Binding agents targeting CD36-expressing tumor cells

Abstract

The present disclosure generally relates to binding agents capable of targeting tumor cells and / or immune cells and their use to treat cancer. The binding agents of the present disclosure comprise one or more antigen-binding domains of a single-domain antibody capable of binding to 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 may be 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 to treat cancer. The binding agents of the present disclosure comprise one or more antigen-binding domains of a single-domain antibody capable of binding to 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 may be monospecific or multispecific and may be in monomeric or multimeric form. [Background technology]

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

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

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

[0005] CD36, also known as fatty acid translocase (FAT), FAT / CD36, SCARB3, GP88, gpIV, and gpIIIb, is a transmembrane protein that plays a key role in lipid metabolism and other cellular functions, such as inflammation, immunological recognition, and apoptosis (Wang, J. et al., Theranostic 9(17), pp. 4893-4908, 2019, the entire contents of which are incorporated herein by reference). CD36 acts as a receptor for various ligands, including apoptotic cells, thrombospondin-1 (TSP-1), and fatty acids (FAs). CD36 promotes tumor metastasis and treatment resistance by promoting lipid uptake and FA oxidation, and inhibits angiogenesis by binding to TSP-1. Chen, Y.J. et al. (Journal of Cancer 12(16), pp. 4762-4773, 2021, the entire contents of which are incorporated herein by reference). CD36 is involved in tumor cell metabolism, anti-angiogenesis, metastasis, therapy resistance and tumor immunoediting.

[0006] The amino acid sequence and structure of human CD36 were first disclosed by Armesilla, A. et al. (The Journal of Biological Chemistry, 269(29), pp. 18895-18991, 1994; GenBank accession number CAA83662.1, Uniprot accession number P16671-1), the entire contents of which are incorporated herein by reference. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] PCT / CA2020 / 051753 [Patent Document 2] WO2022 / 011457 [Patent Document 3] PCT / CA2021 / 050951 [Patent Document 4] U.S. Patent No. 8,326,547 [Patent Document 5] WO2021 / 119832A1 [License 6] U.S. Patent and Trademark Office Publication No. 2019 / 0010242 [License 7] U.S. Patent and Trademark Office Publication No. 20110076275 [License 8] PCT / CA2022 / 050442 [Non-licensed literature]

[0008] [Non-licensed Document 1] De Vliegerら, Antibodies 8(1), pages 1~22, 2019 [Non-licensed Document 2] Wang, Jら, Theranostic 9(17), pages 4893~4908, 2019 [Non-licensed Document 3] Chen, YJら, Journal of Cancer 12(16), pages 4762~4773, 2021 [Non-licensed Document 4] Armesilla, Aら, The Journal of Biological Chemistry, 269(29), pages 18895~18991, 1994 [Non-licensed Document 5] Vincke C. (J. Biol Chem. 2009, 284(5):3273~3284 pages) [Non-licensed Document 6] Kovalenko OVら(J Biol Chem. 2013, 288:17408~17419 pages) [Non-licensed Document 7] Saunders KO (Front. Immunol. 10:1296 pages, 2019) [Non-licensed Document 8] List of Mutations Antibodies (Basel). 2020-11-17;9(4):64 [Non-licensed Document 9] RJ Biol. Chem. 2001, 276, 6591~6604. [Non-Patent Document 10] Proc. Natl. Acad. Sci. USA 2006, 103, 4005~401 [Non-Patent Document 11] Mol. Cancer Ther. 2008, 7, 2517~2527. [Non-Patent Document 12] Cancer Res. 2007, 67, 8882~8890. [Non-Patent Document 13] Breast Cancer Res. 2011, 13, R123. [Non-Patent Document 14] Immunol. Cell Biol. 2020, 98, 287~304. [Non-Patent Document 15] Biotechnol. Bioeng. 2004, 87, 614~622. [Non-Patent Document 16] Chen X et al. (Adv Drug Deliv Rev. 2013;65(10):1357-1369) [Non-Patent Document 17] Angal, S. et al., Mol Immunol 30, 105-108, 1993 [Non-Patent Document 18] Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250. [Non-Patent Document 19] Zhao J. et al., Antibodies 2018, 7, 22;doi 10.3390 / antib7030022 Summary of the Invention [Problem to be solved by the invention]

[0009] The applicant has developed novel binding agents that target tumor cells and / or immune cells for the treatment of cancer. [Means for solving the problem]

[0010] The present disclosure relates generally to binding agents that comprise one or more antigen-binding domains and that have the ability to target, inter alia, tumor cells and / or immune cells.

[0011] In some embodiments, the binding agents of the present disclosure bind to 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 a CD36 protein, eg, human CD36 or a portion thereof.

[0013] In some cases, the anti-CD36 single domain antibodies of the present disclosure may block the binding between CD36 and a CD36 ligand.

[0014] In some cases, the anti-CD36 single domain antibodies of the present disclosure may modulate lipid metabolism.

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

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

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

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

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

[0020] The binding function of an sdAb is conferred by a single polypeptide chain, i.e., the heavy chain variable domain. This unique property provides flexibility in formatting. For example, multiple sdAbs or sdAb antigen-binding domains may be fused into a single polypeptide chain to confer multivalency and / or multispecificity. Additionally, multiple polypeptide chains may be assembled to increase the diversity or avidity of the interaction with CD36. Furthermore, an sdAb antigen-binding domain may be fused to any type of polypeptide chain, including, without limitation, fusions with the heavy and / or light chains of a native antibody or antigen-binding fragment thereof, protein scaffolds, immune cell modulators, etc.

[0021] As such, the present disclosure relates not only to the single domain antibodies disclosed herein, but more generally to binding agents comprising one or more antigen-binding domains of the single domain antibodies disclosed herein.

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

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

[0024] In some instances, the binding agent is capable of binding to CD36 and programmed cell death protein 1 (PD-1). Thus, in some embodiments, the binding agent comprises 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 has the ability to bind to CD36 and cluster of differentiation 47 (CD47). Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain that binds to CD36 and at least one antigen-binding domain that binds to CD47.

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

[0027] In some aspects, the binding agents of the disclosure may bind to human CD36 protein, human PD-1 protein, and / or human CD47 protein and / or cells expressing the human CD36 protein, human PD-1, and / or human CD47.

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

[0029] Thus, a binding agent 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. In accordance with the present disclosure, a binding agent may also comprise additional antigen-binding domains.

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

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

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

[0033] The binding agents of the present disclosure may have the ability to reduce tumor growth and / or cause tumor regression. In some embodiments, the binding agents have anti-tumor activity. In some embodiments, the binding agents can recruit or redirect immune cells.

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

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

[0036] In still other cases, the binding agents of the present disclosure may inhibit fatty acid uptake by tumors.

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

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

[0039] In still other cases, the binding agents of the present disclosure may be used to reduce the resistance of tumor cells to chemotherapeutic agents and / or immune checkpoint inhibitor therapy.

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

[0041] In an exemplary embodiment, the antigen-binding domain is from one or more single-domain antibodies that have the ability to specifically bind to CD36 and have anti-tumor activity.

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

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

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

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

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

[0047] In yet another exemplary embodiment, the antigen-binding domain is from one or more anti-CD36 single domain antibodies that do not significantly couple to platelet activation.

[0048] The binding agents of the present disclosure may also comprise one or more antigen-binding domains of an antibody capable of specifically binding to an immune checkpoint protein.

[0049] For example, a binding agent of the present disclosure may comprise one or more antigen-binding domains of a single-domain antibody capable of specifically binding to and inhibiting PD-1. In some embodiments, a binding agent comprises more than one antigen-binding domain, some of which 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 selected for its ability to block 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 selected for its ability to block PD-L1 and / or PD-L2 interactions.

[0052] In some embodiments, the antigen-binding domain is from an anti-PD-1 single domain antibody selected for its ability to restore T cell effector function.

[0053] In some embodiments, the binding agent comprises one or more antigen-binding domains of an antibody capable of specifically binding to a protein expressed on the surface of an immune cell.

[0054] For example, the antigen-binding domain is from a single domain antibody that has the ability to specifically bind to and inhibit CD47. In some embodiments, the binding agent comprises more than one antigen-binding domain, and some of the antigen-binding domains may be from the same or different single domain antibodies that have the ability to specifically bind to and inhibit CD47.

[0055] In some embodiments, the antigen-binding domain is from an anti-CD47 single domain antibody selected for its ability to block 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 selected for its ability to block 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 selected for its ability to recruit T cells, hi some embodiments, the antigen-binding domain is from an anti-CD47 single domain antibody selected for its ability to enhance macrophage function.

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

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

[0059] In some embodiments, a binding agent may comprise at least one antigen-binding domain 1 (ABD1) and at least one other antigen-binding domain. For example, a binding agent may comprise one or more antigen-binding domains 1 (ABD1) and one or more antigen-binding domains of one or more antibodies.

[0060] In some cases, the binding agent may comprise at least one antigen-binding domain 1 (ABD1) and may also bind to an immune cell. Thus, in some embodiments, the binding agent comprises at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain that binds to an immune cell.

[0061] In some embodiments, the antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1 is antigen-binding domain 2 (ABD2) as described herein. In some embodiments, the antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1 is not limited to antigen-binding domain 2 (ABD2).

[0062] Thus, in some embodiments, the binding agent may comprise at least one antigen-binding domain 2 (ABD2). According to the present disclosure, ABD2 comprises at least the CDRH3 amino acid sequence of an anti-PD-1 single domain antibody disclosed herein. Also according to the present disclosure, ABD2 comprises the CDRH3 amino acid sequence and at least the CDRH1 and / or CDRH2 amino acid sequence of an anti-PD-1 single domain antibody disclosed herein. In some embodiments, ABD2 comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of an anti-PD-1 single domain antibody disclosed herein. In some 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 an anti-PD-1 single domain antibody disclosed herein.

[0063] In some embodiments, the binding agent may comprise at least one antigen binding domain 2 (ABD2) and at least one other antigen binding domain.

[0064] In some embodiments, the antigen-binding domain capable of binding to CD47 or a cell expressing CD47 is antigen-binding domain 3 (ABD3) as described herein. In some embodiments, the antigen-binding domain capable of binding to CD47 or a cell expressing CD47 is not limited to ABD3.

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

[0066] In some embodiments, the binding agent may comprise at least one antigen binding domain 3 (ABD3) and at least one other antigen binding domain.

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

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

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

[0070] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains comprises: antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) and / or Antigen-binding domain 3 (ABD3) is selected from.

[0071] In some embodiments, a binding agent may comprise an antigen-binding domain that targets an epitope other than that recognized by ABD1, ABD2 and ABD3, and thus may comprise an antigen-binding domain with a specificity that is the same as or different from that of ABD1, ABD2 and / or ABD3.

[0072] In some embodiments, the binding agent may comprise more than one antigen-binding domain.

[0073] For example, in some embodiments, the binding agent may comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, seven or more antigen-binding domains, eight or more antigen-binding domains, nine or more antigen-binding domains, ten or more antigen-binding domains.

[0074] In some embodiments, the binding agent may comprise 1 to 12 antigen-binding domains.

[0075] In some embodiments, the binding agents of the present disclosure may be monospecific.

[0076] In some embodiments, binding agents of the present disclosure may be multispecific.

[0077] In some embodiments, the binding agents of the present disclosure may be monovalent.

[0078] In some embodiments, binding agents of the present disclosure may be multivalent.

[0079] In some embodiments, the binders of the present disclosure may be in monomeric form.

[0080] In some embodiments, binding agents of the present disclosure may be in the form of dimers or higher order forms such as trimers, tetramers, pentamers, and the like (eg, multimers).

[0081] In some cases, the antigen-binding domain of the binding agent originates from a heavy chain antibody. In some cases, the heavy chain antibody may be obtained by immunization of a camelid or a transgenic animal.

[0082] The binding agents of the present disclosure are essentially polypeptidic. In some embodiments, the binding agents of the present disclosure consist essentially of one, two, or more than two polypeptide chains. However, the binding agents may also include non-polypeptidic moieties, such as small molecules, sugars, radiolabels, etc.

[0083] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in an N-terminal to C-terminal manner, an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD-1, and an antigen-binding domain that binds CD47.

[0084] In some embodiments, the antigen binding domains are on one or more polypeptide chains.

[0085] In some embodiments, the antigen binding domains are on the same polypeptide chain.

[0086] In some embodiments, the binding agent comprises a single polypeptide chain.

[0087] In some embodiments, the binding agent comprises two polypeptide chains, and thus may be in the form of a dimer.

[0088] In some embodiments, a binding agent comprises more than two polypeptide chains, e.g., three or more polypeptide chains, four or more polypeptide chains, five or more polypeptide chains, six or more polypeptide chains, seven or more polypeptide chains, eight or more polypeptide chains, nine or more polypeptide chains, ten or more polypeptide chains. Thus, a binding agent may be in the form of a multimer. For example, a binding agent comprising three polypeptide chains is referred to herein as a trimer, a binding agent comprising four polypeptide chains is referred to herein as a tetramer, etc.

[0089] In some embodiments, the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises one or more antigen-binding domains.

[0090] It should be understood herein that the polypeptide chains of the present disclosure may alternatively be expressed in tandem.

[0091] In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen-binding domain.

[0092] In some embodiments, the two polypeptide chains are capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen-binding domain.

[0093] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1).

[0094] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 2 (ABD2).

[0095] In some embodiments, each polypeptide chain comprises an identical antigen-binding domain 3 (ABD3).

[0096] In some exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1) and an identical antigen-binding domain 2 (ABD2).

[0097] In another exemplary embodiment, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1) and an identical antigen-binding domain 3 (ABD3).

[0098] In still further exemplary embodiments, the binding agent comprises at least two polypeptide chains that assemble to form a dimer, and each polypeptide chain comprises an identical antigen-binding domain 1 (ABD1), an identical antigen-binding domain 2 (ABD2), and an identical antigen-binding domain 3 (ABD3).

[0099] 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 in each of the two polypeptide chains.

[0100] In some cases, antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), and / or antigen-binding domain 3 (ABD3) may occupy different positions in each of the two polypeptide chains.

[0101] In some embodiments, each polypeptide chain of the binding agent is the same.

[0102] In some embodiments, each polypeptide chain of the binding agent is different.

[0103] In some embodiments, the two polypeptide chains are capable of assembling to form a homodimer.

[0104] In some embodiments, the two polypeptide chains are capable of assembling to form a heterodimer.

[0105] In some embodiments, the antigen binding domains are separated by an amino acid sequence.

[0106] In some embodiments, the amino acid sequence is a linker.

[0107] Binding agents of the disclosure include, for example, an antigen-binding domain disclosed herein, a single polypeptide chain disclosed herein, a dimer of polypeptide chains disclosed herein, or a multimer of polypeptide chains disclosed herein.

[0108] Thus, binding agents of the present disclosure may have the format of antibodies and antigen-binding fragments thereof, antibody-like molecules (Fc, CH3 fusions, etc.), protein scaffolds and fusions with immune cell modulating agents, etc.

[0109] Advantageously, the binders of the present disclosure may have a format as disclosed herein as Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII, etc. in PCT / CA2020 / 051753, filed December 18, 2020, and published June 24, 2021 under WO2021 / 119832 (the entire contents of which are incorporated herein by reference), or as Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII.

[0110] In some embodiments, the binding agent is comprised of a polypeptide chain comprising one or more antigen-binding domains and a dimerization domain that allows at least two polypeptide chains to form a dimer.

[0111] In some embodiments, the binding agents of the present disclosure have the structure of Formula I in N-terminal to C-terminal fashion: X-[(Ab a )-(L b )]m -(DD)-[(L c )-(Ab d )] n -Y and may comprise one or more polypeptide chains each independently comprising the amino acid sequence of m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain.

[0112] 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, Ab a or Ab d At least one of them is antigen-binding domain 2 (ABD2).

[0113] 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, Ab a or Ab d At least one of them is antigen-binding domain 3 (ABD3).

[0114] 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, Ab a or Ab d At least one of them is antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

[0115] In some embodiments, the polypeptide chains comprise two or more antigen-binding domains, three or more antigen-binding domains, four or more antigen-binding domains, five or more antigen-binding domains, six or more antigen-binding domains, etc.

[0116] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen-binding domains.

[0117] In some embodiments, the binding agent comprises one polypeptide chain.

[0118] In some embodiments, the binding agent comprises two polypeptide chains, three polypeptide chains, four polypeptide chains, five polypeptide chains, six polypeptide chains, seven polypeptide chains, eight polypeptide chains, nine polypeptide chains, ten polypeptide chains, or more than ten polypeptide chains.

[0119] In some embodiments, the polypeptide chains may be covalently linked.

[0120] In some embodiments, the polypeptide chains may be non-covalently linked.

[0121] In some embodiments, the polypeptide chains may associate through electrostatic interactions.

[0122] In some embodiments, where m is an integer greater than or equal to 2, [(Ab a )-(L b )] units may be the same or different.

[0123] In some embodiments, where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units may be the same or different.

[0124] In some embodiments where m is an integer greater than or equal to 2, each Ab a are the same or different.

[0125] In some embodiments where n is an integer greater than or equal to 2, each Ab d are the same or different.

[0126] In some embodiments, Ab a represents ABD1, ABD2 or ABD3.

[0127] In some embodiments, Ab d represents ABD1, ABD2 or ABD3.

[0128] In some embodiments, Ab d represents ABD1, ABD2 or ABD3.

[0129] In embodiments, one or more polypeptide chains further comprise a hinge region of an antibody or antigen-binding fragment thereof. For example, in some embodiments, L b is the hinge region of an antibody or antigen-binding fragment thereof.

[0130] In some embodiments, the hinge region is a native hinge region from an IgG1, IgG2, IgG3, or IgG4.

[0131] In some embodiments, the hinge region is a mutated hinge region having at least 70% identity to a native hinge region from IgG1, IgG2, IgG3, or IgG4.

[0132] In some embodiments, each of the one or more linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0133] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0134] 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.

[0135] In some embodiments, the rigid linker comprises multiple PA repeats as described herein.

[0136] In some embodiments, the helical linker comprises one or more EAAAK units as described herein.

[0137] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0138] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0139] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0140] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by formula V: X-(Ab a1 )-(L b2)-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0141] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0142] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has the formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0143] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has a structure represented by Formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Abd3 )-Y (Formula VIII).

[0144] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently comprises an antigen-binding domain.

[0145] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 each independently represents an antigen-binding domain.

[0146] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 At least one of them is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0147] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , or Ab d3 At least one of the domains is not antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3).

[0148] In some embodiments, L b1 comprises one or more linkers and / or a hinge region of an antibody or antigen-binding fragment thereof. In some embodiments, the hinge region is a native hinge region as disclosed herein. In other embodiments, the hinge region is a mutant hinge region as disclosed herein.

[0149] In some embodiments, L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers as disclosed herein.

[0150] In some embodiments, the polypeptide chain comprises an antigen binding domain ABD1 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0151] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD2 as disclosed herein and an antigen binding domain that binds to a tumor cell.

[0152] In some embodiments, the polypeptide chain comprises the antigen binding domain ABD3 and an antigen binding domain that binds to a tumor cell as disclosed herein.

[0153] In some embodiments, the polypeptide chain comprises an antigen binding domain ABD3 as disclosed herein and an antigen binding domain that binds to an immune cell.

[0154] In some embodiments, the polypeptide chain comprises at least one antigen-binding domain selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0155] In some embodiments, the polypeptide chain comprises at least two antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0156] In some embodiments, the polypeptide chain comprises at least three antigen-binding domains selected from ABD1, ABD2, and ABD3 as disclosed herein.

[0157] In some embodiments, the dimerization domain comprises an immunoglobulin dimerization domain. Other dimerization domains known to those of skill in the art are contemplated herein, including leucine zippers and the like.

[0158] In some embodiments, the dimerization domain comprises an IgG, IgM, IgA, IgD, or IgE dimerization domain (from human or animal IgG, IgM, IgA, IgD, or IgE).

[0159] In some embodiments, the dimerization domain comprises a CH3 domain of an antibody. The dimerization domain may also comprise a CH2 domain of an antibody.

[0160] In some exemplary embodiments, the dimerization domain comprises a native CH3 domain.

[0161] In some exemplary embodiments, the dimerization domain comprises a mutated CH3 domain.

[0162] In some embodiments, the dimerization domain comprises a native CH2 and a native CH3 domain.

[0163] In some embodiments, the dimerization domain comprises a native CH2 and a mutated CH3 domain.

[0164] In some embodiments, the dimerization domain comprises a mutated CH2 and a mutated CH3 domain.

[0165] In some embodiments, the dimerization domain comprises a mutated CH2 and a native CH3 domain.

[0166] In some embodiments, the dimerization domain does not include a CH1 domain.

[0167] In some embodiments, the dimerization domain does not comprise a CH4 domain.

[0168] In some embodiments, the dimerization domain comprises an Fc region of an antibody or portion thereof.

[0169] In some embodiments, the polypeptide chain comprises at least two or at least three antigen-binding domains and a dimerization domain that allows assembly of the two polypeptide chains to form a multivalent and / or multispecific binding agent.

[0170] In embodiments, the antigen binding domain comprises or consists of the antigen binding domain of a single domain antibody (sdAb).

[0171] In embodiments, the antigen-binding domain comprises a heavy chain variable region (VH or VHH).

[0172] In some embodiments, the VHH is derived from a human, mouse, rat, etc.

[0173] In some embodiments, the VHH is from a transgenic mouse or rat capable of expressing a camelized mouse or rat VHH, a VHH from another species (such as a human), or a camelized VHH from another species (such as a camelized human VHH).

[0174] In some embodiments, the binding agent does not comprise a light chain variable region (VL or VLL).

[0175] In embodiments, the binding agent comprises a light chain variable region (VL or VLL).

[0176] In embodiments, the antigen-binding domain is a single-chain variable fragment (ScFv).

[0177] In embodiments, the antigen-binding domain is a V NAR It is from a fragment.

[0178] In other embodiments, the antigen-binding domain of the polypeptide chain is a single domain antibody (sdAb), a heavy chain variable region (VH or VHH), a light chain variable region (VL or VLL), a single chain variable fragment (ScFv), and / or a V NAR The fragments may include any combination of antigen-binding domains.

[0179] In some embodiments, the sdAb or VHH is from a camelid antibody.

[0180] In embodiments, the camelid antibody is from a dromedary, camel, llama, alpaca, etc.

[0181] In other embodiments, the sdAb or VHH is from a cartilaginous fish antibody.

[0182] In an embodiment, the cartilaginous fish antibody is a shark antibody.

[0183] In some embodiments, each antigen-binding domain specifically binds to a different epitope.

[0184] In other embodiments, each antigen-binding domain specifically binds to a different antigen.

[0185] In yet other embodiments, each antigen-binding domain specifically binds to a different protein.

[0186] It should be understood herein that a given antigen-binding domain may bind to epitopes present in different proteins. Thus, in some embodiments, an antigen-binding domain, or a binding agent comprising the same, may bind to more than one protein. In some embodiments, an antigen-binding domain or a binding agent may have affinity for more than one protein.

[0187] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof.

[0188] In other embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof.

[0189] In other embodiments, the binding agent comprises the antigen-binding domain of an antibody or antigen-binding fragment thereof.

[0190] In some embodiments, the binding agent is a single domain antibody.

[0191] In other embodiments, the binding agent comprises a single domain antibody.

[0192] In other embodiments, the binding agent comprises the antigen-binding domain of a single domain antibody.

[0193] In some embodiments, the binding agent is a single chain antibody (eg, an scFv).

[0194] In other embodiments, the binding agent comprises a single chain antibody (eg, an scFv).

[0195] In some embodiments, the binding agent comprises at least two polypeptide chains capable of forming a dimer.

[0196] In some embodiments, the two polypeptide chains are the same.

[0197] In some embodiments, the two polypeptide chains are different.

[0198] In some embodiments, the polypeptide chains are the same and the binding agent is a homodimer.

[0199] In some embodiments, the polypeptide chains are different and the binding agent is a heterodimer.

[0200] In some embodiments, the binding agent is multispecific.

[0201] In some embodiments, the binding agents are bispecific, trispecific, or tetraspecific.

[0202] In some embodiments, the binding agent comprises one or more polypeptide chains that are multispecific.

[0203] In some embodiments, the binding agent comprises one or more polypeptide chains that are bispecific, trispecific, or tetraspecific.

[0204] In some embodiments, one or more polypeptide chains have the same valency and specificity.

[0205] In some embodiments, one or more polypeptide chains have different valencies and specificities.

[0206] In some embodiments, each of the one or more polypeptide chains is an antibody heavy chain.

[0207] In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof.

[0208] In some embodiments, the binding agent is a bispecific antibody.

[0209] In some embodiments, the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

[0210] In some embodiments, one or more of the antigen-binding domains are humanized.

[0211] In some embodiments, one or more of the antigen-binding domains are partially humanized.

[0212] In some embodiments, the antigen-binding domain comprises one or more human frameworks.

[0213] In some embodiments, X or Y are independently selected from a linker, a cytokine, a chemokine, a tag, a masking domain, a phage coat protein (pIII, pVI, pV, pVII, or pIX), an antigen-binding domain, or a combination thereof.

[0214] In some embodiments, the polypeptide chain is conjugated to a therapeutic moiety.

[0215] In some embodiments, the polypeptide chain is conjugated to a detectable moiety.

[0216] In some embodiments, the polypeptide chain is conjugated to a protein that allows for an extended half-life.

[0217] In some embodiments, the polypeptide chains are attached to nanoparticles.

[0218] Other aspects and embodiments of the present disclosure relate to compositions comprising at least one of the binding agents disclosed herein.

[0219] In some embodiments, the composition comprises monomers, dimers, and mixtures thereof.

[0220] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as dimers in the composition.

[0221] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as homodimers in the composition.

[0222] In some embodiments, greater than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the first and second polypeptide chains are present as heterodimers in the composition.

[0223] Other aspects and embodiments of the present disclosure relate to pharmaceutical compositions comprising a binding agent disclosed herein and a pharmaceutically acceptable carrier.

[0224] Still other aspects and embodiments of the present disclosure relate to nucleic acids or sets of nucleic acids that encode the polypeptide chains and / or binding agents disclosed herein.

[0225] The nucleic acid may be in the form of a DNA segment as disclosed herein.

[0226] 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.

[0227] Further aspects and embodiments of the present disclosure relate to cells expressing the polypeptide chains or binding agents disclosed herein.

[0228] Additional aspects and embodiments of the present disclosure relate to cells comprising the nucleic acids or vectors disclosed herein.

[0229] Further aspects and embodiments of the present disclosure relate to kits that include the binding agents disclosed herein.

[0230] Still further aspects and embodiments of the present disclosure relate to kits comprising the nucleic acids disclosed herein.

[0231] Still further aspects and embodiments of the present disclosure relate to kits comprising the vectors disclosed herein.

[0232] Still further aspects and embodiments of the present disclosure relate to kits comprising the cells disclosed herein.

[0233] 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.

[0234] Exemplary embodiments of the disease or disorder include cancer, metabolic disease, such as atherosclerosis, diabetes, non-alcoholic fatty liver disease, thrombosis, or inflammation.

[0235] In additional aspects and embodiments, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a binding agent disclosed herein.

[0236] In further aspects and embodiments, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a composition disclosed herein.

[0237] In further aspects and embodiments, the present disclosure relates to a method of treating cancer comprising administering to a subject in need thereof a pharmaceutical composition disclosed herein.

[0238] In some embodiments, the cancer is metastatic.

[0239] In some embodiments, the cancer is leukemia, including without limitation acute myeloid leukemia.

[0240] In some embodiments, the cancer is a solid tumor.

[0241] In some 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., bladder urothelial carcinoma), brain cancer, oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, colon cancer, and colorectal cancer.

[0242] In some embodiments, the cancer originates from fat-rich (adipose) tissue.

[0243] In some 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.

[0244] For example, the binding agents may be used to reduce and / or prevent tumor growth in a subject in need thereof. In other examples, the binding agents may be used to reduce and / or prevent tumor metastasis. In yet other examples, the binding agents may be used to reduce resistance to chemotherapy and / or immune checkpoint inhibitor therapy.

[0245] Thus, the binding agent may be administered to a subject with a 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., bladder urothelial carcinoma), oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, and colorectal cancer.

[0246] In some embodiments, the binding agent is administered to a subject who has failed previous treatment with chemotherapy (chemotherapeutic agent) and / or immune checkpoint therapy.

[0247] In some embodiments, the binding agent is administered to a subject with relapsed leukemia. In some embodiments, the binding agent is administered to a subject with relapsed acute myeloid leukemia. In some embodiments, the binding agent is administered to a subject with cancer that has increased lipid metabolism, such as, without limitation, increased fatty acid uptake and / or increased fatty acid oxidation.

[0248] In some embodiments, the binding agent is administered to a subject with cancer to reduce and / or prevent tumor growth.

[0249] In some embodiments, the binding agent is administered to a subject with cancer to reduce and / or prevent tumor metastasis.

[0250] In some embodiments, the binding agent is administered to a subject with cancer to reduce resistance to chemotherapy and / or immune checkpoint inhibitor therapy.

[0251] In other aspects and embodiments, the present disclosure relates to methods of producing the binding agents disclosed herein, comprising transforming a cell with one or more vectors comprising a nucleic acid disclosed herein.

[0252] In some embodiments, the method may further comprise isolating and / or purifying the binding agent from impurities.

[0253] In other embodiments, the method may further comprise isolating and / or purifying the heterodimer from the monomer and / or homodimer.

[0254] In other embodiments, the method may further comprise isolating and / or purifying the homodimer from the monomer and / or heterodimer.

[0255] In some embodiments, the method further comprises conjugating the binding agent to a therapeutic moiety, a detectable moiety, or a protein that allows for extended half-life, or to a nanoparticle.

[0256] Further scope, applicability, and advantages of the present disclosure will become apparent from the non-limiting detailed description given below. It should be understood, however, that this detailed description, while indicating exemplary embodiments of the present disclosure, is given by way of example only, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0257] [Figure 1] Bar graph illustrating binding of CD36 antibodies to human CD36 protein by ELISA at 10 ug / ml. [Figure 2] Bar graph showing binding of CD36 antibody to OCI-AML3 cells (CD36+) and OCI-AML4 cells (CD36-). [Figure 3] Graph illustrating the inhibitory effect of CD36 antibody on fatty acid oxidation in OCI-AML3 cells at a concentration of 350 nM. [Figure 4]Graph showing the effect of anti-CD36 antibody on platelet activation at 50 ug / ml. [Figure 5] Schematic diagram illustrating the construction of monospecific single domain antibodies in Table 6 (FIG. 5A) and multivalent multispecific binders in Table 7 (FIG. 5B). [Figure 6] Graph showing tumor regression of OCI-AML3 xenograft tumors in the I / O model by anti-CD36 KisoBody KF061. [Figure 7A] Alignment of anti-CD36 VHHs in Table 3 with IMGT CDRs highlighted. [Figure 7B] Alignment of anti-CD36 VHHs in Table 3 with Kabat CDRs highlighted. [Figure 8A] Alignment of selected anti-CD36 VHHs in Table 3. [Figure 8B] Alignment of selected anti-CD36 VHHs in Table 3. [Figure 8C] Alignment of selected anti-CD36 VHHs in Table 3. [Figure 8D] Alignment of selected anti-CD36 VHHs in Table 3. [Figure 9] Alignment of selected anti-PD-1 VHHs in Table 4. DETAILED DESCRIPTION OF THE INVENTION

[0258] definition Unless otherwise indicated, the amino acid numbering indicated for the dimerization domain is according to the EU numbering system.

[0259] The use of the terms "a," "an," and "the" and similar reference words in the context of describing embodiments (particularly in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0260] Unless otherwise stated or apparent from the context, as used herein, the term "or" is understood to be inclusive, covering both "or" and "and."

[0261] The term "and / or", when used herein, should be interpreted as an exclusive disclosure of each of the specified features or components with or without the other.

[0262] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise stated. The term "consisting of" should be construed as closed-ended.

[0263] The term "treatment," for purposes of this disclosure, refers to both therapeutic treatment and prophylactic or tumor preventative measures. Subjects in need of treatment include those who already have cancer, as well as those who are predisposed to having cancer or whose cancer is to be managed.

[0264] As used herein, the term "CD36" refers to a CD36 protein or a polypeptide comprising a CD36 amino acid sequence. The term "CD36" encompasses the human CD36 protein, the CD36 antigen, and any protein having at least 80% identity with the human CD36 protein.

[0265] The term "anti-CD36 antibody" refers to an antibody of any format that binds to the CD36 protein, CD36 antigen, or a portion thereof, including, without limitation, an "anti-CD36 single domain antibody."

[0266] As used herein, the term "PD-1" refers to the PD-1 protein or a polypeptide containing the PD-1 amino acid sequence. The term "PD-1" encompasses the human PD-1 protein, the PD-1 antigen, and any protein having at least 80% identity to the human PD-1 protein.

[0267] The term "anti-PD-1 antibody" refers to an antibody of any format that binds to the PD-1 protein, PD-1 antigen, or a portion thereof, including, without limitation, an "anti-PD-17 single domain antibody."

[0268] As used herein, the term "CD47" refers to the CD47 protein or a polypeptide comprising the CD47 amino acid sequence. The term "CD47" encompasses the human CD47 protein, the CD47 antigen, and any protein having at least 80% identity with the human CD47 protein.

[0269] The term "anti-CD47 antibody" refers to an antibody of any format that binds to the CD47 protein, CD47 antigen, or a portion thereof, including, without limitation, an "anti-CD47 single domain antibody."

[0270] The term "antibody" is used in the broadest sense and encompasses any immunoglobulin that binds to a specific antigen, a variety of antibody formats and structures, including monoclonal antibodies, polyclonal antibodies, bivalent antibodies, monovalent antibodies, diabodies, multispecific (multi-specific) antibodies, conventional antibodies, single domain antibodies, heavy chain-only antibodies, nanobodies, full-length antibodies, humanized antibodies, chimeric antibodies, and any antigen-binding fragment that exhibits the desired antigen-binding activity. Antibodies can be naturally occurring (native) or resulting from recombinant technology.

[0271] The term "subject in need," as used herein, refers to a subject having or suspected of having a disorder or disease associated with CD36, CD36 expression, CD36 overexpression, or CD36 upregulation, which may benefit from treatment with a binding agent that targets CD36. A "subject in need" includes a subject having or suspected of having cancer.

[0272] As used herein, the expressions "single domain antibody" and "heavy chain-only antibody" are used interchangeably. The term "single domain antibody" includes naturally occurring single domain antibodies as well as single domain antibodies in which the naturally occurring constant region has been replaced by another dimerization domain, such as the constant region of a human immunoglobulin (e.g., IgG), and variants such as humanized or chimeric single domain antibodies.

[0273] "Naturally occurring single domain antibodies" include antibodies produced by camelids (camelid antibodies) or sharks. "Naturally occurring single domain antibodies" also encompass antibodies produced by transgenic animals engineered to express heavy chain-only antibodies. Exemplary embodiments of transgenic animals are provided in International Application No. PCT / CA2021 / 050951, filed July 21, 2021, and published January 20, 2022 under the number WO2022 / 011457, the entire contents of which are incorporated herein by reference. While naturally occurring single domain antibodies typically do not include light chains, the present disclosure does not exclude single domain antibodies that include light chains. The terms "naturally occurring antibody" or "naturally occurring single domain antibody" should be understood herein to generally refer to antibodies or single domain antibodies isolated and / or purified from animals by immunization. The term "naturally occurring" is not intended to encompass products that exist in nature without human or instrumental intervention.

[0274] The term "antigen-binding domain" relates to the portion of an antibody involved in antigen binding, including, for example, one or more CDRs, one or more framework regions (FRs), or the entire variable region. The term "antigen-binding domain", in the context of that single domain antibody, relates to the portion of a single domain antibody involved in antigen binding, including, for example, one or more of CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more framework regions FR1, FR2, FR3, FR4, or the entire variable region (VH or VHH). The term "antigen-binding domain", in the context of that native antibody, relates to the portion of a native antibody involved in antigen binding, including, for example, one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light or heavy chain framework regions FR1, FR2, FR3, FR4, or the entire variable region or regions (heavy chain variable domain (VH) and / or light chain variable domain (VL)).

[0275] The term "antigen-binding fragment", when used in the expressions "antibody or antigen-binding fragment thereof" or "single-domain antibody or antigen-binding fragment thereof", refers to a fragment of an antibody or single antibody that includes the antigen-binding domain and may or may not incorporate other portions of the antibody or single-domain antibody, such as amino acid residues of the hinge region, amino acid residues of the constant region, portions of the Fc region, etc. Regardless of structure, an antigen-binding fragment binds to the same antigen that is recognized by the intact antibody (e.g., a single-domain antibody).

[0276] The term "binding agent" refers to a molecule that comprises a polypeptidic portion (e.g., a polypeptide chain) and that has the ability to specifically bind to an antigen, or that comprises a domain, such as an antigen-binding domain, that has the ability to specifically bind to an antigen.

[0277] As used herein, the term "humanized" means that the antigen-binding domain contains human antibody amino acid residues. A "humanized antigen-binding domain" generally has CDRs derived from a non-human animal antibody, FR amino acid residues derived from a human antibody, and, if 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."

[0278] As used herein, the term "fully humanized" means that the amino acid sequences of all framework regions of a given antigen-binding domain are identical to those of a human antibody, e.g., 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, if applicable, constant regions derived from a human antibody. In some cases, a "fully humanized antigen-binding domain" may also include CDR amino acid residues derived from a human antibody.

[0279] As used herein, the term "partially humanized" means that the amino acid sequence of one or more or all of the framework regions of a given antigen-binding domain is not completely identical to that of a human antibody, e.g., a germline human antibody variable region. The term "partially humanized" also applies to antigen-binding domains comprising human framework regions containing backmutations that reintroduce one or more 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, if applicable, constant regions 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.

[0280] The terms "specific binding" or "specifically binds" as used herein refer 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. ≦10 -6 M (e.g., ≦5×10 -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M or ≦10 -9 M, and ≦10 -6 (any value of K) D The value can indicate specific binding between a binding agent (eg, an antibody, eg, a single domain antibody) and CD36 (eg, human CD36).

[0281] As used herein, the term "affinity" refers to the strength of the non-covalent interactions between a binding agent or its antigen-binding domain and an antigen. Affinity can be measured, for example, by the K D value, i.e., the ratio of the dissociation rate to the association rate (k off / k on or k ass / k diss Affinity may be determined by any conventional method known in the art, including, but not limited to, surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (e.g., FACS). Antibodies generally have a affinity of ≦10 -6 M (e.g., ≦5×10 -7 M, ≤ 2 × 10 -7 M, ≤10 -7 M, ≤ 5 × 10 -8 M, ≤ 2 × 10 -8 M, ≤10 -8 M, ≤ 5 × 10-9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M or ≦10 -9 M, and ≦10 -6 (any value of K) D Preferably, the antibody has a densitometric value in the nanomolar range or lower (e.g., ≦9×10 -9 M, ≤8 × 10 -9 M, ≤7 × 10 -9 M, ≤6 × 10 -9 M, ≤ 5 × 10 -9 M, ≤ 4 × 10 -9 M, ≤ 3 × 10 -9 M, ≤ 2 × 10 -9 M, ≤ 1 × 10 -9 M or lower, e.g., ≦1×10 -10 M, ≤ 1 × 10 -11 M, ≤ 1 × 10 -12 M)K D Even more preferably, the antibody has a mAb value in the picomolar range or lower (e.g., ≦9×10 -12 M, ≤8 × 10 -12 M, ≤7 × 10 -12 M, ≤6 × 10 -12 M, ≤ 5 × 10 -12 M, ≤ 4 × 10 -12 M, ≤ 3 × 10 -12 M, ≤ 2 × 10 -12 M, ≤ 1 × 10 -12 M or lower)K D It has a value.

[0282] As used herein, the terms "competing for binding," "compete for binding," "compete(s) with," or "competing with" refer to the ability of a first antibody or antigen-binding fragment thereof (e.g., a single domain antibody or antigen-binding fragment thereof) to inhibit, to any detectable extent, the binding interaction between CD36 and a second antibody to the same CD36, between PD-1 and a second anti-PD-1 antibody, or between CD47 and a second anti-CD47 antibody (e.g., a single domain antibody or antigen-binding fragment thereof).

[0283] As used herein, the term "epitope" refers to a specific group of atoms or amino acid residues on an antigen to which an antibody (e.g., a single domain antibody) binds. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding for that antigen. Epitopes can be linear or conformational (i.e., containing spaced amino acid residues). For example, an antibody or antigen-binding fragment may be considered to bind to the same / closely related epitope as a reference antibody (e.g., a single domain antibody) if it blocks binding of the reference 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 antigen-binding fragment thereof may compete with a single domain antibody of the present disclosure for binding to human DR2.

[0284] As used herein, the term "sequence identity" of the present invention refers to the degree of identity between two nucleic acid or two amino acid sequences, when compared at their best, and when mutations, such as substitutions, insertions, or deletions, are considered appropriate. The sequence identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, and 100%. In some embodiments, framework regions are generally not considered part of the antigen-binding domain. However, some amino acid residues in one or more framework regions may contribute to antigen binding.

[0285] The term "functionally active" in reference to an antigen-binding domain means that the antigen-binding domain has the ability to bind to its target and optionally that the antigen-binding domain has one or more biological activities.

[0286] As used herein, the term "flexible linker" refers to a peptide that includes at least a portion made up of flexible amino acid residues that allows adjacent modules to move relative to one another.

[0287] As used herein, the term "rigid linker" refers to a peptide that includes at least a portion made up of amino acids that presents a rigid structure and maintains the distance between two modules.

[0288] As used herein, the term "helical linker" means a linker composed of amino acid residues that adopt an α-helical conformation.

[0289] As used herein, the term "cleavable linker" refers to a peptide that contains an enzymatic cleavage site that is sensitive to a protease selected from ADAMS, ADAMTS, aspartic proteases, caspases, cysteine ​​cathepsins, cysteine ​​proteinases, metalloproteinases, serine proteases, coagulation factor proteases, type II transmembrane serine proteases (TTSPs), and combinations thereof.

[0290] As used herein, the term "monospecific" with respect to a binding agent refers to a binding agent that binds to a single antigen or epitope. A monospecific binding agent may therefore have one antigen-binding domain or more than one binding domain (the same or different) that have the same specificity for a given antigen or epitope.

[0291] As used herein, the term "multivalent" with respect to a binding agent refers to a binding agent that binds to more than one antigen or epitope.

[0292] The term "multivalent" encompasses "divalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," and the like.

[0293] As used herein, the term "multispecific" with respect to a binding agent refers to a binding agent that binds to at least two different antigens or epitopes.

[0294] The term "multispecific" encompasses "bispecific," "trispecific," "tetraspecific," "pentaspecific," and "hexaspecific," among others.

[0295] As used herein, in the context of binding agents, the term "bispecific" refers to a binding agent that binds to two different antigens or proteins or two epitopes of the same antigen or protein.

[0296] As used herein, a phrase such as "at least two polypeptide chains" in a phrase such as "the binding agent comprises at least two polypeptide chains" refers to a certain number of polypeptide chain species, and does not refer to an absolute numerical value.

[0297] It should be understood herein that expressions referring to ranges of values ​​in the form "A to B," etc., include each individual value and any subranges within and comprising such range. For example, the expression "1 to 10" includes, without limitation, subranges such as "2 to 10," "2 to 9," "3 to 6," "5 to 7," and any individual value within 1 to 10, inclusive, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0298] It should be understood herein that the term "at least" in reference to a given value is intended to include that value and greater values. For example, the term "at least 80%" includes "at least 81%, "at least 82%, "at least 83%, "at least 84%, "at least 85%, "at least 86%, "at least 87%, "at least 88%, "at least 89%, "at least 90%, "at least 91%, "at least 92%, "at least 93%, "at least 94%, "at least 95%, "at least 96%, "at least 97%, "at least 98%, "at least 99%, "at least 99.1%, "at least 99.2%, "at least 99.3%, "at least 99.4%, "at least 99.5%, "at least 99.6%, "at least 99.7%, "at least 99.8%, "at least 99.9%" and 100%.

[0299] The term "about" or "approximately" in reference to a given value means that variation in the value is expected. In some embodiments, the term "about" or "approximately" generally refers to a range within + / - 10 percent, + / - 5 percent, + / - 4 percent, + / - 3 percent, + / - 2 percent, or + / - 1 percent of the given value or range.

[0300] The term "codon-optimized" refers to a sequence in which a codon has been replaced with another codon that encodes the same amino acid but that is preferred or functions better in a given organism (increases expression, minimizes secondary structure in RNA, etc.). "Codon-optimized" sequences may be obtained using publicly available software or through service providers, including GenScript, Inc. (OptimumGene™, U.S. Patent No. 8,326,547).

[0301] The term "single-chain format" in reference to antigen-binding domains means that the domains necessary for antigen binding are present on a single polypeptide chain.

[0302] 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 compound in Table 6 or Table It should be understood herein that the term "binding agent" refers to a binding agent comprising two chains each having the corresponding amino acid sequence as shown in Table 7 (Table 7) or as described herein.

[0303] Antigen-binding domain (Ab) As disclosed herein, a binding agent may comprise one or more antigen-binding domains.

[0304] For example, a binding agent of the present disclosure comprises one or more antigen-binding domains capable of binding to CD36. The binding agent may also comprise one or more antigen-binding domains that bind to one or more other antigens.

[0305] In some cases, the antigen-binding domain may bind, for example, to an immune checkpoint protein.

[0306] In other cases, the antigen-binding domain may bind to a protein expressed on the surface of, for example, an immune cell.

[0307] The antigen-binding domains of the present disclosure may be selected for their ability to bind to a specific target. The antigen-binding domains may also be selected for functional properties or biological effects in vivo and / or in vitro, including, for example, the ability to modulate cellular processes, such as gene expression, signal transduction, cell proliferation, and cell survival.

[0308] For example, a binding agent of the disclosure comprises one or more antigen-binding domains, each independently comprising one or more complementarity-determining regions (CDRs) of an antibody.

[0309] The specificity of the binding agents of the present disclosure may therefore be conferred by their antigen-binding domain.

[0310] A binding agent of the disclosure may comprise an antigen-binding domain capable of binding to CD36 or a cell expressing CD36, an antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1, and / or an antigen-binding domain capable of binding to CD47 or a cell expressing CD47.

[0311] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding to CD36 or a cell expressing CD36, e.g., comprises antigen-binding domain 1 (ABD1). In some embodiments, the cell expressing CD36 comprises a tumor cell.

[0312] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding to PD-1 or a cell expressing PD-1, e.g., comprises antigen-binding domain 2 (ABD2). In some embodiments, the cell expressing PD-1 comprises an immune cell.

[0313] In some embodiments, the antigen-binding domain is an antigen-binding domain capable of binding to CD47 or a cell expressing CD47, e.g., comprises antigen-binding domain 3 (ABD3). In some embodiments, the cell expressing CD47 comprises an immune cell or a tumor cell.

[0314] In some embodiments, the antigen-binding domain targets the same epitope or antigen as ABD1, ABD2 and / or ABD3. In some embodiments, the antigen-binding domain competes with ABD1, ABD2 and / or ABD3 for binding to their respective epitope or antigen.

[0315] In some embodiments, the antigen-binding domain binds to at least one antigen that is not selected from among CD36, PD-1, or CD47.

[0316] In some embodiments, the binding agent may comprise more than one antigen-binding domain.

[0317] For example, in some embodiments, the binding agent may comprise two or more antigen-binding domains.

[0318] In some embodiments, the binding agent may comprise three or more antigen-binding domains.

[0319] In some embodiments, the binding agent may comprise four or more antigen-binding domains.

[0320] In some embodiments, the binding agent may comprise five or more antigen-binding domains.

[0321] In some embodiments, the binding agent may comprise six or more antigen-binding domains.

[0322] In some embodiments, the binder may be 1 to 12, e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 ... to 9, 4 to 10, 4 to 11, 4 to 12, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 9 to 10, 9 to 11, 9 to 12, 10 to 11, 10 to 12, or 11 to 12 antigen-binding domains.

[0323] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is capable of binding to a tumor cell.

[0324] In some embodiments, the binding agent is capable of binding to tumor cells, hi other embodiments, the binding agent is capable of binding to proteins expressed on the surface of tumor cells.

[0325] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is capable of binding to an immune modulator. In some embodiments, at least one of the antigen-binding domains is capable of binding to an immune checkpoint protein. In some embodiments, the immune checkpoint protein is PD-1.

[0326] In some embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is capable of binding to an immune cell, hi some embodiments, at least one of the antigen-binding domains is capable of binding to a protein expressed on the surface of an immune cell (e.g., T cells, NK cells, monocytes, macrophages, etc.).

[0327] In some embodiments, the binding agent is capable of binding to an immune cell, hi other embodiments, the binding agent is capable of binding to a protein expressed on the surface of an immune cell.

[0328] In some embodiments, the binding agents of the present disclosure may be multivalent and may comprise at least one antigen-binding domain that binds to a tumor cell and at least one antigen-binding domain that binds to an immune cell.

[0329] In some embodiments, the protein expressed on the surface of an immune cell is selected from CD47 or CD3.

[0330] In some embodiments, the protein expressed on the surface of an immune cell is CD47.

[0331] In some embodiments, the protein expressed on the surface of an immune cell is CD3.

[0332] The antigen-binding domain may be derived from a natural antibody (of human or animal origin) or a synthetic antibody.

[0333] In some embodiments, the antigen-binding domain of a native antibody is engineered to form a single chain.

[0334] In some embodiments, the antigen-binding domain may be derived from an IgG, such as IgG1, IgG2, IgG3, or IgG4. In particular embodiments, the antigen-binding domain is derived from a human IgG heavy chain.

[0335] In some embodiments, the antigen-binding domain may be derived from a heavy chain-only antibody (HCAb).

[0336] Exemplary embodiments of antigen-binding domains include, but are not limited to, single domain antibodies (sdAbs), heavy chain variable regions (VH or VHH), light chain variable regions (VL or VLL), single chain variable fragments (scFv), V NAR fragments, and combinations thereof.

[0337] In some embodiments, the antigen-binding domain of a binding agent disclosed herein is a VHH.

[0338] In some embodiments, the antigen-binding domain of a binding agent disclosed herein is a VHH comprising the complementarity determining regions of a VHH disclosed herein.

[0339] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to Kabat CDRs.

[0340] In some embodiments, the complementarity determining regions (CDRs) of the VHHs disclosed herein correspond to the IMGT CDRs.

[0341] In certain embodiments, a binding agent of the present disclosure may comprise an antigen-binding domain VHH derived from IgG1, IgG2a, IgG2b, IgG2c, or IgG3, or a combination thereof, of human, mouse, or rat, or a transgenic mouse or rat in which the mouse or rat VHH has been camelized, a human VHH, a camelized human VHH. The antibody may be obtained by immunizing a mouse or rat or transgenic mouse or rat lacking a functional CH1 domain in its heavy chain, IgG1, IgG2a, IgG2b, IgG2c, or IgG3, or a combination thereof, or any of the above VHH combinations, with an antigen of interest.

[0342] In certain embodiments, the polypeptide chains of the disclosure may comprise an antigen-binding domain of a camelid antibody, such as an IgG2 or IgG3 VHH. Camelid antibodies may be obtained by immunizing a dromedary, camel, llama, or alpaca with an antigen of interest.

[0343] In some embodiments, camelid antibodies may originate from so-called Old World camelids, such as the Bactrian camel (Camelus bactrianus), the dromedary (Camelus dromedarius), or New World camelids, such as the alpaca (Lama pacos), the llama (Lama glama), and the vicuna (Lama vicugna).

[0344] In another specific embodiment, the polypeptide chains of the present disclosure comprise an antigen-binding domain of a cartilaginous fish, such as the V of an IgNAR. NAR May contain fragments. V NAR The fragment may originate from a shark antibody.

[0345] If desired, the antigen-binding domain of a non-human antibody may be humanized. For example, the framework regions of a non-human VH, VHH, or HCAb may be modified to make them more human-like. Humanization of camelid antibodies is discussed, for example, in Vincke C. et al. (J. Biol Chem. 2009, 284(5):3273-3284), the entire contents of which are incorporated herein by reference. Humanized camelid antibodies can be prepared, for example, using universal humanized nanobody scaffolds (e.g., h-NbBcII10 disclosed in Vincke C. et al.). FGLA ) may be obtained by CDR grafting onto V NAR Antibodies may be humanized by converting non-CDR residues to those of the human germline Vκ1 sequence DPK9 as discussed in Kovalenko OV et al. (J Biol Chem. 2013, 288:17408-17419), the entire contents of which are incorporated herein by reference. The polypeptide chains of the present disclosure thus include humanized antigen-binding domains.

[0346] In yet another specific embodiment, the antigen-binding domain may comprise a human VH (modified or not). The human VH may, for example, be obtained from a synthetic human VH library. Modified human VHs include those in which some amino acid residues have been modified to make them more camel-like (i.e., by camelization).

[0347] The antigen-binding domain may be a single domain antibody (sdAb), a VHH, a conventional antibody or an antigen-binding fragment thereof, a bispecific antibody, a single-chain Fv-CH3 (scFv-CH3) fusion, a tandem-scFv-CH3 (TaFv-CH3) fusion, a diabody-CH3 (Db-CH3) fusion, a tandem Db-CH3 (TaDb-CH3) fusion, a single-chain Db-CH3 fusion (scDb-CH3), a Fab-CH3 fusion, a single-chain Fab-CH3 fusion, a Fab-scFv-CH3 fusion, a dual affinity retargeting (DART)-CH3 fusion, a Fab- Those skilled in the art will understand that the antibody formats may be incorporated into antibodies, antigen-binding fragments, or antibody-like molecules, including, without limitation, DART-CH3 fusions, single-chain Fv-Fc (scFv-Fc) fusions, tandem-scFv-Fc (TaFv-Fc) fusions, diabody-Fc (Db-Fc) fusions, tandem Db-Fc (TaDb-Fc) fusions, single-chain Db-Fc fusions (scDb-Fc), Fab-Fc fusions, single-chain Fab-Fc fusions, Fab-scFv-Fc fusions, dual affinity retargeting (DART)-Fc fusions, Fab-DART-Fc fusions, etc. These antibody formats may have a native CH3, a mutated CH3 domain, a native CH2-CH3 domain, or a mutated CH2-CH3 domain as disclosed herein.

[0348] Exemplary Embodiments of ABD1 Antigen-binding domain 1 (ABD1) may, for example, be selected from antigen-binding domains that bind to CD36, some non-limiting exemplary embodiments of which are provided herein.

[0349] In some cases, ABD1 has the ability to bind to human CD36 or a portion thereof, or a cell expressing human CD36 or a portion thereof.

[0350] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), comprising heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:1, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:2, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:3.

[0351] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6.

[0352] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10.

[0353] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13.

[0354] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17.

[0355] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20.

[0356] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24.

[0357] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:25, CDRH2 having the amino acid sequence set forth in SEQ ID NO:26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:27.

[0358] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31.

[0359] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34.

[0360] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38.

[0361] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 39, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 41.

[0362] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45.

[0363] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48.

[0364] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52.

[0365] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55.

[0366] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0367] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0368] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0369] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0370] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0371] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0372] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0373] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0374] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0375] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0376] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0377] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0378] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0379] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0380] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0381] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0382] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0383] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0384] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0385] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), including 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.

[0386] In some embodiments, a binding agent of the disclosure may have a CDRH1, CDRH2, CDRH3 combination of ABD1 with 1, 2, 3, 4, or 5 amino acid substitutions in any of CDRH1, CDRH2, or CDRH3. In some embodiments, the total number of amino acid substitutions in any of CDRH1, CDRH2, or CDRH3 is 10 or fewer (a total of 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions). In some embodiments, the total number of amino acid substitutions in CDRH3 is 3 or fewer (1, 2, or 3).

[0387] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 380. QVQLQESGGGLX 2a QPGGSLRLSCAASGX 2b X 2c X 2d SX 2e YAMSWYRQX 2f PGKERELVALITX 2g X 2h GGX 2i TDYADSVKGRFTIX 2j RDDAEKTVSLQMNSLKPEDTAVYYCSAWRAX 2k LX 2l X 2m PYWGQGTQVTVSS (SEQ ID NO: 380) X 2a ~X 2m any one of X is any (e.g., naturally occurring) amino acid residue, or 2a is A or V, and X 2b is L or F, and X 2c is A, T or S, and X 2d is F or V, and X 2e is T or S, and X 2f is A or P, and X 2g is S or T, and X 2h is A or T, and X2i is I or T, and X 2j is A or S, and X 2k is S or T, and X 2l is F, Y or L, and / or X 2m is E or K.

[0388] In some embodiments, the CDR1, CDR2 and / or CDR3 of SEQ ID NO: 380 may independently correspond to the CDR1, CDR2 and / or CDR3 of any one of SEQ ID NOs: 7, 28, 98 or 119, and combinations thereof.

[0389] In some embodiments, the framework regions of SEQ ID NO: 380 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 380 may comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus thereof).

[0390] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 381. QVQLQESGGGLVQPGX 3a SLRLSCX 3b ASGX 3c X 3d X 3e X 3f FTFSX 3g YAMSWYRQX 3h PGKERELVAAX 3i X 3j X 3k X 3l GX 3m X 3n X 3o X 3p YX 3q DSVKGRFX 3r ISRDX 3s AKNTVYLX 3t MNSLKPEDTAX 3u YYCNAVTLQGWX3v EYDYWGQGTQVTVSS (SEQ ID NO: 381) X 3a ~X 3v any one of X is any (e.g., naturally occurring) amino acid residue, or 3a is G or E, and X 3b is A or T, and X 3c does not exist or is F, and X 3d is absent or is T, and X 3e is absent or is S, and X 3f does not exist or is G, and X 3g is S or N, and X 3h is A or T, and X 3i is M or I, and X 3j is Q, P or T, and X 3k is S, H or N, and X 3l is T, A or D, and X 3m is D or G, and X 3n is S or T, and X 3o is T or I, and X 3p is Y or H, and X 3q is A or V, and X 3r is S or T, and X 3s is D or N, and X 3t is Q or L, and X 3u is V or A, and / or X 3v is S or R.

[0391] In some embodiments, X 3c , X 3d , X 3e and X 3f does not exist.

[0392] In some embodiments, the CDR1, CDR2 and / or CDR3 of SEQ ID NO: 381 may independently correspond to the CDR1, CDR2 and / or CDR3 of any one of SEQ ID NOs: 14, 21, 35 or 49, and combinations thereof.

[0393] In some embodiments, the framework regions of SEQ ID NO: 381 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 381 may comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus thereof).

[0394] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 382. QVQLQESGGGLVQPGGSLRLSCAASGFRFSX 4a SIMX 4b WYRQAPX 4c KERELVASISSDSX 4d RTNYAESVKGRFTISRDNEQX 4e X 4f X 4g FLEMNX 4h LKPVDTAVYFCTRPX 4i IRGRDWGQGTQVTVSS (SEQ ID NO: 382) X 4a ~X 4i any one of X is any (e.g., naturally occurring) amino acid residue, or 4a is S or T, and X 4b is S or T, and X 4c is E or G, and X 4d is V or R, and X 4e is R or S, and X 4f is M or T, and X 4g is V or L, and X 4h is S or N, and X 4i is S or N.

[0395] In some embodiments, the CDR1, CDR2 and / or CDR3 of SEQ ID NO: 382 may independently correspond to the CDR1, CDR2 and / or CDR3 of any one of SEQ ID NOs: 56 or 84 and combinations thereof.

[0396] In some embodiments, the framework regions of SEQ ID NO: 382 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 382 may comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus thereof).

[0397] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 383. QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYPMGWFRQAPGKEREFVAAIDWSGDITYYADSVKGRFAISRDNAKNMVYLQMNSLKPEDTAVYYCNAGPLDSWERNDFPSWX 5a QGTQVTVSS (SEQ ID NO: 383) X 5a is any (e.g., naturally occurring) amino acid residue, or X 5a is G or D.

[0398] 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 NOs: 105 or 112, and combinations thereof.

[0399] In some embodiments, the framework regions of SEQ ID NO: 383 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 383 may comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus thereof).

[0400] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7.

[0401] In some embodiments, antigen-binding domain 1 (ABD1) comprises the amino acid sequence set forth in SEQ ID NO:7, with 1 to 10 amino acid substitutions, additions, and / or deletions.

[0402] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:7.

[0403] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and CDRs identical to the Kabat CDRs of SEQ ID NO:7.

[0404] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7 and CDRs identical to the IMGT CDRs of SEQ ID NO:7.

[0405] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:7.

[0406] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0407] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:14.

[0408] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:14.

[0409] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14 and CDRs identical to the Kabat CDRs of SEQ ID NO: 14.

[0410] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14 and CDRs identical to the IMGT CDRs of SEQ ID NO: 14.

[0411] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0412] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21.

[0413] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:21.

[0414] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:21.

[0415] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21 and CDRs identical to the Kabat CDRs of SEQ ID NO:21.

[0416] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21 and CDRs identical to the IMGT CDRs of SEQ ID NO:21.

[0417] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:21.

[0418] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28.

[0419] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:28.

[0420] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:28.

[0421] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28 and CDRs identical to the Kabat CDRs of SEQ ID NO:28.

[0422] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28 and CDRs identical to the IMGT CDRs of SEQ ID NO:28.

[0423] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:28.

[0424] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35.

[0425] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:35.

[0426] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:35.

[0427] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35 and CDRs identical to the Kabat CDRs of SEQ ID NO: 35.

[0428] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35 and CDRs identical to the IMGT CDRs of SEQ ID NO: 35.

[0429] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:35.

[0430] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42.

[0431] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:42.

[0432] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:42.

[0433] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42 and CDRs identical to the Kabat CDRs of SEQ ID NO: 42.

[0434] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42 and CDRs identical to the IMGT CDRs of SEQ ID NO: 42.

[0435] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:42.

[0436] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49.

[0437] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:49.

[0438] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:49.

[0439] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49 and CDRs identical to the Kabat CDRs of SEQ ID NO:49.

[0440] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49 and CDRs identical to the IMGT CDRs of SEQ ID NO:49.

[0441] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:49.

[0442] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56.

[0443] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:56.

[0444] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:56.

[0445] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56 and CDRs identical to the Kabat CDRs of SEQ ID NO: 56.

[0446] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56 and CDRs identical to the IMGT CDRs of SEQ ID NO: 56.

[0447] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:56.

[0448] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:77.

[0449] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:77.

[0450] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:77.

[0451] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 77 and CDRs identical to the Kabat CDRs of SEQ ID NO: 77.

[0452] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 77 and CDRs identical to the IMGT CDRs of SEQ ID NO: 77.

[0453] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:77.

[0454] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:84.

[0455] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:84.

[0456] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:84.

[0457] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 84 and CDRs identical to the Kabat CDRs of SEQ ID NO: 84.

[0458] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 84 and CDRs identical to the IMGT CDRs of SEQ ID NO: 84.

[0459] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:84.

[0460] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:91.

[0461] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence set forth in SEQ ID NO:91 having 1 to 10 amino acid substitutions, additions, and / or deletions.

[0462] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:91.

[0463] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 91 and CDRs identical to the Kabat CDRs of SEQ ID NO: 91.

[0464] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:91 and CDRs identical to the IMGT CDRs of SEQ ID NO:91.

[0465] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:91.

[0466] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:98.

[0467] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence set forth in SEQ ID NO:98 having 1 to 10 amino acid substitutions, additions, and / or deletions.

[0468] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:98.

[0469] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 98 and CDRs identical to the Kabat CDRs of SEQ ID NO: 98.

[0470] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 98 and CDRs identical to the IMGT CDRs of SEQ ID NO: 98.

[0471] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO:98.

[0472] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105.

[0473] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:105.

[0474] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:105.

[0475] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105 and CDRs identical to the Kabat CDRs of SEQ ID NO: 105.

[0476] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105 and CDRs identical to the IMGT CDRs of SEQ ID NO: 105.

[0477] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 105.

[0478] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:112.

[0479] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:112.

[0480] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:112.

[0481] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112 and CDRs identical to the Kabat CDRs of SEQ ID NO: 112.

[0482] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112 and CDRs identical to the IMGT CDRs of SEQ ID NO: 112.

[0483] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 112.

[0484] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:119.

[0485] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:119.

[0486] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:119.

[0487] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119 and CDRs identical to the Kabat CDRs of SEQ ID NO: 119.

[0488] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119 and CDRs identical to the IMGT CDRs of SEQ ID NO: 119.

[0489] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 119.

[0490] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126.

[0491] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:126.

[0492] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:126.

[0493] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126 and CDRs identical to the Kabat CDRs of SEQ ID NO: 126.

[0494] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126 and CDRs identical to the IMGT CDRs of SEQ ID NO: 126.

[0495] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 126.

[0496] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133.

[0497] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:133.

[0498] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:133.

[0499] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133 and CDRs identical to the Kabat CDRs of SEQ ID NO: 133.

[0500] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133 and CDRs identical to the IMGT CDRs of SEQ ID NO: 133.

[0501] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 133.

[0502] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140.

[0503] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:140.

[0504] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:140.

[0505] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140 and CDRs identical to the Kabat CDRs of SEQ ID NO: 140.

[0506] In some embodiments, antigen-binding domain 1 (ABD1) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140 and CDRs identical to the IMGT CDRs of SEQ ID NO: 140.

[0507] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises the amino acid sequence set forth in SEQ ID NO: 140.

[0508] Exemplary Embodiments of ABD2 Antigen-binding domain 2 (ABD2) may be selected, for example, from antigen-binding domains that bind to PD-1, some non-limiting exemplary embodiments of which are provided herein.

[0509] In some 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.

[0510] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2), comprising heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO:57, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO:58, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO:59.

[0511] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62.

[0512] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 275, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 276, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 277.

[0513] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0514] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0515] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0516] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0517] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0518] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: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.

[0519] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0520] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0521] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0522] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0523] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0524] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0525] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0526] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0527] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0528] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0529] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0530] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0531] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0532] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0533] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0534] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0535] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0536] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0537] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 2 (ABD2), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 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.

[0538] In some embodiments, a binding agent of the disclosure may have a CDRH1, CDRH2, CDRH3 combination of ABD2 with 1, 2, 3, 4, or 5 amino acid substitutions in any of CDRH1, CDRH2, or CDRH3. In some embodiments, the total number of amino acid substitutions in any of CDRH1, CDRH2, or CDRH3 is 10 or fewer (a total of 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions). In some embodiments, the total number of amino acid substitutions in CDRH3 is 3 or fewer (1, 2, or 3).

[0539] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 379, and wherein X 1a ~X 1k any one of X is any (e.g., naturally occurring) amino acid residue, or 1a is L or E, and X 1b is A or P, and X 1c is D or G, and X 1d is N or S, and X 1e is S or T, and X 1f is K or R, and X 1g is D or G, and X 1h is K or Q, and X 1j is T or A, and / or X 1k is L or V. QVQLQESGGGX 1a VQX 1b GX 1c SLRLSCAASGFTFSX 1d YGMSWVRQAPGEGLEWVSSIDSX 1e GGTTX 1f YAX 1g SVX 1h GRFTISRDNAKNX 1j X 1k YLQMNSLKPEDTAVYYCAKDFLSWMPRGQGTQVTVSS (SEQ ID NO: 379)

[0540] In some embodiments, the CDR1, CDR2 and / or CDR3 of SEQ ID NO: 379 may independently correspond to the CDR1, CDR2 and / or CDR3 of any one of SEQ ID NOs: 288, 295, 302, 309, 323, 330, 337, 344, 351 or 358 and combinations thereof.

[0541] In some embodiments, the framework regions of SEQ ID NO: 379 may be modified to include amino acid substitutions, additions, and / or deletions. In some embodiments, the framework regions of SEQ ID NO: 379 may comprise amino acid residues of a human antibody (e.g., a human germline antibody sequence or a consensus thereof).

[0542] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63.

[0543] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:63.

[0544] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:63.

[0545] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63 and CDRs identical to the Kabat CDRs of SEQ ID NO: 63.

[0546] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63 and CDRs identical to the IMGT CDRs of SEQ ID NO: 63.

[0547] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:63.

[0548] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:281.

[0549] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence set forth in SEQ ID NO:281 having 1 to 10 amino acid substitutions, additions, and / or deletions.

[0550] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:281.

[0551] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 281 and CDRs identical to the Kabat CDRs of SEQ ID NO: 281.

[0552] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:281 and CDRs identical to the IMGT CDRs of SEQ ID NO:281.

[0553] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:281.

[0554] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:288.

[0555] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence set forth in SEQ ID NO:288 having 1 to 10 amino acid substitutions, additions, and / or deletions.

[0556] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:288.

[0557] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 288 and CDRs identical to the Kabat CDRs of SEQ ID NO: 288.

[0558] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:288 and CDRs identical to the IMGT CDRs of SEQ ID NO:288.

[0559] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:288.

[0560] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:295.

[0561] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:295.

[0562] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:295.

[0563] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 295 and CDRs identical to the Kabat CDRs of SEQ ID NO: 295.

[0564] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 295 and CDRs identical to the IMGT CDRs of SEQ ID NO: 295.

[0565] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO:295.

[0566] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:302.

[0567] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:302.

[0568] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:302.

[0569] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 302 and CDRs identical to the Kabat CDRs of SEQ ID NO: 302.

[0570] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 302 and CDRs identical to the IMGT CDRs of SEQ ID NO: 302.

[0571] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 302.

[0572] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:309.

[0573] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:309.

[0574] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:309.

[0575] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 309 and CDRs identical to the Kabat CDRs of SEQ ID NO: 309.

[0576] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 309 and CDRs identical to the IMGT CDRs of SEQ ID NO: 309.

[0577] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 309.

[0578] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:316.

[0579] In some embodiments, antigen-binding domain 2 (ABD2) comprises the amino acid sequence of SEQ ID NO: 316 with 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence.

[0580] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:316.

[0581] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 316 and CDRs identical to the Kabat CDRs of SEQ ID NO: 316.

[0582] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 316 and CDRs identical to the IMGT CDRs of SEQ ID NO: 316.

[0583] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 316.

[0584] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 323.

[0585] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:323.

[0586] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:323.

[0587] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 323 and CDRs identical to the Kabat CDRs of SEQ ID NO: 323.

[0588] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 323 and CDRs identical to the IMGT CDRs of SEQ ID NO: 323.

[0589] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 323.

[0590] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 330.

[0591] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:330.

[0592] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:330.

[0593] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 330 and CDRs identical to the Kabat CDRs of SEQ ID NO: 330.

[0594] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 330 and CDRs identical to the IMGT CDRs of SEQ ID NO: 330.

[0595] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 330.

[0596] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 337.

[0597] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:337.

[0598] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:337.

[0599] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 337 and CDRs identical to the Kabat CDRs of SEQ ID NO: 337.

[0600] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 337 and CDRs identical to the IMGT CDRs of SEQ ID NO: 337.

[0601] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 337.

[0602] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:344.

[0603] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:344.

[0604] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:344.

[0605] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 344 and CDRs identical to the Kabat CDRs of SEQ ID NO: 344.

[0606] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 344 and CDRs identical to the IMGT CDRs of SEQ ID NO: 344.

[0607] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 344.

[0608] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:351.

[0609] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence set forth in SEQ ID NO:351 having 1 to 10 amino acid substitutions, additions, and / or deletions.

[0610] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:351.

[0611] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 351 and CDRs identical to the Kabat CDRs of SEQ ID NO: 351.

[0612] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 351 and CDRs identical to the IMGT CDRs of SEQ ID NO: 351.

[0613] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 351.

[0614] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:358.

[0615] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:358.

[0616] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:358.

[0617] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 358 and CDRs identical to the Kabat CDRs of SEQ ID NO: 358.

[0618] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 358 and CDRs identical to the IMGT CDRs of SEQ ID NO: 358.

[0619] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 358.

[0620] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 365.

[0621] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:365.

[0622] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:365.

[0623] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 365 and CDRs identical to the Kabat CDRs of SEQ ID NO: 365.

[0624] In some embodiments, antigen-binding domain 2 (ABD2) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63 and CDRs identical to the IMGT CDRs of SEQ ID NO: 365.

[0625] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises the amino acid sequence set forth in SEQ ID NO: 365.

[0626] Exemplary Embodiments of ABD3 Antigen-binding domain 3 (ABD3) may be selected, for example, from antigen-binding domains that bind to CD47, some non-limiting exemplary embodiments of which are provided herein.

[0627] In some cases, ABD3 has the ability to bind to human CD47 or a portion thereof or a cell expressing human CD47 or a portion thereof.

[0628] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3), comprising a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66.

[0629] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, at least one of which is antigen-binding domain 3 (ABD3), including CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69.

[0630] In some embodiments, a binding agent of the disclosure may have a CDRH1, CDRH2, CDRH3 combination of ABD3 with 1, 2, 3, 4, or 5 amino acid substitutions in any of CDRH1, CDRH2, or CDRH3. In some embodiments, the total number of amino acid substitutions in any of CDRH1, CDRH2, or CDRH3 is 10 or fewer (a total of 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions). In some embodiments, the total number of amino acid substitutions in CDRH3 is 3 or fewer (1, 2, or 3).

[0631] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70.

[0632] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:70.

[0633] In some embodiments, the amino acid variation may be located in one or more framework regions of SEQ ID NO:70.

[0634] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 and CDRs identical to the Kabat CDRs of SEQ ID NO: 70.

[0635] In some embodiments, antigen-binding domain 3 (ABD3) comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70 and CDRs identical to the IMGT CDRs of SEQ ID NO: 70.

[0636] In some exemplary embodiments, a binding agent of the present disclosure may comprise one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises the amino acid sequence set forth in SEQ ID NO:70.

[0637] Exemplary Embodiments of Other Antigen-Binding Domains In some embodiments, the binding agent comprises one or more antigen-binding domains, at least one of which is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3), and at least one antigen-binding domain binds to a different antigen.

[0638] In some embodiments, the binding agent may therefore comprise an antigen-binding domain that binds to CD3. Such antigen-binding domains include those known to those skilled in the art. An exemplary embodiment of an antigen-binding domain that binds to CD3 is provided in SEQ ID NO: 197.

[0639] Thus, in some embodiments, the binding agent may comprise an antigen-binding domain that binds to CD36, PD-1, and / or CD3. In other embodiments, the binding agent may comprise an antigen-binding domain that binds to CD36 and / or CD3. In yet other embodiments, the binding agent may comprise an antigen-binding domain that binds to PD-1 and / or CD3.

[0640] In some embodiments, the binding agent comprises one or more antigen-binding domains, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), and at least one of the antigen-binding domains is selected from the group consisting of, but not limited 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, beta1 integrin, tetraspanin 8, CD164, CD59, CD63, CD44, CD166, cWF, TNF, IL-17A, IL17-F, IL-6R, BCMA, TNF, RANKL, ADAMTS5, VEGF, Ang2, CX3CR1, CXCR4, TfR1 (CD71), CXCR2, CD3, PD1, PDL-1, CTLA-4, CD8, LAG-3, OX40, CD27, CD122 / IL2RB, TLR8 / CD288, TIM-3, ICOS / CD278, NKG2A, A2AR, B7-H the antigen-binding domain is selected from the group consisting of antigen-binding domains that specifically bind to 3, B7-H4, GITR / TNFRSF18, 4-IBB / CD137, KIR2DL1, KIR3DL2, SIRPα, CD47, VISTA, CD40, CD112, CD96, TOGOT, BTLA, TIGIT, CD4, VEGFR2, CD19, IGFR1, EpCAM, EGFR, DLL3, CGRP, CD79b, CD28, CCR5, ErbB3, ErbB2, TGFβ1, TGFβ2, TGFβ3, TGFβR1, TGFβR2, IDO1, IDO2, TLR-4, TLR-7, TLR-8, TLR-9, NOX2, and SIGLEC-7.

[0641] Polypeptide chain The binding agents of the present disclosure comprise one or more polypeptide chains.

[0642] A segment of DNA encoding a desired polypeptide chain sequence may be synthesized in vitro. Different DNA modules are assembled in an organized and oriented manner into a single piece that is then cloned into an expression vector. The resulting polypeptide chain is thus composed of the different modules that form a single chain.

[0643] Polypeptide chains of the present disclosure include, for example and without limitation, antigen binding domains, linkers, and dimerization domains that promote assembly of at least two polypeptide chains.

[0644] In some embodiments, the polypeptide chains of the present disclosure may be monospecific.

[0645] In some embodiments, the polypeptide chains of the present disclosure may be multispecific.

[0646] In some embodiments, the polypeptide chains of the present disclosure may be monovalent.

[0647] In some embodiments, the polypeptide chains of the present disclosure may be multivalent.

[0648] In some cases, the polypeptide chain does not comprise a dimerization domain.

[0649] In some cases, the polypeptide chain comprises a dimerization domain.

[0650] In some aspects of the present disclosure, the polypeptide chains may be monospecific.

[0651] An exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising one antigen-binding domain. Another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but where the antigen-binding domains have the same CDRs and framework regions. Yet another exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising more than one antigen-binding domain, but where the antigen-binding domains have the same CDRs and different framework regions. A further exemplary embodiment of a monospecific polypeptide chain includes a polypeptide chain comprising antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs (e.g., conservative substitutions in one or more CDRs) without affecting their ability to bind to the same antigen or epitope.

[0652] In some aspects of the present disclosure, the polypeptide chains may be multispecific, including, for example, bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, biparatopic, and multiparatopic polypeptide chains.

[0653] In exemplary configurations, one or more antigen-binding domains may be located N-terminal, C-terminal, or on each side of the dimerization domain.

[0654] In another exemplary configuration, the polypeptide chain may comprise at least one antigen binding domain N-terminal to the dimerization domain and at least one antigen binding domain C-terminal to the dimerization domain.

[0655] In a further exemplary configuration, the polypeptide chain may comprise one antigen-binding domain N-terminal to the dimerization domain and at least two antigen-binding domains C-terminal to the dimerization domain.

[0656] In a still further exemplary configuration, the polypeptide chain may comprise two antigen-binding domains N-terminal to the dimerization domain and two antigen-binding domains C-terminal to the dimerization domain.

[0657] The polypeptide chain has the formula I in N-terminal to C-terminal fashion: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y may comprise the amino acid sequence of m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain.

[0658] In some embodiments, L b does not have to exist.

[0659] In some embodiments, L c does not have to exist.

[0660] In some embodiments, L b and L c Both do not have to be present.

[0661] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0662] In some embodiments, where m is an integer greater than or equal to 2, [(Ab a )-(L b )] units are the same.

[0663] In other embodiments where m is an integer greater than or equal to 2, the [(Ab a )-(L b )] units are different.

[0664] In other embodiments where m is an integer greater than 2, the [(Ab a )-(L b ) units may include the same and different units.

[0665] In some embodiments, where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units are the same.

[0666] In other embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units are different.

[0667] In other embodiments where n is an integer greater than or equal to 2, [(L c )-(Ab d )] units include same and different units.

[0668] In some embodiments where m is an integer greater than or equal to 2, each Ab a are the same or different.

[0669] In some embodiments where n is an integer greater than or equal to 2, each Ab d are the same or different.

[0670] In some embodiments, Ab a represents ABD1.

[0671] In some embodiments, Abd represents ABD2.

[0672] In some embodiments, Ab d represents ABD3.

[0673] In some embodiments, m is 1 and Ab a represents ABD1.

[0674] In some embodiments, n is 2 and Ab d One of them represents ABD2.

[0675] In some embodiments, n is 2 and Ab d One of them represents ABD3.

[0676] In some embodiments, n is 2 and Ab d One of them represents ABD2, and the other Ab d represents ABD3.

[0677] In some embodiments, m is 2, 3, 4, 5, or an integer greater than 5.

[0678] In some embodiments, m is 2.

[0679] In other embodiments, m is 3.

[0680] In yet other embodiments, m is 4.

[0681] In a further embodiment, m is 5.

[0682] In other embodiments, m is an integer greater than 5.

[0683] In some embodiments, n is 2, 3, 4, 5, or an integer greater than 5.

[0684] In some embodiments, n is 2.

[0685] In other embodiments, n is 3.

[0686] In additional embodiments, n is 4.

[0687] In a further embodiment, n is 5.

[0688] In other embodiments, n is an integer greater than five.

[0689] In embodiments, one or more of the polypeptide chains further comprises a hinge region of an antibody or antigen-binding fragment thereof.

[0690] In some embodiments, the hinge region is N-terminal to the dimerization domain.

[0691] In some embodiments, L b is the hinge region of an antibody or antigen-binding fragment thereof.

[0692] In some embodiments, the hinge region is a native human IgG1 hinge region. In other embodiments, the hinge region is a mutated human IgG1 hinge region.

[0693] In other embodiments, the hinge region is a native human IgG2 hinge region. In other embodiments, the hinge region is a mutated human IgG2 hinge region.

[0694] In other embodiments, the hinge region is a native human IgG3 hinge region. In other embodiments, the hinge region is a mutated human IgG3 hinge region.

[0695] In yet other embodiments, the hinge region is a native human IgG4 hinge region. In other embodiments, the hinge region is a mutated human IgG4 hinge region.

[0696] In some embodiments, each of the one or more linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0697] In some embodiments, each of the one or more linkers is independently a flexible linker, a helical linker, or a rigid linker.

[0698] In some embodiments, the linker L c is a rigid linker.

[0699] In some embodiments, Lc comprises a non-cleavable linker. In other embodiments, Lc consists of a non-cleavable linker.

[0700] In some embodiments, one or more linkers comprise a flexible linker.

[0701] In some embodiments, the one or more linkers comprise a rigid linker.

[0702] In some embodiments, the flexible linker is a GS linker.

[0703] In some embodiments, the flexible linker comprises one or more GGGGS units.

[0704] In some embodiments, the flexible linker comprises at least 2, 3, 4, 5, or more GGGGS units.

[0705] In some embodiments, the rigid linker comprises multiple PA repeats.

[0706] 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.

[0707] In some embodiments, the helical linker comprises one or more EAAAK units.

[0708] In some embodiments, the helical linker is selected from AEAAAKEAAAKA (SEQ ID NO: 191); AEAAAKEAAAKEAAAKA (SEQ ID NO: 192); AEAAAKEAAAKEAAAKEAAAKEAAAKA (SEQ ID NO: 193); or a combination thereof.

[0709] In some embodiments, the dimerization domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:198.

[0710] In some embodiments, the dimerization domain further comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:199.

[0711] In another exemplary embodiment, the polypeptide chain has the formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II) Includes:

[0712] In yet another exemplary embodiment, the polypeptide chain has the formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) Includes:

[0713] In a further exemplary embodiment, the polypeptide chain has Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV) Includes:

[0714] In additional exemplary embodiments, the polypeptide chain has the formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y(Formula V) Includes:

[0715] In a further exemplary embodiment, the polypeptide chain has Formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y(Formula VI) Includes:

[0716] In still further exemplary embodiments, the polypeptide chain has the formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y(Formula VII) Includes:

[0717] In another exemplary embodiment, the polypeptide chain has the formula VIII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1)-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y(Formula VIII) Includes:

[0718] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each independently comprises an antigen-binding domain.

[0719] In some embodiments, Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each independently represents an antigen-binding domain.

[0720] In some embodiments, at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1).

[0721] In some embodiments, the polypeptide chain also comprises antigen binding domain 2 (ABD2) or antigen binding domain 3 (ABD3).

[0722] In some embodiments, the polypeptide chain also comprises at least one antigen binding domain 2 (ABD2) and at least one antigen binding domain 3 (ABD3).

[0723] In some embodiments, L b1 comprises one or more linkers and / or hinge regions of an antibody or antigen-binding fragment thereof. b1 L b is.

[0724] In some embodiments, L b2 , L b3 , L c1 , Lc2 , and L c3 each independently comprises one or more linkers.

[0725] In some embodiments, L b1 , L b2 and / or L b3 One or more of may be absent.

[0726] In some embodiments, L b2 and / or L b3 does not have to exist.

[0727] In some embodiments, L c1 , L c2 , and / or L c3 One or more of may be absent.

[0728] In some embodiments, L c2 and / or L c3 does not have to exist.

[0729] In some embodiments, L c1 is a rigid linker.

[0730] In some embodiments, L c2 is a rigid linker.

[0731] In some embodiments, L c3 is a rigid linker.

[0732] In some embodiments, L c1 , and L c2 is a rigid linker.

[0733] In some embodiments, L c1 , L c2 and L c3 is a rigid linker.

[0734] In some embodiments, L c1 , L c2 and / or L c3 does not have to exist.

[0735] In some embodiments, L c2 , and / or L c3 does not have to exist.

[0736] In some embodiments, L c1 , and / or L c3 does not have to exist.

[0737] In some embodiments, L c2 and / or L c3 does not have to exist.

[0738] In some embodiments, L c1 , L c2 , and L c3 does not have to exist.

[0739] The polypeptide chains of the present disclosure comprise an antigen-binding domain that is functionally active as a single chain or when part of a binding agent disclosed herein.

[0740] For example, the antigen-binding domain of a polypeptide chain may bind to its target and may be biologically active.

[0741] In some embodiments, the biological activity of an antigen-binding domain includes, for example and without limitation, blocking the binding of a target to its natural receptor or ligand. Alternatively, the biological activity of an antigen-binding domain includes its ability to sequester a target. Further, the biological activity of an antigen-binding domain includes its ability to induce signal transduction.

[0742] A polypeptide chain that contains more than one antigen-binding domain is characterized as being multivalent.

[0743] The polypeptide chains of the present disclosure may include additional amino acid sequences (defined by X and Y, respectively, in the formulas disclosed herein) at their N- or C-terminus or both termini.

[0744] 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.

[0745] In some embodiments, the amino acid sequence at the N-terminus (defined by X) or the amino acid sequence at the C-terminus (defined by Y) may independently comprise a linker, a cytokine, a chemokine, a tag (e.g., a His tag (e.g., SEQ ID NO: 216)), a masking domain, a phage coat protein, an antigen-binding domain, or a combination thereof.

[0746] Exemplary embodiments of multispecific polypeptide chains include polypeptide chains comprising at least two antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs, leading to different binding specificities.

[0747] A polypeptide chain may more particularly be characterized as bispecific if it binds to two different epitopes or antigens. A polypeptide chain may be characterized as trispecific if it binds to three different epitopes or antigens. A polypeptide chain may be characterized as tetraspecific if it binds to four different epitopes or antigens. A polypeptide chain may be characterized as pentaspecific if it binds to five different epitopes or antigens. A polypeptide chain may be characterized as hexaspecific if it binds to six different epitopes or antigens.

[0748] A polypeptide chain containing two antigen-binding domains that bind to two non-overlapping epitopes on the same target is characterized as being biparatopic. A polypeptide chain containing three, four, or more epitopes on the same target is characterized as being multiparatopic.

[0749] The antigen-binding domains of a given polypeptide chain are selected based on the intended use, e.g., detection, diagnostic, and / or therapeutic use. Each of the antigen-binding domains of a particular polypeptide chain may be selected to produce an additive or synergistic effect.

[0750] For example, the polypeptide chains of the present disclosure may comprise at least one antigen-binding domain that specifically binds to an antigen expressed by a tumor cell or the tumor cell environment (ie, a tumor-specific antigen-binding domain).

[0751] In other aspects and embodiments of the present disclosure, the polypeptide chain may comprise at least one antigen-binding domain that specifically binds to an immunomodulator.

[0752] For example, the polypeptide chain may include one or more antigen-binding domains (e.g., immunospecific antigen-binding domains) that bind to an immune checkpoint protein, a cytokine, a chemokine, or an immune receptor or co-receptor, etc.

[0753] In some embodiments, the polypeptide chain comprises, in N-terminal to C-terminal manner, an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD-1, and an antigen-binding domain that binds CD47.

[0754] In some exemplary embodiments, the antigen-binding domain may bind to CD36.

[0755] In some exemplary embodiments, the antigen-binding domain may bind to PD1.

[0756] In some exemplary embodiments, the antigen-binding domain may bind to CD47.

[0757] In exemplary embodiments, the polypeptide chains of the present disclosure may comprise at least one tumor-specific antigen-binding domain and at least one immunospecific antigen-binding domain.

[0758] In some embodiments, the tumor-specific antigen-binding domain may be located N-terminal to the dimerization domain.

[0759] In some embodiments, the tumor-specific antigen-binding domain may be located C-terminal to the dimerization domain.

[0760] In some embodiments, the tumor-specific antigen-binding domain may be located both N-terminal and C-terminal to the dimerization domain.

[0761] In some embodiments, multiple immunospecific antigen-binding domains may be located N-terminal to the dimerization domain.

[0762] In some embodiments, the immunospecific antigen-binding domain may be located C-terminal to the dimerization domain.

[0763] In some embodiments, the immunospecific antigen-binding domains may be located both N-terminal and C-terminal to the dimerization domain.

[0764] In exemplary, non-limiting embodiments, a polypeptide chain or binding agent may comprise two immunospecific antigen-binding domains C-terminal to a dimerization domain, hi some embodiments, the immunospecific antigen-binding domains immediately adjacent to the C-terminal portion of the dimerization domain may be linked via a non-cleavable linker.

[0765] Dimerization domain (DD) In some embodiments, the polypeptide chains of the present disclosure comprise a dimerization domain, such that two polypeptide chains may assemble to form a binding agent. Exemplary embodiments of binding agents include homodimers and heterodimers.

[0766] The dimerization domain may comprise, for example and without limitation, an immunoglobulin constant region, including, for example, the Fc, CH2 and / or CH3 domains of a heavy chain immunoglobulin. The constant region of an immunoglobulin heavy chain is particularly contemplated.

[0767] In certain embodiments and aspects of the present disclosure, the dimerization domain may have a sequence identical to that of a native IgG1, IgG2, IgG3, or IgG4 constant region or their corresponding CH2 and / or CH3 domains.

[0768] Dimerization domains having sequences identical to those of natural human antibodies are specifically encompassed by the present disclosure. Exemplary embodiments of dimerization domains include, for example, the CH2-CH3 domains of natural human heavy chains.

[0769] Thus, in some embodiments, the dimerization domain comprises a native constant region of an antibody, such as a native human IgG1 constant region, a native human IgG2 constant region, a native human IgG3 constant region, or a native human IgG4 constant region.

[0770] Thus, in some embodiments, the dimerization domain comprises a native CH3 domain.

[0771] In an exemplary embodiment, the dimerization domain comprises a native human CH3 domain.

[0772] In some embodiments, the dimerization domain comprises a native CH2 domain and a native CH3 domain.

[0773] 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).

[0774] In some embodiments, the native CH3 domain is a native IgG2 CH3 domain, hi other embodiments, the native CH3 domain is a native human IgG2 CH3 domain.

[0775] In some embodiments, the native CH3 domain is a native IgG3 CH3 domain, hi other embodiments, the native CH3 domain is a native human IgG3 CH3 domain.

[0776] In some embodiments, the native CH3 domain is a native IgG4 CH3 domain. In some embodiments, the native CH3 domain is a native human IgG4 CH3 domain.

[0777] When two polypeptide chains of a binding agent are composed of the same amino acid sequence, the binding agent forms a homodimer. However, co-expression of polypeptide chains having the CH2-CH3 domains of a natural antibody but different amino acid sequences can result in a mixture of homodimers and heterodimers. Different binding agents present in the mixture can be separated by methods known in the art, including, for example, size exclusion chromatography.

[0778] Exemplary heterodimers of the present disclosure therefore include those formed by two polypeptide chains having the CH3 domain or CH2-CH3 domains of a natural antibody and having different sequences or configurations.

[0779] In some embodiments, the polypeptide chain may have a mutant dimerization domain that includes, for example, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 amino acid substitutions compared to the native or wild-type sequence.

[0780] In exemplary embodiments, the mutant dimerization domain may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. The amino acid substitutions may be conservative or non-conservative as outlined in Table 2 (Table 2).

[0781] In exemplary embodiments, the polypeptide chain may have a mutant dimerization domain with a sequence that is 80% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domain. Polypeptide chains encompassed by the present disclosure include those that contain a mutant dimerization domain that is 85% to 99% identical, 90% to 99% identical, or 95% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 constant region or CH2 and / or CH3 domain.

[0782] In some embodiments, polypeptide chains of the present disclosure may comprise mutated dimerization domains comprising amino acid substitutions that favor heterodimer formation. Heterodimers of the present disclosure may therefore be formed by polypeptide chains comprising such mutations.

[0783] Thus, in some embodiments, the dimerization domain comprises a mutated constant region of an antibody, such as a mutated human IgG1 constant region, a mutated human IgG2 constant region, a mutated human IgG3 constant region, or a mutated human IgG4 constant region, etc. The mutated constant region may have one or more amino acid substitutions, insertions, or deletions compared to the native constant region.

[0784] Thus, in some embodiments, the dimerization domain comprises a mutated CH3 domain, which may have one or more amino acid substitutions, insertions, or deletions compared to the native CH3 domain.

[0785] In some embodiments, the dimerization domain comprises a native CH2 and a mutated CH3 domain.

[0786] In some embodiments, the dimerization domain comprises a mutant CH2 and a mutant CH3 domain. The mutant CH2 domain may have one or more amino acid substitutions, insertions, or deletions compared to the native CH2 domain.

[0787] In some embodiments, the mutated CH3 domain is a mutated IgG1 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG1 CH3 domain.

[0788] In some embodiments, the mutated CH3 domain is a mutated IgG2 CH3 domain, hi other embodiments, the mutated CH3 domain is a mutated human IgG2 CH3 domain.

[0789] In some embodiments, the mutated CH3 domain is a mutated IgG3 CH3 domain. In other embodiments, the mutated CH3 domain is a mutated human IgG3 CH3 domain.

[0790] In some embodiments, the mutated CH3 domain is a mutated IgG4 CH3 domain, hi other embodiments, the mutated CH3 domain is a mutated human IgG4 CH3 domain.

[0791] In some embodiments, the mutated CH2 domain is a mutated IgG1 CH2 domain. In other embodiments, the mutated CH2 domain is a mutated human IgG1 CH2 domain.

[0792] In some embodiments, the mutated CH2 domain is a mutated IgG2 CH2 domain, hi other embodiments, the mutated CH2 domain is a mutated human IgG2 CH2 domain.

[0793] In some embodiments, the mutated CH2 domain is a mutated IgG3 CH2 domain, hi other embodiments, the mutated CH2 domain is a mutated human IgG3 CH2 domain.

[0794] In some embodiments, the mutated CH2 domain is a mutated IgG4 CH2 domain, hi other embodiments, the mutated CH2 domain is a mutated human IgG4 CH2 domain.

[0795] In some embodiments, the Fc region may be modified to prevent glycosylation, increase its half-life, or modulate receptor binding or effector function. Exemplary mutations are discussed in Saunders KO (Front. Immunol. 10:1296, 2019; the entire contents of which are incorporated herein by reference), including, for example, mutation of asparagine 297 (e.g., N297).

[0796] In some embodiments, the dimerization domain comprises an Fc region or portion thereof of an antibody.

[0797] In some embodiments, the dimerization domain comprises an Fc region or portion thereof, with or without Fc modifications.

[0798] In other embodiments, the dimerization domain comprises an altered or mutated Fc region or portion thereof. For example, the Fc region or portion thereof may be altered or mutated to modify one or more characteristics of the binding agent. In exemplary embodiments, the Fc region or portion thereof is aglycosylated. In other exemplary embodiments, the Fc region or portion thereof comprises one or more of the Fc modifications in Table 1.

[0799] In other exemplary embodiments, the Fc region or portion thereof is altered or mutated to increase ADCC activity. For example, defucosylation (e.g., N297 according to the EU numbering system) may result in increased FcRgII binding on NK cells, potentially increasing ADCC. Thus, in some embodiments, the Fc region or portion thereof may have a reduced number of fucose residues. In other embodiments, the binding agent comprises a defucosylated Fc region or portion thereof. In yet other embodiments, the Fc region or portion thereof of the binding agent lacks fucose residues. In additional embodiments, the Fc region or portion thereof of the binding agent lacks core fucose residues. In further embodiments, the Fc region or portion thereof of the binding agent completely lacks core fucose residues.

[0800] Exemplary, non-limiting embodiments of mutations in the constant region (e.g., Fc region) that improve one or more effector functions are encompassed by the present disclosure, exemplary embodiments of which are provided in Table 1 (List of Mutations Antibodies (Basel). 2020 Nov. 17;9(4):64, the entire contents of which are incorporated herein by reference).

[0801] [Table 1]

[0802] According to the present disclosure, the mutant CH3 domain comprises one or more mutations compared to a native CH3 domain. Thus, in some embodiments, the dimerization domain comprises a mutant CH3 domain comprising one or more mutations compared to a native CH3 domain.

[0803] In some embodiments, the dimerization domain comprises a native CH2 domain and a mutated CH3 domain. Exemplary embodiments of mutated CH3 domains are provided in PCT / CA2020 / 051753, filed December 18, 2020, and published June 24, 2021 under number WO2021 / 119832A1, the entire contents of which are incorporated herein by reference. For example, a heterodimer may be formed by co-transfection of a nucleic acid sequence encoding a polypeptide chain (chain A) comprising a mutated CH3 domain set forth in SEQ ID NO:218 and a nucleic acid sequence encoding a polypeptide chain (chain B) comprising a mutated CH3 domain set forth in SEQ ID NO:219.

[0804] Linker (L) The different modules of the polypeptide chains disclosed herein may be associated with each other via linkers.

[0805] In some embodiments, the linker used to join one or more modules of the polypeptide chain is not a cleavable linker.

[0806] In an exemplary embodiment, the linker (Lc) located immediately adjacent to the C-terminus of the dimerization domain does not comprise a cleavable linker.

[0807] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains does not comprise a cleavable linker.

[0808] In other embodiments, the linker used to join one or more modules of the polypeptide chain may comprise a non-cleavable linker.

[0809] In an exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain is a non-cleavable linker.

[0810] In another exemplary embodiment, at least one of the linkers located between the two antigen-binding domains is a non-cleavable linker.

[0811] In a further exemplary embodiment, the linker located immediately adjacent to the C-terminus of the dimerization domain and the linker joining the first two antigen-binding domains located C-terminal to the dimerization domain are non-cleavable linkers.

[0812] In some embodiments, the linker immediately adjacent to the N-terminus of the dimerization domain may preferably comprise the hinge region of an antibody.

[0813] In some embodiments, the hinge region is a native hinge region.

[0814] In some embodiments, the native hinge region is a native IgG1 hinge region. In some embodiments, the native hinge region is a native human IgG1 hinge region.

[0815] In some embodiments, the native hinge region is a native IgG2 hinge region, hi other embodiments, the native hinge region is a native human IgG2 hinge region.

[0816] In some embodiments, the native hinge region is a native IgG3 hinge region, hi other embodiments, the native hinge region is a native human IgG3 hinge region.

[0817] In some embodiments, the native hinge region is a native IgG4 hinge region. In some embodiments, the native hinge region is a native human IgG4 hinge region.

[0818] In some embodiments, the hinge region is a mutated hinge region.

[0819] In some embodiments, the mutated hinge region is a mutated IgG1 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG1 hinge region.

[0820] In some embodiments, the mutated hinge region is a mutated IgG2 hinge region, hi other embodiments, the mutated hinge region is a mutated human IgG2 hinge region.

[0821] In some embodiments, the mutated hinge region is a mutated IgG3 hinge region, hi other embodiments, the mutated hinge region is a mutated human IgG3 hinge region.

[0822] In some embodiments, the mutated hinge region is a mutated IgG4 hinge region. In some embodiments, the mutated hinge region is a mutated human IgG4 hinge region.

[0823] In some embodiments, all modules of the polypeptide chain are linked via a non-cleavable linker.

[0824] Exemplary embodiments of non-cleavable linkers include those that remain substantially intact during protein expression or manufacturing processes. As used herein, "substantially intact" means that linker cleavage occurs in 20% or less, 15% or less, 10% or less, 7.5% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the total polypeptide content of a given solution or composition.

[0825] Other exemplary embodiments of non-cleavable linkers also include linkers that do not contain specific cleavage sites for one or more proteases present in human or animal blood or serum.

[0826] Additional exemplary embodiments of non-cleavable linkers further include linkers that retain their integrity upon administration of the binding agent in an individual for at least 1, 2, 3, 4, 5, 6, 12, 24, 48 hours or longer following administration.

[0827] In a further exemplary embodiment, the linker comprises both a non-cleavable linker and a cleavable linker.

[0828] In some embodiments, the linker is not cleavable.

[0829] In some cases, cleavable linkers may be used for in vivo release of a drug (e.g., a cytostatic molecule, a cytotoxic molecule, a chemotherapeutic agent, etc.) or label attached to a polypeptide chain of the present disclosure.

[0830] Exemplary embodiments of cleavable linkers are provided, for example, in U.S. Patent Application Publication No. 2019 / 0010242, and include linkers that are susceptible to cleavage by proteases, typically extracellular proteases, such as proteases produced by tumors or activated immune effector cells, such as ADAMS, ADAMTS, e.g., ADAMS; ADAMS; ADAM10; ADAM12; ADAM15; ADAM17 / TACE; ADAMDEC1; ADAMTS1; ADAMTS4; ADAMTS5; aspartic proteases, e.g., BACE or renin; aspartic cathepsins, e.g., cathepsin D or cathepsin E; caspases, e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, or caspase 14; cysteine ​​cathepsins, e.g., cathepsin B, cathepsin C, cathepsin K, cathepsin L, cathepsin S, cathepsin V / L2, cathepsin X / Z / P; cysteine ​​proteinases, e.g., cruzipain; legumain; otubain-2; KLK, e.g., KLK4, KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13, or KLK14; metalloproteinase, e.g., meprin; neprilysin; PSMA; BMP-1; MMPs, such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP23, MMP24, MMP26, or MMP27, serine proteases, such as activated protein C, cathepsin A, cathepsin G, chymase, coagulation factor proteases (e.g., FVIIa, FIXa, FXa, FXIa, FXIIa), elastase, granzyme B, guanidinobenzoatase, HtrAl, human neutrophil elastase, lactoferrin, marapsin, NS3 / 4A, PACE4, plasmin, PSA, tPA, thrombin, tryptase, uPA;and those having a site for specific cleavage by a protease selected from a type II transmembrane serine protease (TTSP), such as DESC1, DPP-4, FAP, hepsin, matriptase-2, matriptase, TMPRSS2, TMPRSS3, or TMPRSS4; and any combination thereof. In some embodiments, the polypeptide chains of the present disclosure do not contain such a linker at a position corresponding to Lc.

[0831] Exemplary embodiments of linkers include flexible linkers, rigid linkers, helical linkers, and combinations thereof. Linkers are discussed, for example, in Chen X et al. (Adv Drug Deliv Rev. 2013;65(10):1357-1369), the entire contents of which are incorporated herein by reference.

[0832] In some embodiments, a hinge region or a portion thereof may be used to link a module to a dimerization domain and is considered herein as a linker. The hinge region may be derived from a natural antibody (of human or animal origin) or a synthetic antibody. The hinge region may be obtained, for example, from an IgG, such as IgG1, IgG2, IgG3, or IgG4. Exemplary embodiments of hinge regions are provided in SEQ ID NO: 180, SEQ ID NO: 203, SEQ ID NO: 207, and SEQ ID NO: 211.

[0833] In some cases, the hinge region may have one or more amino acid substitutions, insertions, and / or deletions compared to the native hinge region. A mutant hinge region may, for example, comprise a sequence (mutant hinge region) that is 80% to 99% identical to the sequence of a native IgG1, IgG2, IgG3, or IgG4 hinge region. An exemplary, non-limiting example of a mutant hinge region is the hinge region of IgG4 in which S228 is replaced with P (EU numbering) (Angal, S. et al., Mol Immunol 30, 105-108, 1993). Other exemplary embodiments of mutant hinges are provided in SEQ ID NOs: 118-120, 122-124, 126-128, and 130-132.

[0834] Flexible linkers are typically composed of small polar amino acids, such as threonine or serine, and glycine. An exemplary, non-limiting embodiment of a flexible linker is a GS linker (glycine / serine repeat), such as (GGGS) n (GGGGS) m (SEQ ID NO: 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), and the like, wherein n and m can independently be an integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or greater, for example, 15, 20, or 25.

[0835] SEQ ID NO: 186 has the formula (GGGGS) n (n is an integer selected from 1 to 10) or alternatively represented by the formula GGGGSX1 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 186).

[0836] SEQ ID NO: 271 is a sequence of the formula (GGGS) n(GGGGS) m (n and / or m are independently an integer selected from 1 to 10) or alternatively may be represented by the formula GGGSX1GGGGSX2 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 271, and X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 6 to 10 of SEQ ID NO: 271).

[0837] SEQ ID NO: 272 is a sequence of the formula (GGGS) n (n is an integer selected from 1 to 10) or alternatively may be represented by the formula GGGSX1 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 272).

[0838] SEQ ID NO: 273 is a sequence of the formula (GGSG) n (n is an integer selected from 1 to 10) or alternatively represented by the formula GGSGX1 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 4 of SEQ ID NO: 273).

[0839] SEQ ID NO: 274 is the formula (GGGSS) n (n is an integer selected from 1 to 10) or alternatively may be represented by the formula GGGSSX1 (X1 is absent, or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 274).

[0840] According to the present disclosure, n may be an integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater, for example, 15, 20, or 25.

[0841] According to the present disclosure, m can be an integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater, for example, 15, 20, or 25.

[0842] Specific exemplary, non-limiting embodiments of flexible linkers include those comprising or consisting of the amino acid sequence set forth in SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185 or SEQ ID NO:186.

[0843] Other exemplary, non-limiting embodiments of linkers include those comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 271, 272, 273, or 274.

[0844] SEQ ID NO: 186 has the formula (GGGGS) n (n is an integer selected from 1 to 10) or alternatively represented by the formula GGGGSX1 (X1 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 5 of SEQ ID NO: 186).

[0845] The rigid linkers of the present disclosure typically comprise a proline-rich sequence (XP) n wherein X represents any amino acid, preferably Ala, Lys, or Glu, and n is an integer, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc. (Chen X et al., 2013).

[0846] Specific exemplary, non-limiting embodiments of rigid linkers include those comprising or consisting of the amino acid sequence set forth in SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189 or SEQ ID NO:190.

[0847] SEQ ID NO: 190 is a compound of the formula (X(PAPAP)) n It should be understood that SEQ ID NO: 190 may be represented by the formula (XPAPAP)X2KA, where X may be present or absent and, if present, is A, and X2 is absent or, if present, is 1 to 9 repeats of amino acid residues 1 to 6 of SEQ ID NO: 190.

[0848] Helical linkers may be characterized as rigid, but are separated into distinct linker families herein. Exemplary embodiments of helical linkers are discussed in Chen X et al., 2013, and include, for example, repeats of alanine residues flanked by positively and negatively charged amino acid residues.

[0849] Specific exemplary, non-limiting embodiments of helical linkers include those 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.

[0850] SEQ ID NO: 194 is of the formula X(EAAAK) n It should be understood that X and X2 may be independently present or absent and, when present, preferably A. Alternatively, SEQ ID NO: 194 may be represented by the formula X(EAAAK)X3X2, where X and X2 are independently present or absent and, when present, preferably A; and X3 is absent or, if present, is 1 to 9 repeats of amino acid residues 2 to 6 of SEQ ID NO: 194.

[0851] In an exemplary embodiment, the linker (L in Formulas II-VIII) immediately adjacent to the C-terminus of the dimerization domain c1 The linker (identified as n) may comprise either a flexible linker, a rigid linker, or a helical linker. Linkers that may be specifically selected to occupy this position include, without limitation, linkers comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 182-191, 193, or 194 (where n is 1).

[0852] In an exemplary embodiment, a linker (L in Formulas II-VIII) joining the first two antigen-binding domains located C-terminal to the dimerization domain is c2The linker (identified as n) may comprise either a flexible linker, a rigid linker, or a helical linker. Linkers that may be specifically selected to occupy this position include, without limitation, linkers comprising or consisting of the amino acid sequence set forth in SEQ ID NOs: 182-192 or SEQ ID NO: 194 (where n is 1).

[0853] The present disclosure also provides linkers having the addition of 1 to 10 amino acids (and any range or value between 1 and 10, e.g., 1 to 5, etc.) at one or both of the N-terminus or C-terminus of any of SEQ ID NOs: 182-194. These additional amino acid residues may each be independently selected from any amino acid residue. These additional amino acid residues preferably form a non-cleavable sequence.

[0854] The present disclosure also provides linkers having deletions of 1, 2, 3, 4, or 5 amino acids (and any value between 1 and 5) at one or both of the N-terminus or C-terminus of any of SEQ ID NOs: 182-194.

[0855] Suitable linkers may comprise, for example, an amino acid sequence containing about 3 to about 50, about 3 to about 40, about 3 to about 30, about 3 to about 25, about 3 to about 20, about 3 to about 15, or about 3 to about 10 amino acid residues.

[0856] In exemplary embodiments, the length of each linker can independently be in the range of about 5 to about 50 amino acid residues, including, for example, about 5 to about 40 amino acid residues, about 10 to about 40 amino acid residues, about 20 to about 40 amino acid residues, about 20 to about 35 amino acid residues, about 25 to about 30 amino acid residues, and any subranges within and including such ranges.

[0857] In some embodiments, a linker comprising the amino acid sequence set forth in SEQ ID NO:186, SEQ ID NO:190 or SEQ ID NO:194 may have an "n" value of preferably 1 to 10, more preferably 2 to 5, including 2, 3, 4 or 5.

[0858] Binder Binding agents of the disclosure include, for example, the antigen-binding domains disclosed herein.

[0859] Binding agents of the present disclosure include, for example, the polypeptide chains disclosed herein. Thus, in some embodiments, a binding agent may be composed of a single polypeptide chain. In some embodiments, a binding agent is a polypeptide chain.

[0860] Binding agents of the present disclosure include, for example, dimers of the polypeptide chains disclosed herein.

[0861] Binding agents of the present disclosure include, for example, multimers of the polypeptide chains disclosed herein.

[0862] The binding agents of the present disclosure are capable of binding to CD36 protein or a portion thereof. In some embodiments, the binding agents comprise one or more antigen-binding domains capable of binding to CD36 protein or a portion thereof, or to cells expressing CD36 protein or a portion thereof. According to the present disclosure, the one or more antigen-binding domains are from an anti-CD36 antibody or an antigen-binding fragment thereof. Also according to the present disclosure, the one or more antigen-binding domains are from an anti-CD36 single-domain antibody.

[0863] Exemplary, non-limiting embodiments of antigen-binding domains are provided in Table 3, Table 4, and Table 5.

[0864] The binding agents of the present disclosure may also be capable of binding to immune checkpoint proteins and / or proteins expressed on the surface of immune cells, and thus 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.

[0865] In some embodiments, a binding agent of the present disclosure may comprise an antigen-binding domain that binds to CD36, an antigen-binding domain that binds to PD1, and an antigen-binding domain that binds to CD47. In some embodiments, a binding agent of the present disclosure may be capable of bringing T cells into proximity with CD36-expressing tumor cells, may be capable of restoring T cell function, and / or may be capable of enhancing macrophage function via blockade of SIRPα / CD47 interaction. In some embodiments of the present disclosure, a binding agent may exhibit its anti-tumor activity via antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments of the present disclosure, a binding agent may exhibit its anti-tumor activity via antibody-dependent cellular phagocytosis (ADCP) activity.

[0866] Thus, in some exemplary embodiments, the binding agent may comprise one or more antigen-binding domains capable of binding to a PD-1 protein or portion thereof, or a cell expressing a PD-1 protein or portion thereof, hi other exemplary embodiments, the binding agent may comprise one or more antigen-binding domains capable of binding to a CD47 protein or portion thereof, or a cell expressing a CD47 protein or portion thereof.

[0867] In some embodiments, the binding agents of the present disclosure may have the ability to reduce tumor growth and / or cause tumor regression. In some embodiments, the binding agents have anti-tumor activity. In some embodiments, the binding agents can recruit or redirect immune cells.

[0868] In some cases, the binding agents of the present disclosure may modulate lipid metabolism.

[0869] In other cases, the binding agents of the present disclosure may inhibit fatty acid oxidation.

[0870] In still other cases, the binding agents of the present disclosure may inhibit fatty acid uptake by tumors.

[0871] In other cases, the binding agents of the present disclosure may be capable of inhibiting the growth of tumor cells.

[0872] In still other cases, the binding agents of the present disclosure may be capable of inhibiting tumor metastasis.

[0873] In still other cases, the binding agents of the present disclosure may be capable of reducing the resistance of tumor cells to chemotherapeutic agents and / or immune checkpoint inhibitor therapy.

[0874] Binding agents of the present disclosure may have the format of antibodies and antigen-binding fragments thereof, antibody-like molecules (Fc, CH3 fusions, etc.), protein scaffolds and fusions with immune cell modulating agents, etc.

[0875] In some embodiments, the binding agent comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the binding agent is an antibody or an antigen-binding fragment thereof.

[0876] In some embodiments, the binding agent comprises an antibody-like molecule. In some embodiments, the binding agent is an antibody-like molecule.

[0877] In some embodiments, the binding agent may be fused to the protein scaffold.

[0878] In some embodiments, the binding agent comprises an immune cell modulating agent. In some embodiments, the binding agent is an immune cell modulating agent.

[0879] Thus, in some embodiments, binding agents include antibodies and antigen-binding fragments thereof, such as, but not limited to, single domain antibodies from camelids or sharks, human antibodies including IgG (including human IgG1, human IgG2, human IgG3, human IgG4), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD, animal antibodies including, for example, IgG (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE, and IgD, and the like.

[0880] In some embodiments, the binding agent comprises the antigen-binding domain of a single domain antibody. In yet other embodiments, the binding agent comprises the antigen-binding domain of one or more single domain antibodies.

[0881] For example, the binding agent may comprise the CDRs of one or more single domain antibodies. In another example, the binding agent may comprise the variable regions (VH or VHH) of one or more single domain antibodies.

[0882] In some embodiments, the binding agent comprises a single domain antibody.

[0883] In some embodiments, the binding agent is a single domain antibody.

[0884] In other embodiments, the binding agent comprises an antigen-binding fragment, such as, but not limited to, Fab, Fab', F(ab')2, complementarity determining regions, and variable regions, including VH, VHH, and VL.

[0885] Binding agents also include immune cell modulators, such as dual affinity retargeting molecules (DARTs), chimeric antigen receptor (CAR) constructs, bispecific T cell engager constructs (BiTEs), bispecific killer cell engagers (BiKEs), trispecific killer cell engagers (TriKEs) containing scFvs or VHHs, etc.

[0886] Binding agents also include fusions with protein scaffolds, including ankyrin repeat proteins, the Z-domain of Staphylococcus protein-A, fibronectin type III, and knottins, among others. Exemplary embodiments of binding agents include monospecific, bispecific (symmetric or asymmetric), trispecific, or multispecific antibodies, as well as monovalent, bivalent, trivalent, or multivalent antibodies, single chain FVs (scFVs) and derivatives such as diabodies, triabodies, tetrabodies, tandem di-scFvs, tandem tri-scFvs, scFV-Fc, minibodies (scFV-CH3), tandem diabodies, didiabodies, and bibodies, VHs or VHHs and derivatives such as tandem bispecific or multispecific VHHs, bivalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, bivalent CH3 fusions, VHH pentabodies, and decabodies.

[0887] In some embodiments, the binding agents of the present disclosure may have a format as disclosed in PCT / CA2020 / 051753, filed December 18, 2020, and published June 24, 2021 under WO2021 / 119832, such as Formula Ia, Formula Ib, Formula Ic, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII, or as disclosed herein as Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII.

[0888] The binding agents of the present disclosure may be formed by the assembly of two polypeptide chains having the same composition (having the same or different amino acid sequences) or different compositions, including compositions set forth in Formula I, Formula II, Formula III, Formula IIIa and IIIb, Formula IV, Formula V, Formula VI, Formula VII, or Formula VIII disclosed herein.

[0889] Binding agents of the present disclosure can be, for example, N-terminal to C-terminally represented by Formula I: X-[(Ab a )-(L b )] m -(DD)-[(Lc )-(Ab d )] n -Y and m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain.

[0890] In some embodiments, when m is 1, 2, or an integer greater than 2, Ab a or Ab d One of them is antigen-binding domain 1 (ABD1), and Ab a or Ab d the other of which is selected from antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

[0891] In some embodiments, when n is 1, 2, or an integer greater than 2, Ab a or Ab d One of them is antigen-binding domain 1 (ABD1), and Ab a or Ab d the other of which is selected from antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

[0892] 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, Ab a or Ab dAt least one of the Abs is antigen-binding domain 1 (ABD1), a or Ab d is antigen-binding domain 2 (ABD2), and a or Ab d is antigen-binding domain 3 (ABD3).

[0893] In some embodiments, L b In some embodiments, L b2 and / or L b3 does not have to exist.

[0894] In some embodiments, L c In some embodiments, L c1 , L c2 , and / or L c3 does not have to exist.

[0895] In some embodiments, L b and L c Both do not have to be present.

[0896] In some embodiments, the polypeptide chain comprises more than one antigen-binding domain.

[0897] In some embodiments, the polypeptide chain comprises two or more antigen-binding domains.

[0898] In some embodiments, the polypeptide chain comprises three or more antigen-binding domains.

[0899] In some embodiments, the polypeptide chain comprises four or more antigen-binding domains.

[0900] In some embodiments, the polypeptide chain comprises five or more antigen-binding domains.

[0901] In some embodiments, the polypeptide chain comprises six or more antigen-binding domains.

[0902] In some embodiments, the polypeptide chain comprises between 1 and 12 antigen-binding domains.

[0903] In some embodiments, the binder may be 1 to 12, e.g., 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 11, 1 to 12, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, 3 to 11, 3 to 12, 4 to 5, 4 to 6, 4 to 7, 4 to 8, 4 to 9, 4 ... to 9, 4 to 10, 4 to 11, 4 to 12, 5 to 6, 5 to 7, 5 to 8, 5 to 9, 5 to 10, 5 to 11, 5 to 12, 6 to 7, 6 to 8, 6 to 9, 6 to 10, 6 to 11, 6 to 12, 7 to 8, 7 to 9, 7 to 10, 7 to 11, 7 to 12, 8 to 9, 8 to 10, 8 to 11, 8 to 12, 9 to 10, 9 to 11, 9 to 12, 10 to 11, 10 to 12, or 11 to 12 antigen-binding domains.

[0904] In some embodiments, the binding agent comprises one polypeptide chain.

[0905] In some embodiments, the binding agent comprises two polypeptide chains.

[0906] In some embodiments, the binding agent comprises three polypeptide chains.

[0907] In some embodiments, the binding agent comprises four polypeptide chains.

[0908] In some embodiments, the binding agent comprises five polypeptide chains.

[0909] In some embodiments, the binding agent comprises six polypeptide chains.

[0910] In some embodiments, the binding agent comprises seven polypeptide chains.

[0911] In some embodiments, the binding agent comprises eight polypeptide chains.

[0912] In some embodiments, the binding agent comprises nine polypeptide chains.

[0913] In some embodiments, the binding agent comprises 10 polypeptide chains.

[0914] In some embodiments, the binding agent comprises more than 10 polypeptide chains.

[0915] In some embodiments, the binding agent has Formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula II).

[0916] In some embodiments, the binding agent has Formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0917] In some embodiments, the binding agent has Formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0918] In some embodiments, the binding agent has formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2)-(Ab d2 )-Y (Formula V).

[0919] In some embodiments, the binder has formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0920] In some embodiments, the binder has Formula VII: X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula VII).

[0921] In some embodiments, the binder has 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).

[0922] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Aba1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1).

[0923] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 1 (ABD1), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 One of them represents antigen-binding domain 3 (ABD3).

[0924] In some embodiments, the binding agent has the formula III in N-terminal to C-terminal fashion X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) and one or more polypeptide chains each independently comprising the amino acid sequence of Ab a1 represents antigen-binding domain 1 (ABD1), and Ab d1 represents antigen-binding domain 2 (ABD2), and Ab d2 represents antigen-binding domain 3 (ABD3).

[0925] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in an N-terminal to C-terminal manner, an antigen-binding domain that binds CD36, an antigen-binding domain that binds PD-1, and an antigen-binding domain that binds CD47.

[0926] In some embodiments, the binder is represented in an N-terminal to C-terminal manner by Formula III (Ab a1 is the antigen-binding domain that binds to CD36, and Ab d1 is the antigen-binding domain that binds to PD-1, and Ab d2 is an antigen-binding domain that binds to CD47).

[0927] In some embodiments, the binding agent comprises one or more polypeptide chains each independently comprising, in N-terminal to C-terminal fashion, the amino acid sequence of Formula III, and Ab a1 is the antigen-binding domain that binds to CD36, and Ab d1 is the antigen-binding domain that binds to CD47, and Ab d2 is the antigen-binding domain that binds to PD-1.

[0928] In some embodiments, the binding agent has the formula IIIa in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa) The polypeptide chains comprise one or more polypeptide chains each independently comprising the amino acid sequence of

[0929] In some embodiments, the binding agent has the formula IIIb in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb) The polypeptide chains comprise one or more polypeptide chains each independently comprising the amino acid sequence of

[0930] In some embodiments, the binding agent comprises two identical polypeptide chains.

[0931] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula II (with the same or different amino acid sequences).

[0932] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula III (with the same or different amino acid sequences).

[0933] In some embodiments, both polypeptide chains of the binding agent may have the structure set forth in Formula IIIa (with the same or different amino acid sequences).

[0934] In some embodiments, both polypeptide chains of a binding agent may have the structure set forth in Formula IIIb (with the same or different amino acid sequences).

[0935] In some embodiments, one polypeptide chain may have the structure set forth in Formula IIIa and one polypeptide chain may have the structure set forth in Formula IIIb (with the antigen-binding domains having the same or different amino acid sequences).

[0936] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula II, and the other may have the configuration shown in Formula III.

[0937] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula II, and the other has the configuration shown in Formula IV.

[0938] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula III, and the other has the configuration shown in Formula IV.

[0939] In some embodiments, one of the polypeptide chains may have the configuration shown in Formula IV, and the other has the configuration shown in Formula IV.

[0940] In some embodiments, the binding agents of the present disclosure are monospecific or multispecific.

[0941] Monospecific binding agents include binding agents that are specific for a single epitope on a given antigen.

[0942] Exemplary embodiments of monospecific binding agents include binding agents that comprise one antigen-binding domain. Another exemplary embodiment of a monospecific binding agent includes binding agents that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and framework regions. Yet another exemplary embodiment of a binding agent chain includes binding agents that comprise more than one antigen-binding domain, but the antigen-binding domains have identical CDRs and different framework regions. Further exemplary embodiments of monospecific binding agents include binding agents that comprise antigen-binding domains that differ in the amino acid sequence of one or more of their CDRs (e.g., conservative substitutions in one or more CDRs) without affecting their ability to bind to the same antigen or epitope.

[0943] Multispecific binding agents include binding agents that are specific for more than one epitope or more than one antigen (of the same or different antigens). For example, a multispecific polypeptide chain or binding agent may thus have more than one antigen-binding domain, at least two of which bind to different antigens or epitopes.

[0944] Binding agents of the present disclosure may therefore be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, etc. In some embodiments, each antigen-binding domain may be specific for a given antigen. In some embodiments, two or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, three or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, four or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, five or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. In some embodiments, six or more antigen-binding domains of a given binding agent may be specific for the same or different antigens. Specificity may depend on the number of antigen-binding domains present in a given binding agent.

[0945] Exemplary, non-limiting embodiments of multispecific binding agents include those composed of multispecific polypeptide chains. Other exemplary, non-limiting embodiments of multispecific binding agents include those with two antigen-binding domains of different specificities. Still other exemplary, non-limiting embodiments of multispecific binding agents include those with more than two antigen-binding domains that bind to two different antigens, proteins, or two different epitopes on the same antigen or protein.

[0946] In some embodiments, the binding agents of the present disclosure are monovalent or multivalent.

[0947] Exemplary, non-limiting embodiments of multivalent binding agents include binding agents comprised of multivalent polypeptide chains. Other non-limiting exemplary embodiments of multivalent binding agents include binding agents comprised of more than one monovalent polypeptide chain.

[0948] According to the present disclosure, a bispecific binding agent may be bivalent or multivalent depending on the number of antigen-binding domains it contains. Exemplary non-limiting embodiments of bispecific binding agents include those that comprise two identical bispecific polypeptide chains that form a dimer.

[0949] Exemplary, non-limiting embodiments of binders are provided in Tables 6 and 7.

[0950] Other exemplary embodiments of binding agents include those comprising the amino acid sequence shown in Table 3 fused at the C-terminus to the amino acid sequence shown in SEQ ID NO: 178.

[0951] Still other exemplary embodiments of binding agents include those that comprise the amino acid sequences shown in Table 3 and include a linker, a dimerization domain, and optionally, one or more antigen-binding domains with different amino acid sequences.

[0952] Other exemplary embodiments of binding agents include those comprising the amino acid sequence shown in Table 6 fused at the C-terminus to the amino acid sequence shown in SEQ ID NO: 179.

[0953] Competitive binders Binding agents of the present disclosure also include those that compete with the binding agents disclosed herein or with single domain antibodies having the amino acid sequences disclosed herein.

[0954] Antibodies that compete with one or more single domain antibodies or antigen-binding fragments thereof having the amino acid sequences disclosed herein are provided. In some embodiments, the antibody is a single domain antibody that competes with one or more single domain antibodies or antigen-binding fragments thereof having the amino acid sequences disclosed herein.

[0955] In an exemplary embodiment, the binding agent may comprise one or more antigen-binding domains of an antibody that competes with one or more single-domain antibodies or antigen-binding fragments thereof having the amino acid sequences disclosed herein.

[0956] A competitive binding agent of the present disclosure may comprise an amino acid sequence having at least 75% or more identity to an amino acid sequence shown in any one of Tables 3, 4, 5, 6, and / or 7, or a combination thereof. Alternatively, a competitive binding agent may comprise an amino acid sequence having less than 75% identity to an amino acid sequence shown in any one of Tables 3, 4, 5, 6, and / or 7, or having a different CDR1, CDR2, and / or CDR3, but still capable of binding to the same target (CD36, PD-1, and / or CD47) and remaining functional. Such competitive binding agents may be identified by a competition assay using the target and a pre-identified binding agent (e.g., Table 6 and / or Table 7). The functionality of a competitive binding agent is confirmed by testing its activity, including, but not limited to, its ability to reduce fatty acid oxidation, lipid metabolism and / or fatty acid uptake by cells.

[0957] In exemplary embodiments, competition between binding agents may be determined based on any type of binding assay (FACS, ELISA, SPR, etc.) using recombinant CD36 or a portion thereof or cells expressing CD36 or a portion thereof.

[0958] In an exemplary embodiment, the competing binding agent has the ability to block CD36 or comprises one or more antigen-binding domains that have the ability to block CD36.

[0959] In some embodiments, the binding agent comprises one or more antigen-binding domains of an antibody (competing antibody) that has the ability to compete with a single-domain antibody comprising antigen-binding domain 1 (ABD1) as set forth herein. In other embodiments, the binding agent comprises one or more antigen-binding domains of an antibody that has the ability to compete with an anti-CD36 single-domain antibody that comprises an antigen-binding domain having the CDRH1, CDRH2, and / or CDRH3 amino acid sequences disclosed herein. More particularly, the binding agent comprises one or more antigen-binding domains of an antibody that has the ability to compete with an anti-CD36 single-domain antibody that comprises an antigen-binding domain having the CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[0960] In some embodiments, at least one of the one or more antigen-binding domains of the competing antibody comprises the CDRH1, CDRH2 and / or CDRH3 amino acid sequence of an anti-CD36 single domain antibody in Table 3.

[0961] In some embodiments, at least one of the one or more antigen-binding domains of the competing antibody comprises: a. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 7; b. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 14; c. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 21; d. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 28; e. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 35; f. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 42; g. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 49; h. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 56; i. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 77, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 77; j. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 84, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 84; k. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 91, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 91; l. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 98, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 98; m. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 105; n. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 112; o. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 119; p. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 126; q. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 133; or r. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 140. Includes:

[0962] The competing antibody may be a competing single domain antibody or antigen-binding fragment thereof.

[0963] In some embodiments, the binding agent may also comprise one or more antigen-binding domains of an anti-PD-1 single domain antibody disclosed herein.

[0964] In some embodiments, at least one of the one or more antigen-binding domains is from an anti-PD-1 antibody (competing antibody) that has the ability to compete with a single-domain antibody comprising antigen-binding domain 2 (ABD2) described herein.

[0965] In other embodiments, the binding agent may also comprise one or more antigen-binding domains of an antibody that has the ability to compete with an anti-PD-1 single domain antibody that comprises an antigen-binding domain having the CDRH1, CDRH2, and / or CDRH3 amino acid sequences disclosed herein. More particularly, the binding agent comprises one or more antigen-binding domains of an antibody that has the ability to compete with an anti-PD-1 single domain antibody that comprises an antigen-binding domain having the CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[0966] In some embodiments, the anti-PD-1 antibody comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 63, 281, 288, 295, 302, 309, 316, 323, 330, 337, 344, 351, 358, and / or 365, or has one to ten amino acid substitutions, additions, and / or deletions in any one of SEQ ID NOs: 281, 288, 295, 302, 309, 316, 323, 330, 337, 344, 351, 358, and / or 365.

[0967] In some embodiments, the binding agent may also comprise one or more antigen-binding domains of an anti-CD47 single domain antibody disclosed herein.

[0968] In some embodiments, at least one of the one or more antigen-binding domains is from an anti-CD471 antibody (competing antibody) that has the ability to compete with a single-domain antibody comprising antigen-binding domain 3 (ABD3) as described herein.

[0969] In other embodiments, the binding agent may also comprise one or more antigen-binding domains of an antibody having the ability to compete 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 particularly, the binding agent comprises one or more antigen-binding domains of an antibody having the ability to compete with an anti-CD47 single domain antibody comprising an antigen-binding domain having the CDRH1, CDRH2, and CDRH3 amino acid sequences disclosed herein.

[0970] In yet 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 that has 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO:70.

[0971] In some embodiments, the binding agent comprises one or more antigen-binding domains of an antibody having the ability to compete with an anti-PD-1 single domain antibody disclosed herein or one or more antigen-binding domains of an antibody having the ability to compete with an anti-CD47 single domain antibody disclosed herein, wherein at least one of the one or more antigen-binding domains of the antibody is: a. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:7; b. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 14; c. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 21; d. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 28; e. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 35; f. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 42; g. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO:49; h. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 56; i. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 77; j. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 84; k. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 91; l. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 98; m. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 105; n. An amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 112; o. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 119; p. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 126; q. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 133, or r. an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 140 Includes:

[0972] Exemplary Embodiments of Monospecific Binding Agents As indicated herein, the binding agents of the present disclosure may be monospecific.

[0973] Exemplary, non-limiting, exemplary embodiments of monospecific binding agents are provided herein.

[0974] For example, a binding agent may comprise only one antigen-binding domain. Alternatively, a binding agent may comprise more than one identical antigen-binding domain.

[0975] Exemplary embodiments of monospecific binding agents comprise an antigen-binding domain disclosed herein comprising ABD1, ABD2, ABD3 or a heavy chain disclosed herein.

[0976] In an exemplary embodiment, the monospecific binding agent may comprise an antigen-binding domain and a dimerization domain disclosed herein.

[0977] Exemplary embodiments of dimerization domains are provided throughout this specification and include, without limitation, a CH3 domain (including native CH3 domains and native human CH3 domains, mutated CH3 domains), and may also include a CH2 domain (native CH2 domain or mutated CH2 domain).

[0978] In some embodiments, the dimerization domain is an Fc region of an antibody or portion thereof.

[0979] In some embodiments, the dimerization domain may also comprise a hinge region of an antibody or portion thereof. In some embodiments, the hinge region or portion thereof is a human hinge region or portion thereof. In some cases, a linker or additional amino acid sequence may be added between the antigen-binding domain and the dimerization domain, or at any end thereof.

[0980] In some embodiments, the binding agent may comprise ABD1 and a dimerization domain.

[0981] In some embodiments, the binding agent may comprise ABD2 and a dimerization domain.

[0982] In some embodiments, the binding agent may comprise ABD3 and a dimerization domain.

[0983] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:7.

[0984] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0985] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:21.

[0986] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:28.

[0987] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:35.

[0988] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:42.

[0989] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49.

[0990] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:56.

[0991] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:77.

[0992] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:84.

[0993] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:91.

[0994] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:98.

[0995] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:105.

[0996] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:112.

[0997] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:119.

[0998] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:126.

[0999] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:133.

[1000] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:140.

[1001] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:63.

[1002] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:281.

[1003] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:288.

[1004] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:295.

[1005] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:302.

[1006] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:309.

[1007] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:316.

[1008] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:323.

[1009] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:330.

[1010] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:337.

[1011] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:344.

[1012] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:351.

[1013] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:358.

[1014] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:365.

[1015] In some exemplary embodiments, a binding agent of the present disclosure may comprise an amino acid sequence and dimerization domain that is at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1016] It should be understood herein that a binding agent may optionally comprise one or more linkers or additional amino acid sequences.

[1017] Other exemplary embodiments of monospecific binding agents are provided herein, including, without limitation, single domain antibodies comprising the amino acid sequence of, or of, a single domain antibody in Table 6.

[1018] Exemplary Embodiments of Multispecific Binding Agents As indicated herein, the binding agents of the present disclosure may be multispecific.

[1019] Exemplary, non-limiting exemplary embodiments of multispecific binding agents include those provided herein.

[1020] For example, in some embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein.

[1021] In other embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to a protein expressed on the surface of an immune cell.

[1022] In still other embodiments, the binding agent may comprise more than one antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) as described herein, wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to an immune checkpoint protein, and wherein at least one of the antigen-binding domains is an antigen-binding domain capable of binding to a protein expressed on the surface of a tumor cell.

[1023] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 2 (ABD2).

[1024] In some embodiments, the binding agent comprises at least one antigen binding domain 1 (ABD1) and at least one antigen binding domain 3 (ABD3).

[1025] In some embodiments, the binding agent comprises at least one antigen binding domain 2 (ABD2) and at least one antigen binding domain 3 (ABD3).

[1026] In some embodiments, the binding agent comprises at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2), and at least one antigen-binding domain 3 (ABD3).

[1027] In some embodiments, the binding agent is a multispecific binding agent comprising two polypeptide chains capable of forming a dimer, wherein each polypeptide chain comprises an antigen-binding domain 1 (ABD1) comprising an amino acid sequence 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. ), 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 antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 80%, 85%, 90%, 95% or 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1028] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:7; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1029] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 14; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1030] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 21; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1031] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:28; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1032] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 35; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1033] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 42; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1034] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:49; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO:70.

[1035] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 56; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1036] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 77; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1037] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 84; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1038] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 91; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1039] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 98; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1040] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 105; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1041] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 112; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1042] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 119; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1043] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 126; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1044] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 133; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1045] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 140; at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 63; and at least one of the antigen-binding domains is antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 70.

[1046] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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 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.

[1047] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1048] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1049] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1050] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1051] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1052] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1053] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1054] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1055] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1056] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1057] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1058] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1059] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1060] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1061] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1062] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1063] In some embodiments, the binding agent comprises one or more antigen-binding domains, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises 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.

[1064] In other exemplary embodiments of the binding agent, SEQ ID NO:63 may be replaced with other anti-PD1 VHH sequences, such as, without limitation, SEQ ID NO:281, SEQ ID NO:288, SEQ ID NO:295, or SEQ ID NO:337.

[1065] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 159. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in SEQ ID NO: 159. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99% or 100% identical to the amino acid sequence set forth in SEQ ID NO: 159. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in SEQ ID NO: 159.

[1066] In some embodiments, the binding agent comprises one or more polypeptide chains, wherein at least one of the polypeptide chains has an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 160-177. In some embodiments, the binding agent comprises one polypeptide chain having the amino acid sequence set forth in any one of SEQ ID NOs: 160-177. In other embodiments, the binding agent comprises two polypeptide chains having amino acid sequences at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 160-177. In yet other embodiments, the binding agent comprises two polypeptide chains having the amino acid sequence set forth in any one of SEQ ID NOs: 160-177.

[1067] It should be understood herein that a binding agent may optionally comprise one or more linkers or additional amino acid sequences.

[1068] Other exemplary embodiments of multispecific binding agents are provided herein, including those in Table 7.

[1069] In some embodiments, the sequence of a monospecific binding agent in Table 6 may be inserted at the N-terminal portion of SEQ ID NO: 179 to create a hexavalent trispecific binding agent containing an anti-PD-1 VHH and an anti-CD47 VHH.

[1070] In some embodiments, any of the VHH sequences in Table 3 may be inserted at the N-terminal portion of SEQ ID NO: 178 to generate a hexavalent trispecific binding agent containing an anti-PD-1 VHH and an anti-CD47 VHH.

[1071] variant Variants of the sequences disclosed herein are also encompassed by the present disclosure.

[1072] Variants encompassed by the present disclosure include those that may include an insertion of one or more amino acid residues at one or more positions, a deletion of one or more amino acid residues at one or more positions, or a substitution (conservative or non-conservative substitution) of one or more amino acid residues at one or more positions.

[1073] For example, naturally occurring residues are divided into groups based on common side chain properties. Conservative substitutions may be made by exchanging an amino acid from one of the groups listed below (Groups 1-6) for another amino acid from the same group. Non-conservative substitutions involve exchanging a member of one of these groups for a member of another group. (Group 1) Hydrophobic: norleucine, methionine (Met), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile) (Group 2) Neutral hydrophilic: cysteine ​​(Cys), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), (Group 3) Acidic: Aspartic acid (Asp), glutamic acid (Glu) (Group 4) Basic: histidine (His), lysine (Lys), arginine (Arg) (Group 5) residues that affect chain orientation: glycine (Gly), proline (Pro); and (Group 6) Aromatic: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[1074] Other exemplary embodiments of conservative substitutions are shown in Table 2 (Table 2) under the heading of "preferred substitutions." If such substitutions result in undesirable properties, more substantial changes such as those designated "exemplary substitutions" in Table 2 (Table 2), or as further described below with respect to amino acid classes, may be introduced and the products screened.

[1075] One of ordinary skill in the art will recognize that certain amino acid...

Claims

1. A binding agent comprising 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. 2. The binding agent of claim 1, wherein the binding agent comprises more than one antigen-binding domain, and 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. 2. The binding agent of claim 1, wherein the binding agent comprises more than one antigen-binding domain, and 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. 4. The binding agent of any one of claims 1 to 3, comprising 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 2 to 12 antigen-binding domains.

5. 5. The binding agent of claim 1, comprising 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 antigen-binding domain capable of specifically binding to CD36 is antigen-binding domain 1 (ABD1); a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 3; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO:4, CDRH2 having the amino acid sequence set forth in SEQ ID NO:5, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:6; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO:8, CDRH2 having the amino acid sequence set forth in SEQ ID NO:9, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:10; d. CDRH1 having the amino acid sequence set forth in SEQ ID NO:11, CDRH2 having the amino acid sequence set forth in SEQ ID NO:12, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:13; e. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 15, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 16, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 17; f. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 18, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 19, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 20; g. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 22, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 23, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 24; h. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 25, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 26, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 27; i. CDRH1 having the amino acid sequence set forth in SEQ ID NO:29, CDRH2 having the amino acid sequence set forth in SEQ ID NO:30, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:31; j. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 32, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 33, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 34; k. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 36, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 37, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 38; l. CDRH1 having the amino acid sequence set forth in SEQ ID NO:39, CDRH2 having the amino acid sequence set forth in SEQ ID NO:40, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:41; m. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 43, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 44, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 45; n. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 46, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 47, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 48; o. CDRH1 having the amino acid sequence set forth in SEQ ID NO:50, CDRH2 having the amino acid sequence set forth in SEQ ID NO:51, and CDRH3 having the amino acid sequence set forth in SEQ ID NO:52; p. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 53, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 54, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 55; q. 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 shown 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; oo. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 7; pp. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 14; qq. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 21; rr. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 28; ss. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 35; tt. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 42; uu. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 49 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 49; vv. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 56; ww. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 77 or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 77; xx. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 84, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 84; yy. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 91, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 91; zz. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 98 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 98; aaa. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 105; bbb. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 112; ccc. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119 or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 119; ddd. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 126; eee. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 133; or fff. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 140 6. The binder according to claim 1, comprising:

7. at least one of the one or more antigen-binding domains is antigen-binding domain 2 (ABD2) capable of specifically binding to PD-1; a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 57, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 58, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 59; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 60, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 61, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 62; c. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 275, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 276, and 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 at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 63; ee. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 281 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 281; ff. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 288 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 288; gg. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 295, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 295; hh. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 302, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 302; ii. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 309, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 309; jj. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 316, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 316; kk. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 323 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 323; ll. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 330, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 330; mm. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 337, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 337; nn. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 344, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO: 344; oo. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 351 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 351; pp. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 358 or having 1 to 10 amino acid substitutions, additions and / or deletions in SEQ ID NO: 358; or qq. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 365 or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO:

365.

7. The binder according to claim 1, comprising:

8. at least one of the one or more antigen-binding domains is antigen-binding domain 3 (ABD3) having the ability to specifically bind to CD47; a. a heavy chain complementarity determining region 1 (CDRH1) having the amino acid sequence set forth in SEQ ID NO: 64, a heavy chain complementarity determining region 2 (CDRH2) having the amino acid sequence set forth in SEQ ID NO: 65, and a heavy chain complementarity determining region 3 (CDRH3) having the amino acid sequence set forth in SEQ ID NO: 66; b. CDRH1 having the amino acid sequence set forth in SEQ ID NO: 67, CDRH2 having the amino acid sequence set forth in SEQ ID NO: 68, and CDRH3 having the amino acid sequence set forth in SEQ ID NO: 69; or c. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 70, or having 1 to 10 amino acid substitutions, additions, and / or deletions in SEQ ID NO:

70.

8. The binder according to claim 1, comprising:

9. 8. The binding agent of claim 7, comprising at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 2 (ABD2).

10. 9. The binding agent of claim 8, comprising at least one antigen-binding domain 1 (ABD1) and at least one antigen-binding domain 3 (ABD3).

11. 11. The binding agent of any one of claims 8 to 10, comprising at least one antigen-binding domain 1 (ABD1), at least one antigen-binding domain 2 (ABD2) and at least one antigen-binding domain 3 (ABD3).

12. At least one of the one or more antigen-binding domains is antigen-binding domain 1 (ABD1) and comprises an amino acid sequence 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; and at least one of the one or more antigen-binding domains is antigen-binding domain 2 (ABD2).

12. The binding agent of claim 1, wherein the one or more antigen-binding domains comprise an amino acid sequence 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 antigen-binding domain 3 (ABD3) and comprises an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO:

70.

13. a. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 7, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; b. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 14, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; c. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; d. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; e. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; f. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; g. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 49, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; h. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; i. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 77, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; j. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 84, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; k. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 91, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; l. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 98, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; m. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 105, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; n. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 112, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; o. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 119, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; p. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 126, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; q. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 133, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 70; r. at least one antigen-binding domain 1 (ABD1) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 140, at least one antigen-binding domain 2 (ABD2) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 63, and at least one antigen-binding domain 3 (ABD3) comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

70.

13. The binder of claim 12, comprising:

14. 14. The binding agent of any one of claims 1 to 13, wherein the one or more antigen-binding domains are on one or more polypeptide chains.

15. 15. The binding agent of any one of claims 1 to 14, wherein the one or more antigen-binding domains are on the same polypeptide chain.

16. 16. The binding agent of any one of claims 1 to 15, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises one or more antigen-binding domains.

17. 17. The binding agent of any one of claims 1 to 16, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises a different antigen-binding domain.

18. 17. The binding agent of any one of claims 1 to 16, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain comprises the same antigen-binding domain.

19. 19. The binding agent of any one of claims 1 to 18, wherein the binding agent comprises at least two polypeptide chains capable of assembling to form a dimer, and each polypeptide chain is identical.

20. Each of the polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula I: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y comprising the amino acid sequence m is 0, 1, 2 or an integer greater than 2; n is 0, 1, 2 or an integer greater than 2; m and n are not simultaneously 0; Ab a , Ab d each represent an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1); X or Y are independently present or absent and comprise an amino acid sequence; L b , L c each independently comprises one or more linkers; and DD represents the dimerization domain; 20. The binder according to any one of claims 14 to 19.

21. When m is an integer of 1, 2 or greater than 2, and / or n is an integer of 1, 2 or greater than 2, Ab a or Ab d 21. The binding agent of claim 20, wherein at least one of said domains is antigen-binding domain 2 (ABD2) and / or antigen-binding domain 3 (ABD3).

22. When m is an integer equal to or greater than 2, Ab a or [(Ab a )-(L b 22. The binder of claim 20 or 21, wherein the )] units are the same.

23. When m is an integer equal to or greater than 2, Ab a or [(Ab a )-(L b 22. The binder of claim 20 or 21, wherein the )] units are different.

24. When m is an integer greater than 2, the [(Ab a )-(L b 22. The binder of claim 20 or 21, wherein the )] units comprise the same and different units.

25. When n is an integer greater than or equal to 2, Ab d or the above [(L c )-(Ab d 25. The binder of claim 20, wherein the )] units are the same.

26. When n is an integer greater than or equal to 2, Ab d or the above [(L c )-(Ab d 25. The binder according to claim 20, wherein the )] units are different.

27. When n is an integer of 2 or more, the [(L c )-(Ab d 25. The binder of claim 20, wherein the )] units comprise the same and different units.

28. 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. Ab a represents ABD1, and Ab d represents ABD2, and / or Ab d represents ABD3.

30. m is 1 and Ab a represents ABD1.

31. n is 2 and Ab d 31. The binding agent of any one of claims 20 to 30, wherein one of the following represents ABD2.

32. n is 2 and Ab d 31. The binding agent of any one of claims 20 to 30, wherein one of the following represents ABD3.

33. n is 2 and Ab d One of them represents ABD2 and the other Ab d represents ABD3.

34. L b is the hinge region of an antibody or antigen-binding fragment thereof, and / or L c 34. The binding agent of any one of claims 20 to 33, wherein is a rigid linker.

35. each of said polypeptide chains independently comprises, in N-terminal to C-terminal fashion: 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(form VI); X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y(form VII); X-(Ab a1 )-(L b3 )-(Ab a2 )-(L b2 )-(Ab a3 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y(Formula VIII) comprising any one of the amino acid sequences: Ab a1 , Ab a2 , Ab a3 , Ab d1 , Ab d2 , Ab d3 each of which represents an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1); L b1 comprises one or more linkers and / or hinge regions of an antibody or antigen-binding fragment thereof; L b2 , L b3 , L c1 , L c2 , and L c3 each independently comprises one or more linkers; and DD represents the dimerization domain; 35. The binder according to any one of claims 14 to 34.

36. 36. The binding agent of claim 35, wherein at least one of the antigen-binding domains is antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3).

37. 37. The binding agent of 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. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 represents the antigen-binding domain 1 (ABD1), 38. The binder according to any one of claims 14 to 37.

39. each of said polypeptide chains independently has, in N-terminal to C-terminal fashion, the formula III X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III) comprising the amino acid sequence of Ab a1 , Ab d1 or Ab d2 one of which represents the antigen-binding domain 1 (ABD1), and Ab a1 , Ab d1 or Ab d2 one of which represents the antigen-binding domain 2 (ABD2), and Ab a1 , Ab d1 or Ab d2 represents the antigen-binding domain 3 (ABD3), 38. The binder according to any one of claims 14 to 37.

40. Each of the polypeptide chains independently has the formula IIIa in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD2)-(L c2 )-(ABD3)-Y (Formula IIIa) comprising the amino acid sequence 40. The binder according to any one of claims 14 to 39.

41. Each of the polypeptide chains independently has the formula IIIb in N-terminal to C-terminal fashion: X-(ABD1)-(L b1 )-(DD)-(L c1 )-(ABD3)-(L c2 )-(ABD2)-Y (Formula IIIb) comprising the amino acid sequence 40. The binder according to any one of claims 14 to 39.

42. 42. The binding agent of any one of claims 20 to 41, wherein the dimerization domain comprises an antibody constant region or a portion thereof.

43. 42. The binding agent of any one of claims 20 to 41, wherein the dimerization domain comprises a CH3 domain and optionally a CH2 domain.

44. 42. The binding agent of any one of claims 20 to 41, wherein the dimerization domain comprises a native CH2 domain and a native CH3 domain.

45. 45. The binding agent of claim 44, wherein the native CH2 and / or native CH3 domain is from human IgG1.

46. 46. ​​The binding agent of any one of claims 20 to 45, wherein each of the linkers independently has a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

47. 47. The binding agent of any one of claims 20 to 46, wherein each of the linkers is independently a flexible linker, a helical linker, or a rigid linker.

48. 48. The binding agent of any one of claims 20 to 47, wherein the linker comprises a flexible linker and / or a rigid linker.

49. L c1 , L c2 and / or L c3 is a rigid linker.

50. L b1 is a hinge region of an antibody or antigen-binding fragment thereof.

51. 51. The binding agent of claim 50, wherein the hinge region is from IgG1, IgG2, IgG3, or IgG4.

52. 52. The binding agent of any one of claims 1 to 51, which is multispecific.

53. 53. The binding agent of claim 52, which is bispecific, trispecific, or tetraspecific.

54. 54. The binding agent of any one of claims 1 to 53, which is multivalent.

55. 55. The binding agent of any one of claims 14 to 54, wherein each of the polypeptide chains has the same valency and specificity.

56. 56. The binding agent of any one of claims 14 to 55, wherein each of the polypeptide chains has a different valency and specificity.

57. 57. The binding agent of any one of claims 14 to 56, wherein each of the polypeptide chains is an antibody heavy chain.

58. 58. The binding agent of any one of claims 1 to 57, wherein one or more antigen-binding domains are humanized.

59. 59. The binding agent of any one of claims 20-58, 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. 60. The binding agent of any one of claims 1 to 59, which is or comprises an antibody or antigen-binding fragment thereof.

61. 61. The binding agent of any one of claims 1 to 60, which is a single domain antibody.

62. 62. The binding agent of any one of claims 1 to 61, comprising a single domain antibody.

63. 63. The binding agent of any one of claims 1 to 62, which is a bispecific antibody.

64. 64. The binding agent of claim 63, wherein the bispecific antibody further comprises a first antibody light chain and a second antibody light chain.

65. 65. The binding agent of any one of claims 1 to 64, which is an antibody-like molecule.

66. 66. The binding agent of any one of claims 1 to 65, comprising a protein scaffold.

67. 67. The binding agent of any one of claims 1 to 66, comprising an immune cell modulating agent.

68. a. an amino acid sequence 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 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 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 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 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 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 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 at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 148; i. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 149; j. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 150; k. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 151; l. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 152; m. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 153; n. An amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 154; o. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 155; p. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 156; q. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 157, or r. an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence set forth in SEQ ID NO: 158 68. The binder of any one of claims 1 to 67, comprising:

69. 69. The binding agent of claim 68, comprising an amino acid sequence at the C-terminus that is at least 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence set forth in SEQ ID NO:

179.

70. 7. The binding agent of claim 6, comprising an amino acid sequence at the C-terminus that is at least 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence set forth in SEQ ID NO:

178.

71. 71. The binding agent of any one of claims 1 to 70, comprising an amino acid sequence set forth in SEQ ID NO: 159 or at least 75%, 80%, 85%, 90%, 95%, 99% identical to the amino acid sequence set forth in SEQ ID NO:

159.

72. 72. The binding agent of claim 71, comprising two polypeptide chains each having an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO:

159.

73. 73. The binding agent of any one of claims 6 to 72, wherein the amino acid substitutions, additions or deletions are outside the complementarity determining regions.

74. 74. A binding agent having the ability to compete with the binding agent of any one of claims 1 to 73.

75. the competing binder is a. the amino acid sequence set forth in SEQ ID NO: 380; b. The amino acid sequence set forth in SEQ ID NO: 381; c. The amino acid sequence set forth in SEQ ID NO: 382; d. the amino acid sequence set forth in SEQ ID NO: 383; e. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 7, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 7; f. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 14, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 14; g. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 21, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 21; h. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 28, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 28; i. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 35, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 35; j. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 42, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 42; k. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO:49, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:49; l. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 56, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 56; m. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 77, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 77; n. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 84, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 84; o. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 91, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 91; p. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 98, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 98; q. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 105, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 105; r. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 112, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 112; s. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 119, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 119; t. an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 126, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO: 126; an amino acid sequence at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 133, or having 1 to 10 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 at least 75%, 80%, 85%, 90%, 95% identical to the amino acid sequence set forth in SEQ ID NO: 140, or having 1 to 10 amino acid substitutions, additions, and / or deletions in the amino acid sequence set forth in SEQ ID NO:

140.

75. The binding agent of claim 74, which is an antibody or antigen-binding fragment thereof comprising a heavy chain variable domain comprising:

76. 76. The binding agent of claim 74 or 75, comprising one or more antigen-binding domains of an antibody that specifically binds to a PD-1 protein or portion thereof.

77. 77. The binding agent of any one of Claims 74 to 76, comprising one or more antigen-binding domains of an antibody that specifically binds to CD47.

78. A binding agent that specifically binds to cluster of differentiation 36 (CD36) or a portion thereof or a cell expressing CD36 or a portion thereof, and is a single domain antibody that modulates tumor lipid metabolism and / or inhibits tumor cell growth.

79. A binding agent that specifically binds to CD36 or cells expressing CD36, PD-1 or cells expressing PD-1, and CD47 protein or cells expressing CD47.

80. 80. The binding agent of any one of claims 1 to 79, conjugated to a therapeutic moiety.

81. 81. The binding agent of any one of claims 1 to 80, conjugated to a detectable moiety.

82. 82. The binding agent of any one of claims 1 to 81, conjugated to a protein allowing for an extended half-life.

83. 83. The binding agent of any one of claims 1 to 82, attached to a nanoparticle.

84. 84. The binding agent of any one of claims 1 to 83, having the ability to modulate lipid metabolism of tumor cells.

85. 85. The binding agent of any one of claims 1 to 84, which has antitumor activity.

86. 86. The binding agent of any one of claims 1 to 85, having the ability to reduce tumor growth.

87. 87. The binding agent of any one of claims 1 to 86, which has the ability to cause tumor regression.

88. 88. The binding agent of any one of claims 1 to 87, wherein CD36 is a human CD36 protein or a portion thereof.

89. 89. The binding agent of any one of claims 1 to 88, wherein PD-1 is a human PD-1 protein or a portion thereof.

90. 90. The binding agent of any one of claims 1 to 89, wherein CD47 is a human CD47 protein or a portion thereof.

91. 91. A composition comprising the binder of any one of claims 1 to 90.

92. 91. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 90 and a pharmaceutically acceptable carrier.

93. 93. The pharmaceutical composition of claim 92, comprising a binder having a purity level of 80.0% to 99.9%.

94. 91. A nucleic acid or set of nucleic acids encoding a binding agent according to any one of claims 1 to 90.

95. 95. A vector comprising the nucleic acid of claim 94.

96. 91. A cell expressing the binding agent of any one of claims 1 to 90.

97. 96. A cell comprising the nucleic acid of claim 94 or the vector of claim 95.

98. 91. A kit comprising the binding agent of any one of claims 1 to 90.

99. 98. A kit comprising a nucleic acid according to claim 94, or a vector according to claim 95, or a 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 a binding agent described in any one of claims 1 to 90, a composition described in claim 91 or a pharmaceutical composition described in claim 92 or 93.

101. 94. A method of treating cancer, comprising administering to a subject in need thereof a binding agent of any one of claims 1 to 90, a composition of claim 91, or a pharmaceutical composition of claim 92 or 93.

102. 91. A method of treating cancer in a subject in need thereof, comprising administering a nucleic acid molecule encoding the binding agent of any one of claims 1 to 90 or a composition comprising same.

103. 103. The method of claim 101 or 102, wherein the cancer is leukemia.

104. 104. The method of claim 103, wherein the leukemia is acute myeloid leukemia.

105. 105. The method of any one of claims 101 to 104, wherein the subject in need has relapsed leukemia.

106. 103. The method of claim 101 or 102, wherein the cancer is selected from the group consisting of 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., bladder urothelial carcinoma), brain cancer, oral cancer, head and neck cancer, glioblastoma (e.g., glioblastoma multiforme), adrenocortical carcinoma, colon cancer, and colorectal cancer.

107. 107. The method of any one of claims 101 to 106, wherein the subject in need has failed previous treatment with chemotherapy and / or immune checkpoint inhibitor therapy.

108. 108. The method of any one of claims 101 to 107, wherein the cancer is associated with high CD36 expression.

109. 109. The method of any one of claims 101 to 108, wherein the cancer is associated with increased lipid metabolism.

110. 110. The method of any one of claims 101 to 109, wherein the cancer originates from fat-rich tissue.

111. 95. A method for producing a binding agent according to any one of claims 1 to 89, comprising transforming a cell with one or more vectors comprising the nucleic acid or set of nucleic acids according to claim 94.

112. 112. The method of claim 111, wherein the binding agent is purified from the cells.

113. 113. The method of claim 112, wherein the binder has a purity level of 80.0% to 99.9%.

114. 114. The method of any one of claims 111 to 113, further comprising conjugating the binding agent to a therapeutic moiety, a detectable moiety, a protein allowing for extended half-life, or attaching the binding agent to a nanoparticle.

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