ ID NO: 383 may independently correspond to the CDR1, CDR2, and / or CDR3 of any one of SEQ ID NO: 105 or 112 and combinations thereof.

[0431] In some embodiments, the framework region of SEQ ID NO: 383 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework region of SEQ ID NO: 383 may comprise the amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus sequence thereof).

[0432] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7.

[0433] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 7.

[0434] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 7.

[0435] In some embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:7.

[0436] In some embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO:7.

[0437] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:7.

[0438] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0439] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:14.

[0440] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:14.

[0441] In some embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:14.

[0442] In some embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO:14.

[0443] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0444] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21.

[0445] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 21.

[0446] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 21.

[0447] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO: 21.

[0448] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO: 21.

[0449] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0450] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28.

[0451] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:28.

[0452] In certain embodiments, the amino acid variation can be located in one or more framework regions of SEQ ID NO:28.

[0453] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:28.

[0454] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO:28.

[0455] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:28.

[0456] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35.

[0457] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:35.

[0458] In certain embodiments, the amino acid variation can be located in one or more framework regions of SEQ ID NO:35.

[0459] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:35.

[0460] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO:35.

[0461] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:35.

[0462] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42.

[0463] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:42.

[0464] In certain embodiments, the amino acid variation can be located in one or more framework regions of SEQ ID NO:42.

[0465] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:42.

[0466] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO:42.

[0467] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:42.

[0468] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49.

[0469] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 49.

[0470] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 49.

[0471] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO: 49.

[0472] In certain embodiments, antigen-binding domain 1 (ABD1) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO: 49.

[0473] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 49.

[0474] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56.

[0475] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 56.

[0476] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 56.

[0477] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:56.

[0478] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56, and the CDRs are the same as the IMGT CDRs of SEQ ID NO:56.

[0479] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:56.

[0480] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:77.

[0481] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:77.

[0482] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO:77.

[0483] In certain embodiments, antigen-binding domain 1 (ABD1) 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:77, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:77.

[0484] In certain embodiments, antigen-binding domain 1 (ABD1) 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:77, and the CDRs are the same as the IMGT CDRs of SEQ ID NO:77.

[0485] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:77.

[0486] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:84.

[0487] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:84.

[0488] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO:84.

[0489] In certain embodiments, antigen-binding domain 1 (ABD1) 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:84, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:84.

[0490] In certain embodiments, antigen-binding domain 1 (ABD1) 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:84, and the CDRs are the same as the IMGT CDRs of SEQ ID NO:84.

[0491] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:84.

[0492] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:91.

[0493] In certain embodiments, antigen binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:91.

[0494] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO:91.

[0495] In certain embodiments, antigen binding domain 1 (ABD1) 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:91, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:91.

[0496] In certain embodiments, antigen binding domain 1 (ABD1) 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:91, and the CDRs are the same as the IMGT CDRs of SEQ ID NO:91.

[0497] In certain exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:91.

[0498] In certain exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen binding domains, wherein at least one of the antigen binding domains is antigen binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:98.

[0499] In certain embodiments, antigen binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:98.

[0500] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO:98.

[0501] In certain embodiments, antigen binding domain 1 (ABD1) 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:98, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:98.

[0502] In certain embodiments, antigen-binding domain 1 (ABD1) 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:98, and the CDRs are identical to the IMGT CDRs of SEQ ID NO:98.

[0503] In certain exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:98.

[0504] In certain exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:105.

[0505] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:105.

[0506] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO:105.

[0507] In certain embodiments, antigen-binding domain 1 (ABD1) 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:105, and the CDRs are identical to the Kabat CDRs of SEQ ID NO:105.

[0508] In certain embodiments, antigen-binding domain 1 (ABD1) 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:105, and the CDRs are identical to the IMGT CDRs of SEQ ID NO:105.

[0509] In certain exemplary embodiments, the binding agent of the present disclosure can include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:105.

[0510] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112.

[0511] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 112.

[0512] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 112.

[0513] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 112, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 112.

[0514] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 112, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 112.

[0515] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 112.

[0516] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119.

[0517] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 119.

[0518] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 119.

[0519] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 119, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 119.

[0520] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 119, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 119.

[0521] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 119.

[0522] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126.

[0523] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 126.

[0524] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO: 126.

[0525] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 126, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 126.

[0526] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 126, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 126.

[0527] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 126.

[0528] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133.

[0529] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 133.

[0530] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 133.

[0531] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 133, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 133.

[0532] In certain embodiments, antigen-binding domain 1 (ABD1) 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: 133, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 133.

[0533] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 133.

[0534] In certain exemplary embodiments, the binder of the present disclosure may include one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140.

[0535] In certain embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:140.

[0536] In certain embodiments, the amino acid variation(s) may be located in one or more framework regions of SEQ ID NO:140.

[0537] In certain embodiments, antigen-binding domain 1 (ABD1) 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:140, and the CDRs are the same as the Kabat CDRs of SEQ ID NO:140.

[0538] In certain embodiments, antigen-binding domain 1 (ABD1) 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:140, and the CDRs are the same as the IMGT CDRs of SEQ ID NO:140.

[0539] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:140.

[0540] Exemplary embodiments of ABD2

[0541] Antigen-binding domain 2 (ABD2) may be selected, for example, from antigen-binding domains that bind PD-1, some non-limiting exemplary embodiments of which are provided herein.

[0542] In certain cases, ABD2 is capable of binding to human PD-1 or a portion thereof or a cell expressing human PD-1 or a portion thereof.

[0543] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:57, heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:58, and heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:59.

[0544] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62.

[0545] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 275, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 276, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 277.

[0546] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 278, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 279, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 280.

[0547] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 282, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 283, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 284.

[0548] In certain exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 285, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 286, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 287.

[0549] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 289, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 290, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 291.

[0550] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 292, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 293, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 294.

[0551] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 296, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 297, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 298.

[0552] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 299, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 300, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 301.

[0553] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 303, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 304, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 305.

[0554] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 306, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 307, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 308.

[0555] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 310, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 311, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 312.

[0556] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 313, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 314, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 315.

[0557] In certain exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 317, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 318, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 319.

[0558] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 320, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 321, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 322.

[0559] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 324, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 325, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 326.

[0560] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 327, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 328, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 329.

[0561] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 331, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 332, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 333.

[0562] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 334, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 335, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 336.

[0563] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 338, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 339, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 340.

[0564] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 341, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 342, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 343.

[0565] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 345, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 346, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 347.

[0566] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 348, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 349, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 350.

[0567] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 352, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 353, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 354.

[0568] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises CDRH1 having the amino acid sequence set forth in SEQ ID NO: 355, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 356, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 357.

[0569] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 359, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 360, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 361.

[0570] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 362, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 363, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 364.

[0571] In some embodiments, the binder of the present disclosure can have a combination of CDRH1, CDRH2, and CDRH3 of ABD2, wherein there is one, two, three, four, or five amino acid substitutions in any one of CDRH1, CDRH2, or CDRH3. In some embodiments, the total number of amino acid substitutions in any one of CDRH1, CDRH2, or CDRH3 is ten amino acid substitutions or fewer (nine, eight, seven, six, five, four, three, two, or one in total). In some embodiments, the total number of amino acid substitutions in CDRH3 is three or fewer (one, two, or three).

[0572] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence set forth in SEQ ID NO: 379, wherein X 1a to X 1k is any (e.g., natural) amino acid residue, or wherein X 1a is L or E, wherein X 1b is A or P, wherein X 1c is D or G, wherein X 1d is N or S, wherein X 1e is S or T, wherein X 1f is K or R, wherein X 1g is D or G, wherein X 1h is K or Q, wherein X 1j is T or A, and / or wherein X 1k is L or V.

[0573] QVQLQESGGGX1a VQX 1b GX 1c SLRLSCAASGFTFSX 1d YGMSWVRQAPGEGLEWVSSIDSX 1e GGTTX 1f YAX 1g SVX 1h GRFTISRDNAKNX 1j X 1k YLQMNSLKPEDTAVYYCAKDFLSW MPRGQGTQVTVSS(SEQ ID NO:379)

[0574] In some embodiments, the CDR1, CDR2, and / or CDR3 of SEQ ID NO:379 can independently correspond to the CDR1, CDR2, and / or CDR3 of any one of SEQ ID NO:288, 295, 302, 309, 323, 330, 337, 344, 351, or 358 and combinations thereof.

[0575] In some embodiments, the framework region of SEQ ID NO:379 can be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework region of SEQ ID NO:379 can contain amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus sequence thereof).

[0576] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63.

[0577] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:63.

[0578] In some embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO:63.

[0579] In some embodiments, antigen-binding domain 2 (ABD2) 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 the CDRs are the same as the Kabat CDRs of SEQ ID NO:63.

[0580] In some embodiments, antigen-binding domain 2 (ABD2) 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 the CDRs are identical to the IMGT CDRs of SEQ ID NO: 63.

[0581] In some exemplary embodiments, the binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63.

[0582] In some exemplary embodiments, the binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 281.

[0583] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 281.

[0584] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 281.

[0585] In some embodiments, antigen-binding domain 2 (ABD2) 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: 281, and the CDRs are identical to the Kabat CDRs of SEQ ID NO: 281.

[0586] In some embodiments, antigen-binding domain 2 (ABD2) 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: 281, and the CDRs are identical to the IMGT CDRs of SEQ ID NO: 281.

[0587] In some exemplary embodiments, the binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 281.

[0588] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 288.

[0589] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 288.

[0590] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 288.

[0591] In some embodiments, antigen-binding domain 2 (ABD2) 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: 288, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 288.

[0592] In some embodiments, antigen-binding domain 2 (ABD2) 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: 288, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 288.

[0593] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence set forth in SEQ ID NO: 288.

[0594] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 295.

[0595] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 295.

[0596] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 295.

[0597] In some embodiments, antigen-binding domain 2 (ABD2) 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: 295, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 295.

[0598] In some embodiments, antigen-binding domain 2 (ABD2) 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: 295, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 295.

[0599] In some exemplary embodiments, a binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 295.

[0600] In some exemplary embodiments, a binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 302.

[0601] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 302.

[0602] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 302.

[0603] In some embodiments, antigen-binding domain 2 (ABD2) 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: 302, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 302.

[0604] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 302, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 302.

[0605] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 302.

[0606] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 309.

[0607] In certain embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 309.

[0608] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO: 309.

[0609] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 309, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 309.

[0610] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 309, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 309.

[0611] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 309.

[0612] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 316.

[0613] In certain embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 316.

[0614] In certain embodiments, the amino acid variation can be located in one or more framework regions of SEQ ID NO: 316.

[0615] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 316, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 316.

[0616] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 316, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 316.

[0617] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 316.

[0618] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 323.

[0619] In certain embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 323.

[0620] In certain embodiments, the amino acid variation can be located in one or more framework regions of SEQ ID NO: 323.

[0621] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 323, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 323.

[0622] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 323, and the CDRs are identical to the IMGT CDRs of SEQ ID NO: 323.

[0623] In some exemplary embodiments, the binding agents of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence set forth in SEQ ID NO: 323.

[0624] In some exemplary embodiments, the binding agents of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 330.

[0625] In certain embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 330.

[0626] In certain embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO: 330.

[0627] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 330, and the CDRs are identical to the Kabat CDRs of SEQ ID NO: 330.

[0628] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 330, and the CDRs are identical to the IMGT CDRs of SEQ ID NO: 330.

[0629] In some exemplary embodiments, the binding agents of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence set forth in SEQ ID NO: 330.

[0630] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 337.

[0631] In certain embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 337.

[0632] In certain embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 337.

[0633] In certain embodiments, antigen-binding domain 2 (ABD2) 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: 337 and has CDRs identical to the Kabat CDRs of SEQ ID NO: 337.

[0634] In some embodiments, antigen-binding domain 2 (ABD2) 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: 337, and has CDRs identical to the IMGT CDRs of SEQ ID NO: 337.

[0635] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises the amino acid sequence set forth in SEQ ID NO: 337.

[0636] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 344.

[0637] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 344.

[0638] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 344.

[0639] In some embodiments, antigen-binding domain 2 (ABD2) 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: 344, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 344.

[0640] In some embodiments, antigen-binding domain 2 (ABD2) 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: 344, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 344.

[0641] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 344.

[0642] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 351.

[0643] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 351.

[0644] In some embodiments, the amino acid variations may be located in one or more framework regions of SEQ ID NO: 351.

[0645] In some embodiments, antigen-binding domain 2 (ABD2) 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: 351, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 351.

[0646] In some embodiments, antigen-binding domain 2 (ABD2) 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: 351, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 351.

[0647] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 351.

[0648] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 358.

[0649] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 358.

[0650] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 358.

[0651] In some embodiments, antigen-binding domain 2 (ABD2) 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: 358, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 358.

[0652] In some embodiments, antigen-binding domain 2 (ABD2) 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: 358, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 358.

[0653] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 358.

[0654] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 365.

[0655] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 365.

[0656] In some embodiments, the amino acid variations can be located in one or more framework regions of SEQ ID NO: 365.

[0657] In some embodiments, antigen-binding domain 2 (ABD2) 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: 365, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 365.

[0658] In some embodiments, antigen-binding domain 2 (ABD2) 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 the CDRs are the same as the IMGT CDRs of SEQ ID NO: 365.

[0659] In some exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 365.

[0660] Exemplary embodiments of ABD3

[0661] Antigen-binding domain 3 (ABD3) can be selected, for example, from antigen-binding domains that bind CD47, some non-limiting exemplary embodiments of which are provided herein.

[0662] In certain cases, ABD3 is capable of binding human CD47 or a portion thereof or a cell expressing human CD47 or a portion thereof.

[0663] In some exemplary embodiments, the binding agent of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66.

[0664] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises a CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69.

[0665] In some embodiments, the binder of the present disclosure may have a combination of CDRH1, CDRH2, and CDRH3 of ABD3, wherein there is one, two, three, four, or five amino acid substitutions in any one of CDRH1, CDRH2, or CDRH3. In some embodiments, the total number of amino acid substitutions in any one of CDRH1, CDRH2, or CDRH3 is ten amino acid substitutions or less (a total of nine, eight, seven, six, five, four, three, two, or one). In some embodiments, the total number of amino acid substitutions in CDRH3 is three or less (one, two, or three).

[0666] In some exemplary embodiments, the binder of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), and comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[0667] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence having from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 70.

[0668] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO: 70.

[0669] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70, and the CDRs are the same as the Kabat CDRs of SEQ ID NO: 70.

[0670] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70, and the CDRs are the same as the IMGT CDRs of SEQ ID NO: 70.

[0671] In some exemplary embodiments, the binder of the present disclosure can comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[0672] Exemplary embodiments of other antigen-binding domains

[0673] In some embodiments, the binder comprises one or more antigen-binding domains, 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), and wherein at least one antigen-binding domain binds to a different antigen.

[0674] In some embodiments, the binder can thus comprise an antigen-binding domain that binds CD3. Such antigen-binding domains include those known to those of skill in the art. An exemplary embodiment of an antigen-binding domain that binds CD3 is provided in SEQ ID NO: 197.

[0675] Thus, in some embodiments, the binder can comprise an antigen-binding domain that binds CD36, PD-1, and / or CD3. In other embodiments, the binder can comprise an antigen-binding domain that binds CD36 and / or CD3. In still other embodiments, the binder can comprise an antigen-binding domain that binds PD-1 and / or CD3.

[0676] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and wherein at least one of the antigen-binding domains is selected from antigen-binding domains that specifically bind to CD3, CD36, DRD1, DRD2, DRD3, DRD4, DRD5, TROP2, CD147, MCT1, IL1RAP, AMIGO2, PTK7, MCT2, MCT4, NHE1, H+ / K+-ATPase, LAP, HLA-I A2, CD73, CD98, CEACAM5 / 6, ICAM-1, MCSP, fibronectin, β1 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-H3, 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.

[0677] Polypeptide chain

[0678] The binder of the present disclosure comprises one or more polypeptide chains.

[0679] The DNA segment encoding the required polypeptide chain sequence can be synthesized in vitro. Different DNA modules are assembled into a single fragment in an orderly and oriented manner and then cloned into an expression vector. Thus, the resulting polypeptide chain consists of different modules that form a single chain.

[0680] The polypeptide chains of the present disclosure include, for example but not limited to, antigen-binding domains, linkers, and dimerization domains that facilitate the assembly of at least two polypeptide chains.

[0681] In some embodiments, the polypeptide chains of the present disclosure can be monospecific.

[0682] In some embodiments, the polypeptide chains of the present disclosure can be multispecific.

[0683] In some embodiments, the polypeptide chains of the present disclosure can be monovalent.

[0684] In some embodiments, the polypeptide chains of the present disclosure can be multivalent.

[0685] In certain cases, the polypeptide chain does not contain a dimerization domain.

[0686] In certain cases, the polypeptide chain contains a dimerization domain.

[0687] In some aspects of the present disclosure, the polypeptide chain can be monospecific.

[0688] An exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising one antigen-binding domain. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising multiple antigen-binding domains, but these antigen-binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising multiple antigen-binding domains, but these antigen-binding domains have the same CDRs and different framework regions. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising antigen-binding domains in which there are differences (e.g., conservative substitutions in one or more of the CDRs) in their amino acid sequences but which do not affect their ability to bind the same antigen or epitope.

[0689] In some aspects of the present disclosure, the polypeptide chain can be multispecific. These polypeptide chains can include, for example, bispecific polypeptide chains, trispecific polypeptide chains, tetraspecific polypeptide chains, pentaspecific polypeptide chains, hexaspecific polypeptide chains, bispecificity polypeptide chains, multispecificity polypeptide chains, etc.

[0690] In an exemplary configuration, one or more antigen-binding domains can be located at the N-terminus, C-terminus, or on both sides of the dimerization domain.

[0691] In another exemplary configuration, the polypeptide chain can include at least one antigen-binding domain at the N-terminus of the dimerization domain and at least one antigen-binding domain at the C-terminus of the dimerization domain.

[0692] In a further exemplary configuration, the polypeptide chain can include one antigen-binding domain at the N-terminus of the dimerization domain and at least two antigen-binding domains at the C-terminus of the dimerization domain.

[0693] In another further exemplary configuration, the polypeptide chain may comprise two antigen-binding domains at the N-terminus of the dimerization domain and two antigen-binding domains at the C-terminus of the dimerization domain.

[0694] The polypeptide chain may sequentially comprise the amino acid sequence of formula I in the form from the N-terminus to the C-terminus:

[0695] X - [(Ab a ) - (L b )] m - (DD) - [(L c ) - (Ab d )] n - Y

[0696] wherein m is 0, 1, 2 or an integer greater than 2;

[0697] wherein n is 0, 1, 2 or an integer greater than 2;

[0698] wherein m and n are not both 0;

[0699] wherein 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);

[0700] wherein X or Y independently exists or is absent and comprises an amino acid sequence;

[0701] wherein L b , L c each independently comprise one or more linkers; and

[0702] wherein DD represents a dimerization domain.

[0703] In some embodiments, L b may be absent.

[0704] In some embodiments, L c may be absent.

[0705] In some embodiments, both L b and L c may be absent.

[0706] In some embodiments, the polypeptide chain comprises multiple antigen-binding domains.

[0707] In some embodiments, when m is 2 or an integer greater than 2, the [(Ab a ) - (L b )] units are the same.

[0708] In other embodiments, when m is an integer of 2 or greater than 2, the [(Ab a )-(L b )] units of the polypeptide chain or binder are different.

[0709] In other embodiments, when m is an integer greater than 2, the [(Ab a )-(L b )] units of the polypeptide chain or binder can include identical and different units.

[0710] In some embodiments, when n is an integer of 2 or greater than 2, the [(L c )-(Ab d )] units are identical.

[0711] In other embodiments, when n is an integer of 2 or greater than 2, the [(L c )-(Ab d )] units are different.

[0712] In other embodiments, when n is an integer of 2 or greater than 2, the [(L c )-(Ab d )] units include identical and different units.

[0713] In some embodiments, when m is an integer of 2 or greater than 2, each Ab a is identical or different.

[0714] In some embodiments, when n is an integer of 2 or greater than 2, each Ab d is identical or different.

[0715] In some embodiments, Ab a represents ABD1.

[0716] In some embodiments, Ab d represents ABD2.

[0717] In some embodiments, Ab d represents ABD3.

[0718] In some embodiments, m is 1 and Ab a represents ABD1.

[0719] In some embodiments, n is 2 and one of the Ab d represents ABD2.

[0720] In some embodiments, n is 2 and one of the Ab d represents ABD3.

[0721] In some embodiments, n is 2 and where Ab d one of which represents ABD2 and the other Ab d represents ABD3.

[0722] In some embodiments, m is an integer of 2, 3, 4, 5 or greater than 5.

[0723] In some embodiments, m is 2.

[0724] In other embodiments, m is 3.

[0725] In some other embodiments, m is 4.

[0726] In further embodiments, m is 5.

[0727] In other embodiments, m is an integer greater than 5.

[0728] In some embodiments, n is an integer of 2, 3, 4, 5 or greater than 5.

[0729] In some embodiments, n is 2.

[0730] In other embodiments, n is 3.

[0731] In additional embodiments, n is 4.

[0732] In further embodiments, n is 5.

[0733] In other embodiments, n is an integer greater than 5.

[0734] In embodiments, one or more polypeptide chains further comprise the hinge region of an antibody or an antigen-binding fragment thereof.

[0735] In some embodiments, the hinge region is located at the N-terminus of the dimerization domain.

[0736] In some embodiments, L b is the hinge region of an antibody or an antigen-binding fragment thereof.

[0737] In some embodiments, the hinge region is a native human IgG1 hinge region. In other embodiments, the hinge region is a mutant human IgG1 hinge region.

[0738] In other embodiments, the hinge region is a native human IgG2 hinge region. In other embodiments, the hinge region is a mutant human IgG2 hinge region.

[0739] In other embodiments, the hinge region is a native human IgG3 hinge region. In other embodiments, the hinge region is a mutant human IgG3 hinge region.

[0740] In some embodiments, the hinge region is a native human IgG4 hinge region. In other embodiments, the hinge region is a mutant human IgG4 hinge region.

[0741] In some embodiments, one or more linkers each independently have a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0742] In some embodiments, one or more linkers each independently are a flexible linker, a helical linker, or a rigid linker.

[0743] In some embodiments, linker L c is a rigid linker.

[0744] In some embodiments, L c comprises a non-cleavable linker. In other embodiments, L c consists of a non-cleavable linker.

[0745] In some embodiments, one or more linkers comprise a flexible linker.

[0746] In some embodiments, one or more linkers comprise a rigid linker.

[0747] In some embodiments, the flexible linker is a GS linker.

[0748] In some embodiments, the flexible linker comprises one or more GGGGS units.

[0749] In some embodiments, the flexible linker comprises at least 2, 3, 4, 5, or more GGGGS units.

[0750] In some embodiments, the rigid linker comprises multiple PA repeats.

[0751] In some embodiments, the rigid linker is selected from PAPAPKA (SEQ ID NO:187), APAPAPAPAPKA (SEQ ID NO:188), APAPAPAPAPAPAPAPAPAPKA (SEQ ID NO:189), or a combination thereof.

[0752] In some embodiments, the helical linker comprises one or more EAAAK units.

[0753] In some embodiments, the helical joint is selected from AEAAAKEAAAKA (SEQ ID NO:191), AEAAAKEAAAKEAAAKA (SEQ ID NO:192), AEAAAKEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO:193), or a combination thereof.

[0754] In some embodiments, the dimerization domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:198.

[0755] In some embodiments, the dimerization domain further comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:199.

[0756] In other exemplary embodiments, the polypeptide chain comprises Formula II:

[0757] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0758] In additional exemplary embodiments, the polypeptide chain comprises Formula III:

[0759] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0760] In further exemplary embodiments, the polypeptide chain comprises Formula IV:

[0761] X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0762] In additional exemplary embodiments, the polypeptide chain comprises Formula V:

[0763] X-(Ab a1 )-(L b2 )-(Ab a2)-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0764] In a further exemplary embodiment, the polypeptide chain comprises Formula VI:

[0765] 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).

[0766] In a further exemplary embodiment, the polypeptide chain comprises Formula VII:

[0767] 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).

[0768] In other exemplary embodiments, the polypeptide chain comprises Formula VIII:

[0769] 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).

[0770] In some embodiments, Ab a1 , Ab a2 , Aba3 、Ab d1 、Ab d2 、Ab d3 Each independently comprises an antigen-binding domain.

[0771] In some embodiments, Ab a1 、Ab a2 、Ab a3 、Ab d1 、Ab d2 、Ab d3 Each independently represents an antigen-binding domain.

[0772] In some embodiments, at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1).

[0773] In some embodiments, the polypeptide chain further comprises antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3).

[0774] In some embodiments, the polypeptide chain further comprises at least one antigen-binding domain 2 (ABD2) and at least one antigen-binding domain 3 (ABD3).

[0775] In some embodiments, L b1 comprises one or more linkers and / or the hinge region of an antibody or an antigen-binding fragment thereof. In some embodiments, L b1 is L b .

[0776] In some embodiments, L b2 、L b3 、L c1 、L c2 and L c3 Each independently comprises one or more linkers.

[0777] In some embodiments, one or more of L b1 、L b2 and / or L b3 may be absent.

[0778] In some embodiments, L b2 and / or L b3 may be absent.

[0779] In some embodiments, one or more of L c1 、L c2 and / or L c3 may be absent.

[0780] In some embodiments, L c2 and / or L c3 may be absent.

[0781] In some embodiments, L c1 is a rigid joint.

[0782] In some embodiments, L c2 is a rigid joint.

[0783] In some embodiments, L c3 is a rigid joint.

[0784] In some embodiments, L c1 and L c2 are rigid joints.

[0785] In some embodiments, L c1 、L c2 and L c3 are rigid joints.

[0786] In some embodiments, L c1 、L c2 and / or L c3 may be absent.

[0787] In some embodiments, L c2 and / or L c3 may be absent.

[0788] In some embodiments, L c1 and / or L c3 may be absent.

[0789] In some embodiments, L c2 and / or L c3 may be absent.

[0790] In some embodiments, L c1 、L c2 and L c3 may be absent.

[0791] The polypeptide chain of the present disclosure comprises an antigen-binding domain that is functionally active as a single chain or when part of a binder as disclosed herein.

[0792] For example, the antigen-binding domain of the polypeptide chain can bind its target and can have biological activity.

[0793] In some embodiments, the biological activity of the antigen-binding domain includes, for example but not limited to, blocking the binding of the target to its natural receptor or ligand. Alternatively, the biological activity of the antigen-binding domain includes its ability to sequester the target. Additionally, the biological activity of the antigen-binding domain includes its ability to induce signal transduction.

[0794] A polypeptide chain comprising multiple antigen-binding domains is characterized as multivalent.

[0795] The polypeptide chains of the present disclosure may comprise additional amino acid sequences at their N-terminus or C-terminus or both (defined by X and Y in the formulas disclosed herein).

[0796] 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: 215.

[0797] In some embodiments, the amino acid sequence at the N-terminus (defined by X) or C-terminus (defined by Y) may independently include a linker, a cytokine, a chemokine, a tag (e.g., His tag (e.g., SEQ ID NO: 216)), a masking domain, a phage coat protein, an antigen-binding domain, or a combination thereof.

[0798] An exemplary embodiment of a multispecific polypeptide chain comprises a polypeptide chain comprising at least two antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs, resulting in different binding specificities.

[0799] When a polypeptide chain binds two different epitopes or antigens, it can be more specifically characterized as bispecific. When a polypeptide chain binds three different epitopes or antigens, it can be characterized as trispecific. When a polypeptide chain binds four different epitopes or antigens, it can be characterized as tetra-specific. When a polypeptide chain binds five different epitopes or antigens, it can be characterized as penta-specific. When a polypeptide chain binds six different epitopes or antigens, it can be characterized as hexa-specific.

[0800] A polypeptide chain comprising antigen-binding domains that bind non-overlapping epitopes on the same target can be characterized as bispecific. A polypeptide chain comprising antigen-binding domains that bind three, four, or more epitopes on the same target can be characterized as multispecific.

[0801] The antigen-binding domains of a given polypeptide chain will be selected according to the intended use (e.g., detection, diagnostic, and / or therapeutic use). Each antigen-binding domain of a specific polypeptide chain can be selected to produce an additive or synergistic effect.

[0802] For example, the polypeptide chains of the present disclosure may comprise at least one antigen-binding domain that specifically binds an antigen expressed by a tumor cell or by the tumor cell environment (i.e., a tumor-specific antigen-binding domain).

[0803] In other aspects and embodiments of the present disclosure, the polypeptide chain may comprise at least one antigen-binding domain that specifically binds an immunomodulator.

[0804] For example, the polypeptide chain can comprise one or more antigen-binding domains (e.g., immunospecific antigen-binding domains) that bind to immune checkpoint proteins, cytokines, chemokines, or immune receptors or co-receptors, etc.

[0805] In some embodiments, the polypeptide chain comprises, in an N-terminal to C-terminal fashion, an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD-1, and an antigen-binding domain that binds CD47.

[0806] In some exemplary embodiments, the antigen-binding domain can bind CD36.

[0807] In some exemplary embodiments, the antigen-binding domain can bind PD-1.

[0808] In some exemplary embodiments, the antigen-binding domain can bind CD47.

[0809] In one exemplary embodiment, the polypeptide chain of the present disclosure can comprise at least one tumor-specific antigen-binding domain and at least one immunospecific antigen-binding domain.

[0810] In some embodiments, the tumor-specific antigen-binding domain can be located at the N-terminus of the dimerization domain.

[0811] In some embodiments, the tumor-specific antigen-binding domain can be located at the C-terminus of the dimerization domain.

[0812] In some embodiments, the tumor-specific antigen-binding domain can be located at the N-terminus and C-terminus of the dimerization domain.

[0813] In some embodiments, the more immunospecific antigen-binding domain can be located at the N-terminus of the dimerization domain.

[0814] In some embodiments, the immunospecific antigen-binding domain can be located at the C-terminus of the dimerization domain.

[0815] In some embodiments, the immunospecific antigen-binding domain can be located at the N-terminus and C-terminus of the dimerization domain.

[0816] In exemplary and non-limiting embodiments, the polypeptide chain or binder can comprise two immunospecific antigen-binding domains at the C-terminus of the dimerization domain. In some embodiments, the immunospecific antigen-binding domain adjacent to the C-terminal portion of the dimerization domain can be linked by a non-cleavable linker.

[0817] Dimerization domain (DD)

[0818] In some embodiments, the polypeptide chains of the present disclosure include a dimerization domain. Accordingly, two polypeptide chains can assemble to form a binder. Exemplary embodiments of the binder include homodimers and heterodimers.

[0819] The dimerization domain can include, for example but not limited to, the constant regions of immunoglobulins, including, for example, the Fc, CH2, and / or CH3 domains of the heavy chain immunoglobulin. The constant regions of the immunoglobulin heavy chain are specifically contemplated.

[0820] In certain embodiments and aspects of the present disclosure, the dimerization domain can have a sequence identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or its corresponding CH2 and / or CH3 domains.

[0821] The present disclosure specifically includes a dimerization domain having a sequence identical to the sequence of a native human antibody. Exemplary embodiments of the dimerization domain include, for example, the CH2-CH3 domains of a native human heavy chain.

[0822] Accordingly, in some embodiments, the dimerization domain includes a native constant region of an antibody, e.g., 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.

[0823] Accordingly, in some embodiments, the dimerization domain includes a native CH3 domain.

[0824] In an exemplary embodiment, the dimerization domain includes a native human CH3 domain.

[0825] In some embodiments, the dimerization domain includes a native CH2 domain and a native CH3 domain.

[0826] 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: 198).

[0827] 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.

[0828] 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.

[0829] In some embodiments, the native CH3 domain is the native IgG4 CH3 domain. In some embodiments, the native CH3 domain is the native human IgG4 CH3 domain.

[0830] When the two polypeptide chains of a binder consist of the same amino acid sequence, the binder will form a homodimer. However, co-expression of polypeptide chains having native antibody CH2-CH3 domains but different amino acid sequences may result in a mixture of homodimers and heterodimers. The different binders in the mixture can be separated by methods known in the art and including, for example, size exclusion chromatography.

[0831] Thus, exemplary heterodimers of the present disclosure include those having CH3 domains or CH2-CH3 domains of native antibodies and formed by two polypeptide chains having different sequences or configurations.

[0832] In some embodiments, the polypeptide chain may have a mutant dimerization domain, e.g., containing 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3 amino acid substitutions compared to the native or wild-type sequence.

[0833] In an exemplary embodiment, the mutant dimerization domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. The amino acid substitutions may be conservative or non-conservative substitutions as described in Table 2.

[0834] In an exemplary embodiment, the polypeptide chain may have a mutant dimerization domain having a sequence that is 80% to 99% identical to the sequence of the native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domains. Polypeptide chains included in the present disclosure include those having mutant dimerization domains that are 85% to 99% identical, 90% to 99% identical, 95% to 99% identical to the sequence of the native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domains.

[0835] In certain embodiments, the polypeptide chains of the present disclosure may contain a mutant dimerization domain that contains amino acid substitutions that favor heterodimer formation. Thus, the heterodimers of the present disclosure can be formed by polypeptide chains containing such mutations.

[0836] Thus, in certain embodiments, the dimerization domain comprises a mutant constant region of an antibody, such as a mutant human IgG1 constant region, a mutant human IgG2 constant region, a mutant human IgG3 constant region, or a mutant human IgG4 constant region. The mutant constant region may comprise one or more amino acid substitutions, amino acid insertions, or amino acid deletions compared to the native constant region.

[0837] Thus, in certain embodiments, the dimerization domain comprises a mutant CH3 domain. The mutant CH3 domain may comprise one or more amino acid substitutions, amino acid insertions, or amino acid deletions compared to the native CH3 domain.

[0838] In certain embodiments, the dimerization domain comprises a native CH2 and a mutant CH3 domain.

[0839] In certain embodiments, the dimerization domain comprises a mutant CH2 and a mutant CH3 domain. The mutant CH2 domain may comprise one or more amino acid substitutions, amino acid insertions, or amino acid deletions compared to the native CH2 domain.

[0840] In certain embodiments, the mutant CH3 domain is a mutant IgG1 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG1 CH3 domain.

[0841] In certain embodiments, the mutant CH3 domain is a mutant IgG2 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG2 CH3 domain.

[0842] In certain embodiments, the mutant CH3 domain is a mutant IgG3 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG3 CH3 domain.

[0843] In certain embodiments, the mutant CH3 domain is a mutant IgG4 CH3 domain. In other embodiments, the mutant CH3 domain is a mutant human IgG4 CH3 domain.

[0844] In certain embodiments, the mutant CH2 domain is a mutant IgG1 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG1 CH2 domain.

[0845] In certain embodiments, the mutant CH2 domain is a mutant IgG2 CH2 domain. In other embodiments, the mutant CH2 domain is a mutant human IgG2 CH2 domain.

[0846] In certain 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.

[0847] In certain 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.

[0848] In certain embodiments, the Fc region can be modified to prevent glycosylation, extend its half-life, modulate receptor binding, or effector function. Exemplary mutations are discussed in Saunders K.O. (Front. Immunol. 10:1296, 2019, the full text of which is incorporated herein by reference) and include, for example, mutations at asparagine 297 (e.g., N297).

[0849] In certain embodiments, the dimerization domain comprises the Fc region of the antibody or a portion thereof.

[0850] In certain embodiments, the dimerization domain comprises the Fc region of the antibody or a portion thereof with or without an Fc modification.

[0851] In other embodiments, the dimerization domain comprises an altered or mutated Fc region or a portion thereof. For example, the Fc region or a portion thereof can be altered or mutated to modify one or more properties of the binder. In an exemplary embodiment, the Fc region or a portion thereof is non-glycosylated. In other exemplary embodiments, the Fc region or a portion thereof comprises one or more Fc modifications in Table 1.

[0852] In other exemplary embodiments, the Fc region or a portion thereof is altered or mutated to increase ADCC activity. For example, defucosylation (e.g., N297 according to the EU numbering system) can result in increased binding of FcRgII on NK cells and can significantly increase ADCC. Thus, in certain embodiments, the Fc region or a portion thereof can have a reduced number of fucose residues. In other embodiments, the binder comprises a defucosylated Fc region or a portion thereof. In still other embodiments, the Fc region or a portion thereof of the binder lacks fucose residues. In additional embodiments, the Fc region or a portion thereof of the binder lacks core fucose residues. In further embodiments, the Fc region or a portion thereof of the binder completely lacks core fucose residues.

[0853] The present disclosure encompasses exemplary and non-limiting embodiments of mutations in the constant region (e.g., Fc region) that improve one or more effector functions. Exemplary embodiments are provided in Table 1 (List of Mutations Antibodies (Basel). 2020 Nov 17;9(4):64, which is incorporated herein by reference in its entirety).

[0854] Table 1

[0855]

[0856]

[0857] According to the present disclosure, the mutated CH3 domain contains one or more mutations compared to the native CH3 domain. Thus, in certain embodiments, the dimerization domain contains a mutated CH3 domain that contains one or more mutations compared to the native CH3 domain.

[0858] In certain embodiments, the dimerization domain contains a native CH2 domain and a mutated CH3 domain. Exemplary embodiments of the mutated CH3 domain are provided in PCT / CA2020 / 051753, which was filed on Dec 18, 2020 and published on Jun 24, 2021 with publication number WO2021 / 119832A1, which is incorporated herein by reference in its entirety. For example, a heterodimer can be formed by co-transfecting a nucleic acid sequence encoding a polypeptide chain containing the mutated CH3 domain described in SEQ ID NO:218 (chain A) and a nucleic acid sequence encoding a polypeptide chain containing the mutated CH3 domain described in SEQ ID NO:219 (chain B).

[0859] Linker (L)

[0860] The different modules of the polypeptide chains disclosed herein can be associated with each other through linkers.

[0861] In certain embodiments, the linker for connecting one or more modules of the polypeptide chain is not a cleavable linker.

[0862] In one exemplary embodiment, the linker (Lc) adjacent to the C-terminus of the dimerization domain does not contain a cleavable linker.

[0863] In another exemplary embodiment, at least one of the linkers between the two antigen-binding domains does not contain a cleavable linker.

[0864] In other embodiments, the linker for connecting one or more modules of the polypeptide chain can include a non-cleavable linker.

[0865] In one exemplary embodiment, the linker immediately adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.

[0866] In another exemplary embodiment, at least one of the linkers between the two antigen-binding domains is a non-cleavable linker.

[0867] In a further exemplary embodiment, the linker immediately adjacent to the C-terminus of the dimerization domain and the linker connecting the first two antigen-binding domains located at the C-terminus of the dimerization domain are non-cleavable linkers.

[0868] In certain embodiments, the linker immediately adjacent to the N-terminus of the dimerization domain may preferably comprise the hinge region of an antibody.

[0869] In certain embodiments, the hinge region is a native hinge region.

[0870] In certain embodiments, the native hinge region is a native IgG1 hinge region. In certain embodiments, the native hinge region is a native human IgG1 hinge region.

[0871] In certain 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.

[0872] In certain 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.

[0873] In certain embodiments, the native hinge region is a native IgG4 hinge region. In certain embodiments, the native hinge region is a native human IgG4 hinge region.

[0874] In certain embodiments, the hinge region is a mutant hinge region.

[0875] In certain embodiments, the mutant hinge region is a mutant IgG1 hinge region. In certain embodiments, the mutant hinge region is a mutant human IgG1 hinge region.

[0876] In certain embodiments, the mutant hinge region is a mutant IgG2 hinge region. In other embodiments, the mutant hinge region is a mutant human IgG2 hinge region.

[0877] In certain embodiments, the mutant hinge region is a mutant IgG3 hinge region. In other embodiments, the mutant hinge region is a mutant human IgG3 hinge region.

[0878] In certain embodiments, the mutant hinge region is a mutant IgG4 hinge region. In certain embodiments, the mutant hinge region is a mutant human IgG4 hinge region.

[0879] In certain embodiments, all modules of the polypeptide chain are linked by non-cleavable linkers.

[0880] Exemplary embodiments of non-cleavable linkers include those that remain substantially intact during protein expression or manufacturing processes. As used herein, "remain substantially intact" means that linker cleavage occurs at 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.

[0881] Other exemplary embodiments of non-cleavable linkers also include linkers that do not contain one or more protease-specific cleavage sites present in human or animal blood or serum.

[0882] Additional exemplary embodiments of non-cleavable linkers also include linkers that maintain their integrity for at least one hour, two hours, three hours, four hours, five hours, six hours, twelve hours, twenty-four hours, forty-eight hours, or longer after administration of the binder to an individual.

[0883] In further exemplary embodiments, the linker contains both a non-cleavable linker and a cleavable linker.

[0884] In certain embodiments, the linker is non-cleavable.

[0885] In some cases, cleavable linkers can be used to release drugs in vivo (such as molecules that inhibit cell growth, cytotoxic molecules, chemotherapeutic drugs, etc.) or tags attached to the polypeptide chains of the present disclosure.

[0886] Exemplary embodiments of cleavable linkers are provided, for example, in US2019 / 0010242, including linkers sensitive to cleavage by proteases (generally extracellular proteases such as those produced by tumors or activated immune effector cells), and including those having specific cleavage sites for proteases selected from ADAMS, ADAMTS (such as 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 proteases (such as Cruzipain; Legumain; Otubain-2), KLK (such as KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 or KLK14), metalloproteases (such as transmembrane peptidase; Neprilysin; PSMA; BMP-1), MMP (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 (such as FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidobenzoate esterase, HtrA1, human neutrophil elastase, lactoferrin, Marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, trypsin, uPA; type II transmembrane serine proteases (TTSP) (such as DESC1, DPP-4, FAP, Hepsin, Matriptase-2, Matriptase, TMPRSS2, TMPRSS3 or TMPRSS4), and any combination thereof. In certain embodiments, the polypeptide chain of the present disclosure does not include such a linker at the position corresponding to Lc.

[0887] 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 content of which is incorporated herein by reference.

[0888] In certain embodiments, the hinge region or a portion thereof can be used to connect the module to the dimerization domain and is considered a linker herein. The hinge region can be derived from a natural antibody (from human or animal sources) or from a synthetic antibody. The hinge region can be obtained, for example, from IgG such as IgG1, IgG2, IgG3, or IgG4. Exemplary embodiments of the hinge region are provided in SEQ ID NO:180, SEQ ID NO:203, SEQ ID NO:207, and SEQ ID NO:211.

[0889] In some cases, the hinge region can have one or more amino acid substitutions, amino acid insertions, and / or amino acid deletions compared to the native hinge region. Mutant hinge regions include, for example, sequences that are 80% to 99% identical to the sequence of the native IgG1, IgG2, IgG3, or IgG4 hinge region (mutant hinge regions). An exemplary and non-limiting embodiment of a mutant hinge region includes the hinge region of IgG4 in which S228 is replaced by P (EU numbering) (Angal, S. et al., Mol Immunol 30, 105-108, 1993). Other exemplary embodiments of mutant hinge regions are provided in SEQ ID NO:118-120, 122-124, 126-128, and 130-132.

[0890] Flexible linkers are typically composed of small polar amino acids such as threonine or serine and glycine. Exemplary and non-limiting embodiments of flexible linkers include GS linkers (glycine / serine repeats), for example, (GGGS) n (GGGGS) m (SEQ ID NO:271), (GS) n , (GGS) n , (GGGS) n (SEQ ID NO:272), (GGGGS) n (SEQ ID NO:186), (GGSG) n (SEQ ID NO:273), (GGGSS) n (SEQ ID NO:274), where n and m can independently be integers, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, such as 15, 20, or 25.

[0891] It should be understood that SEQ ID NO:186 can be represented by the formula (GGGGS) n , where n is an integer selected from 1 to 10, or alternatively represented by the formula GGGGSX1, where X1 is absent or, if present, is 1 to 9 repetitions of amino acid residues 1 to 5 of SEQ ID NO:186.

[0892] It should be understood that SEQ ID NO:271 can be represented by the formula (GGGS) n (GGGGS) m , where n and / or m are independently integers selected from 1 to 10, or alternatively represented by the formula GGGSX1GGGGSX2, where X1 is absent or, if present, is 1 to 9 repetitions of amino acid residues 1 to 4 of SEQ ID NO:271, and where X2 is absent or, if present, is 1 to 9 repetitions of amino acid residues 6 to 10 of SEQ ID NO:271.

[0893] It should be understood that SEQ ID NO:272 can be represented by the formula (GGGS) n , where n is an integer selected from 1 to 10, or alternatively represented by the formula GGGSX1, where X1 is absent or, if present, is 1 to 9 repetitions of amino acid residues 1 to 4 of SEQ ID NO:272.

[0894] It should be understood that SEQ ID NO:273 can be represented by the formula (GGSG) n , where n is an integer selected from 1 to 10, or alternatively represented by the formula GGSGX1, where X1 is absent or, if present, is 1 to 9 repetitions of amino acid residues 1 to 4 of SEQ ID NO:273.

[0895] It should be understood that SEQ ID NO:274 can be represented by the formula (GGGSS) n , where n is an integer selected from 1 to 10, or alternatively represented by the formula GGGSSX1, where X1 is absent or, if present, is 1 to 9 repetitions of amino acid residues 1 to 5 of SEQ ID NO:274.

[0896] According to the present disclosure, n can be an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 15, 20 or 25.

[0897] According to the present disclosure, m can be an integer such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, such as 15, 20 or 25.

[0898] Specific exemplary and non-limiting embodiments of the flexible linker include those linkers that comprise or consist of the amino acid sequences set forth in SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, or SEQ ID NO:186.

[0899] Other exemplary and non-limiting embodiments of the linker include those linkers that comprise or consist of the amino acid sequences set forth in SEQ ID NO:271, 272, 273, or 274.

[0900] It should be understood that SEQ ID NO:186 can be represented by the formula (GGGGS) n , where n is an integer selected from 1 to 10, or alternatively represented by the formula GGGGSX1, where X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO:186.

[0901] The rigid linkers of the present disclosure generally consist of proline-rich sequences (XP) n where 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).

[0902] Specific exemplary and non-limiting embodiments of the rigid linker include those linkers that comprise or consist of the amino acid sequences set forth in SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, or SEQ ID NO:190.

[0903] It should be understood that SEQ ID NO:190 can be represented by the formula (X(PAPAP)) n KA, where n is an integer selected from 1 to 10, where X is present or absent and, if present, is A, or alternatively, SEQ ID NO:190 can be represented by the formula (XPAPAP)X2KA, where X can be present or absent and, if present, is A; and where X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 6 of SEQ ID NO:190.

[0904] Helical linkers are sometimes also characterized as being rigid, but are classified herein as a separate linker family. Exemplary embodiments of helical linkers are discussed in Chen X et al., 2013 and include, for example, repeating sequences of alanine residues flanked by positively and negatively charged amino acid residues.

[0905] Specific exemplary and non-limiting embodiments of the helical linker include those linkers comprising or consisting of the amino acid sequences set forth in SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, and SEQ ID NO:194.

[0906] It is understood that SEQ ID NO:194 can be represented by the formula X(EAAAK) n X2, where n is an integer selected from 1 to 10, more preferably 2 - 5, where X and X2 are independently present or absent and, if present, are preferably A. Alternatively, SEQ ID NO:194 can be represented by the formula X(EAAAK)X3X2, where X and X2 are independently present or absent and, if present, are preferably A; and X3 is absent or, if present, is 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO:194.

[0907] In one exemplary embodiment, the linker immediately C-terminal to the dimerization domain (designated L in Formulas II - VIII) c1 ) can comprise a flexible linker, a rigid linker, or a helical linker. Particularly selected linkers to occupy this position include, for example but not limited to, linkers comprising or consisting of the amino acid sequences set forth in SEQ ID NO:182 - 191, SEQ ID NO:193, or SEQ ID NO:194 and where n is 1.

[0908] In one exemplary embodiment, the linker connecting the first two antigen-binding domains C-terminal to the dimerization domain (designated L in Formulas II - VIII) c2 ) can comprise a flexible linker, a rigid linker, or a helical linker. Particularly selected linkers to occupy this position include, for example but not limited to, linkers comprising or consisting of the amino acid sequences set forth in SEQ ID NO:182 - 192, or SEQ ID NO:194 and where n is 1.

[0909] The present disclosure also provides linkers having 1 to 10 amino acids (and any range or value between 1 and 10, such as 1 to 5) added to one or both of the N-terminus and C-terminus of any of SEQ ID NO:182 to 194. These additional amino acid residues can each independently be selected from any amino acid residue. These additional amino acid residues preferably form a non-cleavable sequence.

[0910] The present disclosure also provides linkers having 1, 2, 3, 4, or 5 amino acids (and any value between 1 and 5) deleted from one or both of the N-terminus and C-terminus of any of SEQ ID NO:182 to 194.

[0911] Suitable linkers can comprise, for example, an amino acid sequence comprising from about 3 to about 50, from about 3 to about 40, from about 3 to about 30, from about 3 to about 25, from about 3 to about 20, from about 3 to about 15, or from about 3 to about 10 amino acid residues.

[0912] In exemplary embodiments, the length of each linker can independently range from about 5 to about 50 amino acid residues, including, for example, from about 5 to about 40 amino acid residues, from about 10 to about 40 amino acid residues, from about 20 to about 40 amino acid residues, from about 20 to about 35 amino acid residues, from about 25 to about 30 amino acid residues, and any subranges subsumed therein and including these ranges.

[0913] In some embodiments, the "n" value of a linker comprising the amino acid sequence of SEQ ID NO:186, SEQ ID NO:190, or SEQ ID NO:194 is preferably from 1 to 10, more preferably from 2 to 5, including 2, 3, 4, or 5.

[0914] Binding agent

[0915] The binding agents of the present disclosure comprise, for example, an antigen-binding domain disclosed herein.

[0916] The binding agents of the present disclosure comprise, for example, a polypeptide chain disclosed herein. Thus, in some embodiments, a binding agent can consist of a single polypeptide chain. In some embodiments, a binding agent is a polypeptide chain.

[0917] The binding agents of the present disclosure comprise, for example, a dimer of a polypeptide chain disclosed herein.

[0918] The binding agents of the present disclosure comprise, for example, a multimer of a polypeptide chain disclosed herein.

[0919] The binding agents of the present disclosure are capable of binding to the CD36 protein or a portion thereof. In some embodiments, a binding agent comprises one or more antigen-binding domains capable of binding to the CD36 protein or a portion thereof or to a cell expressing the CD36 protein or a portion thereof. According to the present disclosure, one or more antigen-binding domains are from an anti-CD36 antibody or an antigen-binding fragment thereof. Also according to the present disclosure, one or more antigen-binding domains are from an anti-CD36 single-domain antibody.

[0920] Exemplary and non-limiting embodiments of antigen-binding domains are provided in Tables 3, 4, and 5.

[0921] The binder of the present disclosure may also be capable of binding to immune checkpoint proteins and / or proteins expressed on the surface of immune cells. Accordingly, the binder may comprise one or more antigen-binding domains capable of binding to immune checkpoint proteins and / or one or more antigen-binding domains capable of binding to proteins expressed on the surface of immune cells.

[0922] In some embodiments, the binder of the present disclosure may comprise an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD1, and an antigen-binding domain that binds CD47. In some embodiments, the binder of the present disclosure may be capable of bringing T cells to the vicinity of tumor cells expressing CD36, may be capable of restoring T cell function and / or may be capable of enhancing macrophage function by blocking the SIRPα / CD47 interaction. In some embodiments of the present disclosure, the binder may exert its anti-tumor activity through antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments of the present disclosure, the binder may exert its anti-tumor activity through antibody-dependent cell phagocytosis (ADCP) activity.

[0923] Accordingly, in some exemplary embodiments, the binder may comprise one or more antigen-binding domains capable of binding to the PD-1 protein or a portion thereof or a cell expressing the PD-1 protein or a portion thereof. In other exemplary embodiments, the binder may comprise one or more antigen-binding domains capable of binding to the CD47 protein or a portion thereof or a cell expressing the CD47 protein or a portion thereof.

[0924] In some embodiments, the binder of the present disclosure may be capable of reducing tumor growth and / or causing tumor regression. In some embodiments, the binder has anti-tumor activity. In some embodiments, the binder is capable of recruiting or redirecting immune cells.

[0925] In certain cases, the binder of the present disclosure may modulate lipid metabolism.

[0926] In other cases, the binder of the present disclosure may inhibit fatty acid oxidation.

[0927] In other cases, the binder of the present disclosure may inhibit the uptake of fatty acids by tumors.

[0928] In other cases, the binder of the present disclosure may be capable of inhibiting the growth of tumor cells.

[0929] In other cases, the binder of the present disclosure may be capable of inhibiting tumor metastasis.

[0930] In other cases, the binder of the present disclosure may be capable of reducing the resistance of tumor cells to chemotherapy and / or immune checkpoint inhibitor therapy.

[0931] The binder of the present disclosure can be in the form of an antibody and its antigen-binding fragments, antibody-like molecules (such as Fc-, CH3-fusions, etc.), fusions with protein scaffolds, immunomodulators of immune cells, etc.

[0932] In some embodiments, the binder comprises an antibody or its antigen-binding fragment. In some embodiments, the binder is an antibody or its antigen-binding fragment.

[0933] In some embodiments, the binder comprises an antibody-like molecule. In some embodiments, the binder is an antibody-like molecule.

[0934] In some embodiments, the binder can be fused with a protein scaffold.

[0935] In some embodiments, the binder comprises an immunomodulator of immune cells. In some embodiments, the binder is an immunomodulator of immune cells.

[0936] Thus, in some embodiments, the binder includes an antibody and its antigen-binding fragments, such as but not limited to, single-domain antibodies from camelids or sharks, human antibodies including IgG (including human IgG1, human IgG2, human IgG3, human IgG4), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD, and animal antibodies including, for example, IgG (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE, and IgD.

[0937] In some embodiments, the binder comprises the antigen-binding domain of a single-domain antibody. In other embodiments, the binder comprises the antigen-binding domains of one or more single-domain antibodies.

[0938] For example, the binder can comprise one or more CDRs of a single-domain antibody. In another example, the binder can comprise one or more variable regions (VH or VHH) of a single-domain antibody.

[0939] In some embodiments, the binder comprises a single-domain antibody.

[0940] In some embodiments, the binder is a single-domain antibody.

[0941] In other embodiments, the binder includes antigen-binding fragments, such as but not limited to, Fab, Fab’, F(ab’)2, complementarity-determining regions, variable regions including VH, VHH, VL, etc.

[0942] The binder further includes immunomodulators, such as, but not limited to, bispecific 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) comprising scFv or VHH.

[0943] The binder further includes fusions with protein scaffolds, including ankyrin repeat proteins, the Z domain of staphylococcal protein A, type III fibronectin, knottin, etc. Exemplary embodiments of the binder include monospecific, bispecific (symmetric or asymmetric), trispecific or multispecific antibodies, as well as monovalent, bivalent, trivalent or multivalent antibodies, single-chain FV (scFV) and its derivatives such as diabodies, triabodies, tetra-bodies, tandem di-scFV, tandem tri-scFV, scFV-Fc, miniantibodies (scFV-CH3), tandem diabodies, bis-diabodies, bisomes, etc., VH or VHH and its derivatives such as tandem bispecific or multispecific VHH, bivalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, bivalent CH3 fusions, VHH pentabodies, decabodies, etc.

[0944] In some embodiments, the binder of the present disclosure may adopt the form of Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII disclosed in PCT / CA2020 / 051753, filed on December 18, 2020 (published as WO2021 / 119832A1 on June 24, 2021), or the form of Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII disclosed herein.

[0945] The binder of the present disclosure may be formed by the assembly of two polypeptide chains having the same configuration (having the same or different amino acid sequences) or having different configurations, where the same or different configurations include the configurations described 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.

[0946] The binder of the present disclosure may, for example, comprise one or more polypeptide chains independently comprising the amino acid sequence of Formula I in the form from the N-terminus to the C-terminus:

[0947] X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y

[0948] where m is an integer of 0, 1, 2, or greater than 2;

[0949] where n is an integer of 0, 1, 2, or greater than 2;

[0950] where m and n are not both 0 at the same time;

[0951] where Ab a and Ab d each represent an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1);

[0952] where X or Y independently exists or is absent and contains an amino acid sequence;

[0953] where L b and L c each independently contain one or more linkers; and

[0954] where DD represents a dimerization domain.

[0955] In some embodiments, when m is an integer of 1, 2, or greater than 2, one of Ab a or Ab d is antigen-binding domain 1 (ABD1), and the other of Ab a or Ab d is selected from antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

[0956] In some embodiments, when n is an integer of 1, 2, or greater than 2, one of Ab a or Ab d is antigen-binding domain 1 (ABD1), and the other of Ab a or Ab d is selected from antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

[0957] In some embodiments, when m is an integer of 1, 2, or greater than 2 and / or when n is an integer of 1, 2, or greater than 2, at least one of Ab a or Ab d is antigen-binding domain 1 (ABD1), at least one of Ab a or Ab d is antigen-binding domain 2 (ABD2), and at least one of Ab a or Ab d is antigen-binding domain 3 (ABD3).

[0958] In some embodiments L bmay be absent. In some embodiments, L b2 and / or L b3 may be absent.

[0959] In some embodiments, L c may be absent. In some embodiments, L c1 、L c2 and / or L c3 may be absent.

[0960] In some embodiments, L b and L c may be absent simultaneously.

[0961] In some embodiments, the polypeptide chain comprises a plurality of antigen-binding domains.

[0962] In some embodiments, the polypeptide chain comprises two or more antigen-binding domains.

[0963] In some embodiments, the polypeptide chain comprises three or more antigen-binding domains.

[0964] In some embodiments, the polypeptide chain comprises four or more antigen-binding domains.

[0965] In some embodiments, the polypeptide chain comprises five or more antigen-binding domains.

[0966] In some embodiments, the polypeptide chain comprises six or more antigen-binding domains.

[0967] In some embodiments, the polypeptide chain comprises from one to twelve antigen-binding domains.

[0968] In some embodiments, the binder comprises from one to twelve antigen-binding domains, such as one to two, one to three, one to four, one to five, one to six, one to seven, one to eight, one to nine, one to ten, one to eleven, one to twelve, two to three, two to four, two to five, two to six, two to seven, two to eight, two to nine, two to ten, two to eleven, two to twelve, three to four, three to five, three to six, three to seven, three to eight, three to nine, three to ten, three to eleven, three to twelve, four to five, four to six, four to seven, four to eight, four to nine, four to ten, four to eleven, four to twelve, five to six, five to seven, five to eight, five to nine, five to ten, five to eleven, five to twelve, six to seven, six to eight, six to nine, six to ten, six to eleven, six to twelve, seven to eight, seven to nine, seven to ten, seven to eleven, seven to twelve, eight to nine, eight to ten, eight to eleven, eight to twelve, nine to ten, nine to eleven, nine to twelve, ten to eleven, ten to twelve, or eleven to twelve.

[0969] In some embodiments, the binder comprises a single polypeptide chain.

[0970] In some embodiments, the binder comprises two polypeptide chains.

[0971] In some embodiments, the binder comprises three polypeptide chains.

[0972] In some embodiments, the binder comprises four polypeptide chains.

[0973] In some embodiments, the binder comprises five polypeptide chains.

[0974] In some embodiments, the binder comprises six polypeptide chains.

[0975] In some embodiments, the binder comprises seven polypeptide chains.

[0976] In some embodiments, the binder comprises eight polypeptide chains.

[0977] In some embodiments, the binder comprises nine polypeptide chains.

[0978] In some embodiments, the binder comprises ten polypeptide chains.

[0979] In some embodiments, the binder comprises more than ten polypeptide chains.

[0980] In certain embodiments, the binder comprises at least one polypeptide chain having Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1)-(Ab d1 )-Y (Formula II).

[0981] In certain embodiments, the binder comprises at least one polypeptide chain having Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0982] In certain embodiments, the binder comprises at least one polypeptide chain having Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0983] In certain embodiments, the binder comprises at least one polypeptide chain having Formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0984] In certain embodiments, the binder comprises at least one polypeptide chain having 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).

[0985] In certain embodiments, the binder comprises at least one polypeptide chain having Formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(Lc1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0986] In certain embodiments, the binder comprises at least one polypeptide chain having 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).

[0987] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, in an N-terminal to C-terminal fashion, the amino acid sequence of Formula III

[0988] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0989] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 1 (ABD1).

[0990] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, in an N-terminal to C-terminal fashion, the amino acid sequence of Formula III

[0991] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0992] wherein one of Ab a1 , Ab d1 or Ab d2 represents antigen-binding domain 1 (ABD1), Aba1 , Ab d1 or Ab d2 represents an antigen - binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 represents an antigen - binding domain 3 (ABD3).

[0993] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N - terminus to the C - terminus, an amino acid sequence of Formula III

[0994] X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III);

[0995] wherein Ab a1 represents an antigen - binding domain 1 (ABD1), Ab d1 represents an antigen - binding domain 2 (ABD2) and Ab d2 represents an antigen - binding domain 3 (ABD3).

[0996] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N - terminus to the C - terminus, an antigen - binding domain that binds CD36, an antigen - binding domain that binds PD - 1, and an antigen - binding domain that binds CD47.

[0997] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N - terminus to the C - terminus, an amino acid sequence of Formula III, wherein Ab a1 is an antigen - binding domain that binds CD36, Ab d1 is an antigen - binding domain that binds PD - 1, and Ab d2 is an antigen - binding domain that binds CD47.

[0998] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N - terminus to the C - terminus, an amino acid sequence of Formula III, wherein Ab a1 is an antigen - binding domain that binds CD36, Ab d1 is an antigen - binding domain that binds CD47, and Ab d2 is an antigen - binding domain that binds PD - 1.

[0999] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N-terminus to the C-terminus, an amino acid sequence of Formula IIIa:

[1000] X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa).

[1001] In certain embodiments, the binder comprises one or more polypeptide chains, each independently comprising, from the N-terminus to the C-terminus, an amino acid sequence of Formula IIIb:

[1002] X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb).

[1003] In certain embodiments, the binder comprises two identical polypeptide chains.

[1004] In certain embodiments, the two polypeptide chains of the binder can have the configuration of Formula II (with the same or different amino acid sequences).

[1005] In certain embodiments, the two polypeptide chains of the binder can have the configuration of Formula III (with the same or different amino acid sequences).

[1006] In certain embodiments, the two polypeptide chains of the binder can have the configuration of Formula IIIa (with the same or different amino acid sequences).

[1007] In certain embodiments, the two polypeptide chains of the binder can have the configuration of Formula IIIb (with the same or different amino acid sequences).

[1008] In certain embodiments, one polypeptide chain can have the configuration of Formula IIIa and one polypeptide chain can have the configuration of Formula IIIb (with antigen-binding domains having the same or different amino acid sequences).

[1009] In some embodiments, one of the polypeptide chains can have the configuration of Formula II while the other can have the configuration of Formula III.

[1010] In some embodiments, one of the polypeptide chains can have the configuration of Formula II while the other has the configuration of Formula IV.

[1011] In some embodiments, one of the polypeptide chains can have the configuration described by Formula III, while the other has the configuration described by Formula IV.

[1012] In some embodiments, one of the polypeptide chains can have the configuration described by Formula IV, while the other has the configuration described by Formula IV.

[1013] In some embodiments, the binding agents of the present disclosure are monospecific or multispecific.

[1014] A monospecific binding agent comprises a binding agent that is specific for a single epitope of a given antigen.

[1015] An exemplary embodiment of a monospecific binding agent includes a binding agent comprising one antigen-binding domain. Another exemplary embodiment of a monospecific binding agent includes a binding agent comprising multiple antigen-binding domains but these antigen-binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a monospecific binding agent includes a binding agent comprising multiple antigen-binding domains but these antigen-binding domains have the same CDRs and different framework regions. A further exemplary embodiment of a monospecific binding agent includes a binding agent comprising antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs (e.g., conservative substitutions of one or more CDRs), but do not affect their ability to bind the same antigen or epitope.

[1016] A multispecific binding agent comprises a binding agent that is specific for multiple epitopes (of the same antigen or different antigens) or for multiple antigens. For example, a multispecific polypeptide chain or binding agent can thus have multiple antigen-binding domains, where at least two bind different antigens or epitopes.

[1017] The binding agents of the present disclosure can thus be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc. In some embodiments, each antigen-binding domain can be specific for a given antigen. In some embodiments, two or more antigen-binding domains of a given binding agent can be specific for the same or different antigens. In some embodiments, three or more antigen-binding domains of a given binding agent can be specific for the same or different antigens. In some embodiments, four or more antigen-binding domains of a given binding agent can be specific for the same or different antigens. In some embodiments, five or more antigen-binding domains of a given binding agent can be specific for the same or different antigens. In some embodiments, six or more antigen-binding domains of a given binding agent can be specific for the same or different antigens. Specificity can depend on the number of antigen-binding domains present in a given binding agent.

[1018] Exemplary non-limiting embodiments of the multispecific binding agent include those consisting of a multispecific polypeptide chain. Other exemplary non-limiting embodiments of the multispecific binding agent include those having two different antigen-binding domains of specificity. Other exemplary non-limiting embodiments of the multispecific binding agent include those having more than two antigen-binding domains that bind two different antigens, proteins, or two different epitopes on the same antigen or protein.

[1019] In some embodiments, the binding agents of the present disclosure are monovalent or multivalent.

[1020] Exemplary non-limiting embodiments of the multivalent binding agent include binding agents consisting of a multivalent polypeptide chain. Other non-limiting exemplary embodiments of the multivalent binding agent include binding agents consisting of more than one monovalent polypeptide chain.

[1021] According to the present disclosure, a bispecific binding agent can be bivalent or multivalent, depending on the number of antigen-binding domains it contains. Exemplary non-limiting embodiments of the bispecific binding agent include those comprising two identical bispecific polypeptide chains that form dimers.

[1022] Exemplary and non-limiting embodiments of the binding agent are provided in Tables 6 and 7.

[1023] Other exemplary embodiments of the binding agent include those comprising the amino acid sequence described in Table 3 and fused at the C-terminus to the amino acid sequence of SEQ ID NO: 178.

[1024] Other exemplary embodiments of the binding agent include those comprising the amino acid sequence described in Table 3 and comprising a linker, a dimerization domain, and optionally one or more antigen-binding domains having different amino acid sequences.

[1025] Other exemplary embodiments of the binding agent include those comprising the amino acid sequence described in Table 6 and fused at the C-terminus to the amino acid sequence of SEQ ID NO: 179.

[1026] Competitive binding agent

[1027] The binding agents of the present disclosure also include those that compete with the binding agents disclosed herein or single-domain antibodies having the amino acid sequences disclosed herein.

[1028] Antibodies are provided that compete with one or more single-domain antibodies having the amino acid sequences disclosed herein or antigen-binding fragments thereof. In some embodiments, the antibody is a single-domain antibody that competes with one or more single-domain antibodies having the amino acid sequences disclosed herein or antigen-binding fragments thereof.

[1029] In one exemplary embodiment, the binding agent can comprise one or more antigen-binding domains of an antibody that compete with one or more single-domain antibodies having the amino acid sequences disclosed herein or antigen-binding fragments thereof.

[1030] The competitive binding agents of the present disclosure can comprise an amino acid sequence having at least 75% or higher identity to the amino acid sequences described in any one of Tables 3, 4, 5, 6, and / or 7 or a combination thereof. Alternatively, the competitive binding agent can comprise an amino acid sequence having an identity of less than 75% to the amino acid sequences described in any one of Tables 3, 4, 5, 6, and / or 7, or having different CDR1, CDR2, and / or CDR3, but still capable of binding the same targets (CD36, PD-1, and / or CD47) and maintaining functionality. Such competitive binding agents can be identified by a competition assay using the target and a pre-identified binding agent (such as in Tables 6 and / or 7). The functionality of the competitive binding agent is confirmed by testing its activity, including, for example, but not limited to, its ability to reduce cellular fatty acid oxidation, lipid metabolism, and / or fatty acid uptake.

[1031] In one exemplary embodiment, competition between binding agents can be determined using recombinant CD36 or a portion thereof or cells expressing CD36 or a portion thereof based on any type of binding assay (FACS, ELISA, SPR, etc.).

[1032] In an exemplary embodiment, the competitive binding agent is capable of blocking CD36 or comprises one or more antigen-binding domains capable of blocking CD36.

[1033] In some embodiments, the binding agent comprises one or more antigen-binding domains of an antibody that can compete with a single-domain antibody comprising antigen-binding domain 1 (ABD1) described herein (competitive antibody). In other embodiments, the binding agent comprises one or more antigen-binding domains of an antibody that can compete with a anti-CD36 single-domain antibody comprising CDRH1, CDRH2, and / or CDRH3 amino acid sequences disclosed herein. More specifically, the binding agent comprises one or more antigen-binding domains of an antibody that can compete with a anti-CD36 single-domain antibody comprising CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[1034] In some embodiments, at least one of the one or more antigen-binding domains of the competitive antibody comprises the CDRH1, CDRH2, and / or CDRH3 amino acid sequences of the anti-CD36 single-domain antibody in Table 3.

[1035] In some embodiments, at least one of the one or more antigen-binding domains of the competitive antibody comprises:

[1036] a. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:7;

[1037] b. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:14;

[1038] c. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:21;

[1039] d. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:28;

[1040] e. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:35;

[1041] f. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:42;

[1042] g. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:49;

[1043] h. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:56;

[1044] i. 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:77 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:77;

[1045] j. 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: 84 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 84;

[1046] k. 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: 91 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 91;

[1047] l. 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: 98 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 98;

[1048] m. 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: 105 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 105;

[1049] n. 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: 112 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 112;

[1050] o. 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: 119 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 119;

[1051] p. 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: 126 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 126;

[1052] q. 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: 133 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 133; or

[1053] 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: 140 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 140.

[1054] A competitive antibody can be a competitive single-domain antibody or an antigen-binding fragment thereof.

[1055] In some embodiments, the binding agent can further comprise one or more antigen-binding domains of the anti-PD-1 single-domain antibodies disclosed herein.

[1056] In some embodiments, at least one of the one or more antigen-binding domains is from an anti-PD-1 antibody that is capable of competing (competitive antibody) with a single-domain antibody comprising the antigen-binding domain 2 (ABD2) described herein.

[1057] In other embodiments, the binding agent can further comprise one or more antigen-binding domains of an antibody that is capable of competing with an anti-PD-1 single-domain antibody comprising an antigen-binding domain having the CDRH1, CDRH2, and / or CDRH3 amino acid sequences disclosed herein. More specifically, the binding agent comprises one or more antigen-binding domains of an antibody that is capable of competing with an anti-PD-1 single-domain antibody comprising an antigen-binding domain having the CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[1058] In some embodiments, the anti-PD-1 antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to any one of SEQ ID NO: 63, 281, 288, 295, 302, 309, 316, 323, 330, 337, 344, 351, 358, and / or 365 or has from one to ten amino acid substitutions, additions, and / or deletions in any one of the amino acid sequences of SEQ ID NO: 281, 288, 295, 302, 309, 316, 323, 330, 337, 344, 351, 358, and / or 365.

[1059] In some embodiments, the binding agent can further comprise one or more antigen-binding domains of the anti-CD47 single-domain antibodies disclosed herein.

[1060] In some embodiments, at least one of the one or more antigen-binding domains is from an anti-CD47 antibody that is capable of competing (competitive antibody) with a single-domain antibody comprising the antigen-binding domain 3 (ABD3) described herein.

[1061] In other embodiments, the binder can further comprise one or more antigen-binding domains of an antibody that is capable of competing with an anti-CD47 single-domain antibody comprising an antigen-binding domain having the CDRH1, CDRH2, and / or CDRH3 amino acid sequences disclosed herein. More specifically, the binder comprises one or more antigen-binding domains of an antibody that is capable of competing with an anti-CD47 single-domain antibody comprising an antigen-binding domain having the CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[1062] In other embodiments, the anti-CD47 antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70 or has one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:70.

[1063] In some embodiments, the binder comprises one or more antigen-binding domains of an antibody that is capable of competing with an anti-PD-1 single-domain antibody disclosed herein or one or more antigen-binding domains of an antibody that is capable of competing with an anti-CD47 single-domain antibody disclosed herein, and at least one of the one or more antigen-binding domains of the antibody comprises:

[1064] a. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:7;

[1065] b. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:14;

[1066] c. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:21;

[1067] d. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:28;

[1068] e. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:35;

[1069] f. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:42;

[1070] g. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:49;

[1071] h. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:56;

[1072] i. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:77;

[1073] j. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 84;

[1074] k. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 91;

[1075] l. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 98;

[1076] m. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 105;

[1077] n. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 112;

[1078] o. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 119;

[1079] p. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 126;

[1080] q. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 133;

[1081] r. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 140.

[1082] Exemplary embodiments of monospecific binders

[1083] As described herein, the binders of the present disclosure can be monospecific.

[1084] Exemplary and non - limiting exemplary embodiments of monospecific binders are provided herein.

[1085] For example, a binder can comprise only one antigen - binding domain. Alternatively, a binder can comprise multiple identical antigen - binding domains.

[1086] Exemplary embodiments of monospecific binders include the antigen - binding domains described herein, including ABD1, ABD2, ABD3 or the heavy chain disclosed herein.

[1087] In an exemplary embodiment, a monospecific binder can comprise a dimerization domain and an antigen - binding domain disclosed herein.

[1088] Exemplary embodiments of dimerization domains are provided throughout the specification, including but not limited to CH3 domains (including native CH3 domains and native human CH3 domains, mutant CH3 domains), and can also include CH2 domains (native CH2 domains or mutant CH2 domains).

[1089] In some embodiments, the dimerization domain is the Fc region of an antibody or a portion thereof.

[1090] In some embodiments, the dimerization domain may further comprise the hinge region of an antibody or a portion thereof. In some embodiments, the hinge region or a portion thereof is a human hinge region or a portion thereof. In some cases, a linker or additional amino acid sequence may be added between or at either end of the antigen-binding domain and the dimerization domain.

[1091] In some embodiments, the binder may comprise an ABD1 and a dimerization domain.

[1092] In some embodiments, the binder may comprise an ABD2 and a dimerization domain.

[1093] In some embodiments, the binder may comprise an ABD3 and a dimerization domain.

[1094] In some exemplary embodiments, the binder 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:7 and a dimerization domain.

[1095] In some exemplary embodiments, the binder 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:14 and a dimerization domain.

[1096] In some exemplary embodiments, the binder 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:21 and a dimerization domain.

[1097] In some exemplary embodiments, the binder 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:28 and a dimerization domain.

[1098] In some exemplary embodiments, the binder 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:35 and a dimerization domain.

[1099] In some exemplary embodiments, the binder 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:42 and a dimerization domain.

[1100] In some exemplary embodiments, the binder 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: 49 and a dimerization domain.

[1101] In some exemplary embodiments, the binder 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: 56 and a dimerization domain.

[1102] In some exemplary embodiments, the binder 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: 77 and a dimerization domain.

[1103] In some exemplary embodiments, the binder 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: 84 and a dimerization domain.

[1104] In some exemplary embodiments, the binder 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: 91 and a dimerization domain.

[1105] In some exemplary embodiments, the binder 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: 98 and a dimerization domain.

[1106] In some exemplary embodiments, the binder 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: 105 and a dimerization domain.

[1107] In some exemplary embodiments, the binder 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: 112 and a dimerization domain.

[1108] In some exemplary embodiments, the binder 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: 119 and a dimerization domain.

[1109] In some exemplary embodiments, the binder 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: 126 and a dimerization domain.

[1110] In some exemplary embodiments, the binder 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: 133 and a dimerization domain.

[1111] In some exemplary embodiments, the binder 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: 140 and a dimerization domain.

[1112] In some exemplary embodiments, the binder 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: 63 and a dimerization domain.

[1113] In some exemplary embodiments, the binder 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: 281 and a dimerization domain.

[1114] In some exemplary embodiments, the binder 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: 288 and a dimerization domain.

[1115] In some exemplary embodiments, the binder 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: 295 and a dimerization domain.

[1116] In some exemplary embodiments, the binder 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: 302 and a dimerization domain.

[1117] In some exemplary embodiments, the binder 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: 309 and a dimerization domain.

[1118] In some exemplary embodiments, the binder 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: 316 and a dimerization domain.

[1119] In some exemplary embodiments, the binder 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: 323 and a dimerization domain.

[1120] In some exemplary embodiments, the binder 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: 330 and a dimerization domain.

[1121] In some exemplary embodiments, the binder 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: 337 and a dimerization domain.

[1122] In some exemplary embodiments, the binder 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: 344 and a dimerization domain.

[1123] In some exemplary embodiments, the binder 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: 351 and a dimerization domain.

[1124] In some exemplary embodiments, the binder 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: 358 and a dimerization domain.

[1125] In some exemplary embodiments, the binder 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: 365 and a dimerization domain.

[1126] In some exemplary embodiments, the binder 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: 70 and a dimerization domain.

[1127] It should be understood that, herein, the binder may optionally comprise one or more linkers or additional amino acid sequences.

[1128] Other exemplary embodiments of monospecific binders are provided herein, including but not limited to the single domain antibodies in Table 6 or amino acid sequences comprising the single domain antibodies in Table 6.

[1129] Exemplary embodiments of multispecific binders

[1130] As described herein, the binders of the present disclosure may be multispecific.

[1131] Exemplary and non-limiting embodiments of multispecific binders are provided herein.

[1132] For example, in some embodiments, the binder may comprise multiple antigen-binding domains, wherein at least one of the antigen-binding domains is the antigen-binding domain 1 (ABD1) described herein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein.

[1133] In other embodiments, the binder may comprise multiple antigen-binding domains, wherein at least one of the antigen-binding domains is the antigen-binding domain 1 (ABD1) described herein, at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding a protein expressed on the surface of an immune cell.

[1134] In other embodiments, the binder may comprise multiple antigen-binding domains, wherein at least one of the antigen-binding domains is the antigen-binding domain 1 (ABD1) described herein, at least one of the antigen-binding domains is an antigen-binding domain capable of binding an immune checkpoint protein, and at least one of the antigen-binding domains is an antigen-binding domain capable of binding a protein expressed on the surface of a tumor cell.

[1135] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 2 (ABD2).

[1136] In some embodiments, the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 3 (ABD3).

[1137] In some embodiments, the binder comprises at least one antigen-binding domain 2 (ABD2) and at least one antigen-binding domain 3 (ABD3).

[1138] In some embodiments, the binder 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).

[1139] In some embodiments, the binder is a multispecific binder comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and antigen-binding domain 3 (ABD3), ABD1 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:105, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:126, SEQ ID NO:133, or SEQ ID NO:140, ABD2 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:63, SEQ ID NO:281, SEQ ID NO:288, SEQ ID NO:295, SEQ ID NO:302, SEQ ID NO:309, SEQ ID NO:316, SEQ ID NO:323, SEQ ID NO:330, SEQ ID NO:337, SEQ ID NO:344, SEQ ID NO:351, SEQ ID NO:358, or SEQ ID NO:365, and ABD3 comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1140] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1141] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1142] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1143] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1144] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1145] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1146] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1147] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1148] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:77; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1149] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:84; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1150] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 91; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1151] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 98; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1152] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 105; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1153] In certain embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 112; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1154] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 119; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1155] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 126; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1156] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 133; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1157] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 140; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to 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 an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1158] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence of SEQ ID NO: 7; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence of SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence of SEQ ID NO: 70.

[1159] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 14; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1160] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 21; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1161] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 28; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1162] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 35; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1163] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 42; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1164] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 49; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1165] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 56; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1166] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 77; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1167] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:84; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[1168] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:91; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[1169] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:98; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[1170] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:105; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[1171] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 112; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1172] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 119; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1173] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 126; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1174] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 133; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1175] In some embodiments, the binder comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 140; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO: 70.

[1176] In other exemplary embodiments of the binder, SEQ ID NO: 63 can be replaced with other anti-PD1 VHH sequences, such as but not limited to SEQ ID NO: 281, SEQ ID NO: 288, SEQ ID NO: 295, or SEQ ID NO: 337.

[1177] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 159. In some embodiments, the binder comprises a polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 159. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 159. In some other embodiments, the binder comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 159.

[1178] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 160 - 177. In some embodiments, the binder comprises a polypeptide chain having any one of the amino acid sequences set forth in SEQ ID NOs: 160 - 177. In other embodiments, the binder comprises two polypeptide chains that have an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 160 - 177. In some other embodiments, the binder comprises two polypeptide chains having any one of the amino acid sequences set forth in SEQ ID NOs: 160 - 177.

[1179] It should be understood that, herein, the binder may optionally comprise one or more linkers or additional amino acid sequences.

[1180] Other exemplary embodiments of the multispecific binders are provided herein, including the content in Table 7.

[1181] In some embodiments, the sequences of the monospecific binders of Table 6 can be inserted into the N-terminal portion of SEQ ID NO: 179 to prepare a hexavalent and trispecific binder comprising an anti-PD-1 VHH and an anti-CD47 VHH.

[1182] In some embodiments, any VHH sequence of Table 3 can be inserted into the N-terminal portion of SEQ ID NO: 178 to prepare a hexavalent and trispecific binder comprising an anti-PD-1 VHH and an anti-CD47 VHH.

[1183] Variant

[1184] The present disclosure also encompasses variants of the sequences disclosed herein.

[1185] Variants encompassed by the present disclosure include those that may comprise insertion of one or more amino acid residues at one or more positions, deletion of one or more amino acid residues at one or more positions, or substitution of one or more amino acid residues at one or more positions (conservative or non-conservative substitutions).

[1186] For example, naturally occurring amino acid residues are grouped according to common side chain characteristics. Conservative substitutions can be made by exchanging one amino acid in the following groups (Groups 1 to 6) for another amino acid in the same group. Non-conservative substitutions would involve exchanging a member of one of these groups for a member of another group.

[1187] (Group 1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile)

[1188] (Group 2) Neutral hydrophilic: cysteine (Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln)

[1189] (Group 3) Acidic: aspartic acid (Asp), glutamic acid (Glu)

[1190] (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg)

[1191] (Group 5) Residues affecting chain orientation: glycine (Gly), proline (Pro); and

[1192] (Group 6) Aromaticity: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

[1193] Other exemplary embodiments of conservative substitutions are shown under the heading "Preferred Substitutions" in Table 2. If such substitutions result in undesirable properties, substitutions marked as "Exemplary Substitutions" in Table 2 or greater changes as further described below regarding amino acid classifications can be introduced and the product screened.

[1194] Those skilled in the art will recognize that certain amino acids are less positively charged, neutral, negatively charged, or have reduced charge compared to other amino acids. Amino acids can be classified according to their net charge as indicated by their isoelectric point. The isoelectric point is the pH value at which the average net charge of an amino acid molecule is zero. When pH > pI, the amino acid has a net negative charge; when pH < pI, the amino acid has a net positive charge. In some embodiments, the measured pI value of the antibody is from about 3 to 9 (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, and 9) and any value therebetween. In some embodiments, the measured pI value of the antibody is from about 4 to 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) and any value therebetween. Exemplary isoelectric points of amino acids are shown in Table 2 below. Amino acids with generally 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 properties include, for example, Serine (S), Threonine (T), Asparagine (N), Glutamine (Q), Cysteine (C), Tyrosine (Y), and Tryptophan (W). Non-polar amino acids include, for example, Alanine (A),...

Claims

1. An agent that comprises one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains is an antigen-binding domain capable of specifically binding to cluster of differentiation 36 (CD36).

2. The agent according to claim 1, wherein the agent comprises more than one antigen-binding domain, and wherein at least one of the one or more antigen-binding domains is an antigen-binding domain capable of specifically binding to an immune checkpoint protein and / or an antigen-binding domain capable of specifically binding to a protein expressed on the surface of an immune cell.

3. The agent according to claim 1, wherein the agent comprises more than one antigen-binding domain, and wherein at least one of the one or more antigen-binding domains is an antigen-binding domain capable of specifically binding to programmed cell death protein 1 (PD-1) and / or at least one of the one or more antigen-binding domains is an antigen-binding domain capable of specifically binding to cluster of differentiation 47 (CD47).

4. The agent according to any one of the preceding claims, wherein the agent comprises 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, or two to twelve antigen-binding domains.

5. The agent according to any one of the preceding claims, wherein the agent comprises at least two antigen-binding domains capable of specifically binding to CD36 and at least two antigen-binding domains capable of specifically binding to PD-1 and / or at least two antigen-binding domains capable of specifically binding to CD47.

6. The agent according to any one of the preceding claims, wherein the antigen-binding domain capable of specifically binding to CD36 is antigen-binding domain 1 (ABD1) and comprises: a. 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; 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. CDRH1 having the amino acid sequence set forth in SEQ ID NO:71, CDRH2 having the amino acid sequence set forth in SEQ ID NO:72, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:73; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO:74, CDRH2 having the amino acid sequence set forth in SEQ ID NO:75, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:76; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO:78, CDRH2 having the amino acid sequence set forth in SEQ ID NO:79, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:80; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO:81, CDRH2 having the amino acid sequence set forth in SEQ ID NO:82, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:83; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO:85, CDRH2 having the amino acid sequence set forth in SEQ ID NO:86, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:87; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO:88, CDRH2 having the amino acid sequence set forth in SEQ ID NO:89, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:90; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO:92, CDRH2 having the amino acid sequence set forth in SEQ ID NO:93, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:94; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO:95, CDRH2 having the amino acid sequence set forth in SEQ ID NO:96, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:97; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO:99, CDRH2 having the amino acid sequence set forth in SEQ ID NO:100, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:101; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO:102, CDRH2 having the amino acid sequence set forth in SEQ ID NO:103, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:104; aa. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 106, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 107, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 108; bb. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 109, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 110, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 111; cc. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 113, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 114, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 115; dd. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 116, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 117, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 118; ee. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 120, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 121, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 122; ff. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 123, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 124, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 125; gg. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 127, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 128, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 129; hh. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 130, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 131, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 132; ii. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 134, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 135, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 136; jj. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 137, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 138, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 139; kk. The amino acid sequence set forth in SEQ ID NO: 380; ll. The amino acid sequence set forth in SEQ ID NO: 381; mm. The amino acid sequence set forth in SEQ ID NO: 382; nn. The amino acid sequence set forth in SEQ ID NO: 383; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:7; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:14; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:21; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:28; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:35; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:42; 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 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:49; An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:56; 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:77 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:77; 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:84 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:84; 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:91 or has from one to ten amino acid substitutions, additions and / or deletions in SEQ ID NO:91; zz. 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: 98 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 98; aaa. 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: 105 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 105; bbb. 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: 112 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 112; ccc. 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: 119 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 119; ddd. 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: 126 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 126; eee. 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: 133 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 133, or fff. 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: 140 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:

140.

7. The binder according to any one of the preceding claims, wherein at least one of the one or more antigen-binding domains is an antigen-binding domain 2 (ABD2) capable of specifically binding to PD-1 and comprises: 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. A CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62; c. A CDRH1 having the amino acid sequence set forth in SEQ ID NO: 275, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 276, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 277; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 278, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 279, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 280; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 282, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 283, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 284; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 285, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 286, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 287; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 289, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 290, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 291; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 292, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 293, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 294; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 296, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 297, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 298; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 299, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 300, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 301; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 303, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 304, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 305; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 306, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 307, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 308; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 310, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 311, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 312; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 313, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 314, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 315; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 317, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 318, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 319; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 320, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 321, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 322; q. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 324, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 325, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 326; r. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 327, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 328, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 329; s. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 331, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 332, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 333; t. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 334, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 335, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 336; u. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 338, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 339, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 340; v. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 341, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 342, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 343; w. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 345, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 346, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 347; x. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 348, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 349, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 350; y. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 352, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 353, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 354; z. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 355, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 356, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 357; aa. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 359, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 360, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 361; bb. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 362, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 363, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 364; cc. the amino acid sequence set forth in SEQ ID NO: 379; dd. 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 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 63; ee. 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: 281 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 281; ff. 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: 288 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 288; gg. 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: 295 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 295; hh. 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: 302 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 302; ii. 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: 309 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 309; jj. 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: 316 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 316; kk. 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: 323 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 323; ll. 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: 330 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 330; mm. 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: 337 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 337; nn. 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: 344 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 344; oo. 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: 351 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 351; pp. 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: 358 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO: 358; or qq. 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: 365 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:

365.

8. The binder according to any one of the preceding claims, wherein at least one of the one or more antigen-binding domains is an antigen-binding domain 3 (ABD3) capable of specifically binding CD47 and comprises: 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. A CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, a CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and a CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or c. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 or has from one to ten amino acid substitutions, additions, and / or deletions in SEQ ID NO:

70.

9. The binder according to claim 7, wherein the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 2 (ABD2).

10. The binder according to claim 8, wherein the binder comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 3 (ABD3).

11. The binder according to any one of claims 8 to 10, wherein the binder 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).

12. The binder according to any one of the preceding claims, wherein at least one of the one or more antigen-binding domains is an antigen-binding domain 1 (ABD1) and comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:7, SEQ ID NO:14, SEQ ID NO:21, SEQ ID NO:28, SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:77, SEQ ID NO:84, SEQ ID NO:91, SEQ ID NO:98, SEQ ID NO:105, SEQ ID NO:112, SEQ ID NO:119, SEQ ID NO:126, SEQ ID NO:133 or SEQ ID NO:140, wherein at least one of the one or more antigen-binding domains is an antigen-binding domain 2 (ABD2) and comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:63, SEQ ID NO:281, SEQ ID NO:288, SEQ ID NO:295, SEQ ID NO:302, SEQ ID NO:309, SEQ ID NO:316, SEQ ID NO:323, SEQ ID NO:330, SEQ ID NO:337, SEQ ID NO:344, SEQ ID NO:351, SEQ ID NO:358 or SEQ ID NO:365, and wherein at least one of the one or more antigen-binding domains is an antigen-binding domain 3 (ABD3) and comprises an amino acid sequence that is at least 85% identical to the amino acid sequence set forth in SEQ ID NO:

70.

13. The binder according to claim 12, wherein the binder comprises: a. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:7; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70; b. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:14; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70; c. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:21; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70; d. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:28; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70; e. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:35; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:70; f. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 42; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; g. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 49; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; h. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 56; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; i. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 77; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; j. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 84; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; k. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 91; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; l. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 98; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; m. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 105; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; n. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 112; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; o. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 119; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; p. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 126; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; q. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 133; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; r. At least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 140; at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

70.

14. The binder according to any one of the preceding claims, wherein one or more antigen-binding domains are located on one or more polypeptide chains.

15. The binder according to any one of the preceding claims, wherein one or more antigen-binding domains are located on the same polypeptide chain.

16. The binder according to any one of the preceding claims, wherein the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises one or more antigen-binding domains.

17. The binder according to any one of claims 1 to 16, wherein the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises a different antigen-binding domain.

18. The binder according to any one of claims 1 to 16, wherein the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain comprises the same antigen-binding domain.

19. The binder according to any one of the preceding claims, wherein the binder comprises at least two polypeptide chains capable of assembling to form a dimer, and wherein each polypeptide chain is the same.

20. The binder according to any one of claims 14 to 19, wherein the polypeptide chains each independently comprise, in an N-terminal to C-terminal form, an amino acid sequence of Formula I: X - [(Ab a ) - (L b )] m - (DD) - [(L c ) - (Ab d )] n - Y wherein m is an integer of 0, 1, 2 or greater than 2; wherein n is an integer of 0, 1, 2 or greater than 2; wherein m and n are not both 0; wherein Ab a and Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1); wherein X or Y independently is present or absent and comprises an amino acid sequence; wherein L b and L c each independently includes one or more joints; and wherein DD represents a dimerization domain.

21. The binder according to claim 20, wherein when m is an integer of 1, 2 or greater than 2, and / or wherein when n is an integer of 1, 2 or greater than 2, Ab a or Ab d at least one of which is antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

22. The binder according to claim 20 or 21, wherein when m is an integer of 2 or greater than 2, Ab a or [(Ab a )-(L b )] units are the same.

23. The binder according to claim 20 or 21, wherein when m is an integer of 2 or greater than 2, Ab a or [(Ab a )-(L b )] units are different.

24. The binder according to any one of claims 20 or 21, wherein when m is an integer greater than 2, the [(Ab a )-(L b )] units contain identical and different units.

25. The binder according to any one of claims 20 to 24, wherein when n is an integer of 2 or greater than 2, Ab d or [(L c )-(Ab d )] units are the same.

26. The binder according to any one of claims 20 to 24, wherein when n is an integer of 2 or greater than 2, Ab d or [(L c )-(Ab d )] units are different.

27. The binder according to any one of claims 20 to 24, wherein when n is an integer of 2 or greater than 2, the [(L c )-(Ab d )] unit contains identical and different units.

28. The binder according to any one of claims 20 to 27, wherein m is 1 or 2 and / or n is 1 or 2.

29. The binder according to any one of claims 20 to 28, wherein Ab a represents ABD1, Ab d represents ABD2 and / or Ab d represents ABD3.

30. The binder according to any one of claims 20 to 29, wherein m is 1 and Ab a represents ABD1.

31. The binder according to any one of claims 20 to 30, wherein n is 2 and wherein one of Ab d represents ABD2.

32. The binder according to any one of claims 20 to 30, wherein n is 2 and wherein one of Ab d represents ABD3.

33. The binder according to any one of claims 20 to 30, wherein n is 2 and wherein one of Ab d represents ABD2, and the other Ab d represents ABD3.

34. The binder according to any one of claims 20 to 33, wherein L b is the hinge region of an antibody or an antigen-binding fragment thereof and / or L c is a rigid linker.

35. The binder according to any one of claims 14 to 34, wherein each polypeptide chain independently comprises, in the form from the N-terminus to the C-terminus, any one of the following amino acid sequences: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula 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 (Formula VI); 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); 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); Among them, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each represents an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), wherein L b1 comprises one or more linkers and / or the hinge region of an antibody or an antigen-binding fragment thereof; wherein L b2 , L b3 , L c1 , L c2 and L c3 each independently comprises one or more joints; and wherein DD represents a dimerization domain.

36. The binder according to claim 35, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3).

37. The binder according to claim 36, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3).

38. The binder according to any one of claims 14 to 37, wherein each polypeptide chain independently comprises, in the form from the N-terminus to the C-terminus, an amino acid sequence of formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Among them, Ab a1 , Ab d1 or Ab d2 represents an antigen-binding domain 1 (ABD1).

39. The binder according to any one of claims 14 to 37, wherein each polypeptide chain independently comprises, in the form from the N-terminus to the C-terminus, an amino acid sequence of formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III); Wherein Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 1 (ABD1), Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 represents one of the antigen-binding domains 3 (ABD3).

40. The binder according to any one of claims 14 to 39, wherein each polypeptide chain independently comprises, in the form from the N-terminus to the C-terminus, an amino acid sequence of formula IIIa: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa).

41. The binder according to any one of claims 14 to 39, wherein each polypeptide chain independently comprises, in the form from the N-terminus to the C-terminus, an amino acid sequence of formula IIIb: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb).

42. The binder according to any one of claims 20 to 41, wherein the dimerization domain comprises the constant region of an antibody or a portion thereof.

43. The binder according to any one of claims 20 to 41, wherein the dimerization domain comprises a CH3 domain and optionally a CH2 domain.

44. The binder according to any one of claims 20 to 41, wherein the dimerization domain comprises a native CH2 domain and a native CH3 domain.

45. The binder according to claim 44, wherein the native CH2 domain and / or the native CH3 domain is from human IgG1.

46. The binder according to any one of claims 20 to 45, wherein each linker independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues.

47. The binder according to any one of claims 20 to 46, wherein each linker independently is a flexible linker, a helical linker or a rigid linker.

48. The binder according to any one of claims 20 to 47, wherein the linker comprises a flexible linker and / or a rigid linker.

49. The binder according to any one of claims 35 to 48, wherein L c1 , L c2 and / or L c3 is a rigid joint.

50. The binder according to any one of claims 35 to 49, wherein L b1 is the hinge region of an antibody or an antigen-binding fragment thereof.

51. The binder according to claim 50, wherein the hinge region is from IgG1, IgG2, IgG3 or IgG4.

52. The binder according to any one of the preceding claims, wherein the binder is multispecific.

53. The binder according to claim 52, wherein the binder is bispecific, trispecific or tetra-specific.

54. The binder according to any one of the preceding claims, wherein the binder is multivalent.

55. The binder according to any one of claims 14 to 54, wherein each polypeptide chain has the same valence and specificity.

56. The binder according to any one of claims 14 to 55, wherein each polypeptide chain has a different valence and specificity.

57. The binder according to any one of claims 14 to 56, wherein each polypeptide chain is an antibody heavy chain.

58. The binder according to any one of the preceding claims, wherein one or more antigen-binding domains are humanized.

59. The binder according to any one of claims 20 to 58, wherein X or Y is 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.

60. The binder according to any one of the preceding claims, wherein the binder is or comprises an antibody or an antigen-binding fragment thereof.

61. The binder according to any one of the preceding claims, wherein the binder is a single-domain antibody.

62. The binder according to any one of the preceding claims, wherein the binder comprises a single-domain antibody.

63. The binder according to any one of the preceding claims, wherein the binder is a bispecific antibody.

64. The binder according to claim 63, wherein the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

65. The binder according to any one of the preceding claims, wherein the binder is an antibody-like molecule.

66. The binder according to any one of the preceding claims, wherein the binder comprises a protein scaffold.

67. The binder according to any one of the preceding claims, wherein the binder comprises an immunocyte regulator.

68. The binder according to any one of the preceding claims, wherein the binder comprises: a. 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: 141; b. 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: 142; c. 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: 143; d. 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: 144; e. 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: 145; f. 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: 146; g. 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: 147; h. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 148; i. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 149; j. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 150; k. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 151; l. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 152; m. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 153; n. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 154; o. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 155; p. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 156; q. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 157; or r. An amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO:

158.

69. The binder according to claim 68, wherein the binder comprises, at the C-terminus, an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

179.

70. The binder according to claim 6, wherein the binder comprises, at the C-terminus, an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO:

178.

71. The binder according to any one of the preceding claims, wherein the binder comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 99% identical to the amino acid sequence set forth in SEQ ID NO: 159 or the amino acid sequence set forth in SEQ ID NO:

159.

72. The binder according to claim 71, wherein the binder comprises two polypeptide chains, each polypeptide chain having an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

159.

73. The binder according to any one of claims 6 to 72, wherein the amino acid substitutions, additions, or deletions are located outside the complementarity-determining regions.

74. A binder that is capable of competing with the binder as described in any one of the preceding claims.

75. The binder according to claim 74, wherein the competitive binder is an antibody or an antigen-binding fragment thereof comprising a heavy-chain variable region, the heavy-chain variable region comprising: a. The amino acid sequence of SEQ ID NO: 380; b. The amino acid sequence of SEQ ID NO: 381; c. The amino acid sequence of SEQ ID NO: 382; d. The amino acid sequence of SEQ ID NO: 383; e. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 7 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 7; f. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 14 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 14; g. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 21 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 21; h. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 28 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 28; i. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 35 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 35; j. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 42 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 42; k. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 49 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 49; l. An amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence of SEQ ID NO: 56 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence of SEQ ID NO: 56; m. 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:77 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:77; n. 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:84 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:84; o. 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:91 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:91; p. 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:98 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:98; q. 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:105 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:105; r. 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:112 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:112; s. 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:119 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:119; t. 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:126 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:126; u. 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:133 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:133; or v. 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:140 or has from one to ten amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:

140.

76. The binder according to claim 74 or 75, wherein the binder comprises one or more antigen-binding domains of an antibody that specifically binds to the PD-1 protein or a portion thereof.

77. The binder according to any one of claims 74 to 76, wherein the binder comprises one or more antigen-binding domains of an antibody that specifically binds to CD47.

78. A binder that specifically binds to cluster of differentiation 36 (CD36) or a portion thereof or a cell expressing CD36 or a portion thereof, wherein the binder is a single-domain antibody that regulates tumor lipid metabolism and / or inhibits the growth of tumor cells.

79. A binder that specifically binds to CD36 or a cell expressing CD36, PD-1 or a cell expressing PD-1, and the CD47 protein or a cell expressing CD47.

80. The binder according to any one of the preceding claims, wherein the binder is conjugated to a therapeutic moiety.

81. The binder according to any one of the preceding claims, wherein the binder is conjugated to a detectable moiety.

82. The binder according to any one of the preceding claims, wherein the binder is conjugated to a protein that allows for an extended half-life.

83. The binder according to any one of the preceding claims, wherein the binder is attached to a nanoparticle.

84. The binder according to any one of the preceding claims, wherein the binder is capable of regulating the lipid metabolism of tumor cells.

85. The binder according to any one of the preceding claims, wherein the binder has anti-tumor activity.

86. The binder according to any one of the preceding claims, wherein the binder is capable of reducing tumor growth.

87. The binder according to any one of the preceding claims, wherein the binder is capable of causing tumor regression.

88. The binder according to any one of the preceding claims, wherein CD36 is the human CD36 protein or a portion thereof.

89. The binder according to any one of the preceding claims, wherein PD-1 is the human PD-1 protein or a portion thereof.

90. The binder according to any one of the preceding claims, wherein CD47 is the human CD47 protein or a portion thereof.

91. A composition comprising the binder according to any one of the preceding claims.

92. A pharmaceutical composition comprising the binder according to any one of the preceding claims and a pharmaceutically acceptable carrier.

93. The pharmaceutical composition according to claim 92, which comprises a binder at a purity level of 80.0% to 99.9%.

94. A nucleic acid or a set of nucleic acids that encodes the binder according to any one of the preceding claims.

95. A vector comprising the nucleic acid according to claim 94.

96. A cell that expresses the binder according to any one of the preceding claims.

97. A cell that comprises the nucleic acid according to claim 94 or the vector according to claim 95.

98. A kit that comprises the binder according to any one of claims 1-90.

99. A kit that comprises the nucleic acid according to claim 94, or the vector according to claim 95, or the cell according to claim 96 or 97.

100. A method for treating or preventing a disease or disorder associated with CD36, CD36 expression, CD36 overexpression, or CD36 upregulation, comprising administering to a subject in need thereof an agent as described in any one of claims 1-90, a composition as described in claim 91, or a pharmaceutical composition as described in claim 92 or 93.

101. A method for treating cancer, comprising administering to a subject in need thereof an agent as described in any one of claims 1-90, a composition as described in claim 91, or a pharmaceutical composition as described in claim 92 or 93.

102. A method for treating cancer in a subject in need thereof, the method comprising administering a nucleic acid molecule encoding an agent as described in any one of claims 1-90 or a pharmaceutical composition comprising the same.

103. The method according to claim 101 or 102, wherein the cancer is leukemia.

104. The method according to claim 103, wherein the leukemia is acute myeloid leukemia.

105. The method according to any one of claims 101 to 104, wherein the subject in need thereof has recurrent leukemia.

106. The method according to claim 101 or 102, wherein the cancer is endometrial cancer, breast cancer (e.g., triple-negative breast cancer), liver cancer (e.g., hepatocellular carcinoma), lung cancer, prostate cancer, kidney cancer (e.g., renal clear cell carcinoma), ovarian cancer, cervical cancer, pancreatic cancer, gastric cancer, bladder cancer (e.g., urothelial carcinoma of the bladder), brain cancer, oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, colon cancer, and colorectal cancer.

107. The method according to any one of claims 101 to 106, wherein the treatment of the subject in need thereof has failed with chemotherapy and / or immune checkpoint inhibitor therapy.

108. The method according to any one of claims 101 to 107, wherein the cancer is associated with high CD36 expression.

109. The method according to any one of claims 101 to 108, wherein the cancer is associated with increased lipid metabolism.

110. The method according to any one of claims 101 to 109, wherein the cancer originates from adipose-rich tissue.

111. A method for manufacturing an agent as described in any one of claims 1-89, the method comprising transforming a cell with a vector comprising one or more nucleic acids as described in claim 93.

112. The method according to claim 111, wherein the agent is purified from the cell.

113. The method according to claim 112, wherein the purity level of the agent is from 80.0% to 99.9%.

114. The method according to any one of claims 111 to 113, further comprising conjugating or attaching the agent to a therapeutic moiety, a detectable moiety, a protein that allows for an extended half-life, or a nanoparticle.

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