A conjugate that targets TROP2-expressing tumor cells.

Abstract

This disclosure relates to conjugates, such as antibodies and their antigen-binding fragments, that are capable of binding to trophoblast cell surface antigen-2 (TROP2) in general. Similarly, this disclosure also discloses conjugates capable of targeting TROP2-expressing tumor cells and their use for the treatment of cancer. A single-domain antibody that specifically binds to amino acid residues of the extracellular domain of TROP2 is provided.

Description

[Technical Field]

[0001] This disclosure relates to conjugates, such as antibodies and their antigen-binding fragments, that are capable of binding to trophoblast cell surface antigen-2 (TROP2) in general. Similarly, this disclosure also discloses conjugates capable of targeting TROP2-expressing tumor cells and their use for the treatment of cancer. A single-domain antibody that specifically binds to amino acid residues of the extracellular domain of TROP2 is provided. [Background technology]

[0002] Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed at low levels in normal adult tissues. TROP2 has multifaceted functions, including inducing cell signaling and cell-cell junction formation by mediating interactions between extracellular and intracellular proteins. TROP2 has an extracellular epidermal growth factor (EGF)-like domain followed by a 23-amino acid transmembrane domain and a cytoplasmic end. The EGF-like domain contains a cysteine-rich domain, a thyroglobulin type 1 domain, and a cysteine-poor domain. In TROP2 deficiency, its role in normal tissues is thought to be compensated for by the expression of EpCAM, which is highly related to TROP2. TROP2 is highly expressed in a wide range of late-stage epithelial cancers, including breast and pancreatic cancer (Stepan et al., J. Histochem and Cytochem, 2011, the entire text of which is incorporated herein by reference). Overexpression of TROP2 has been shown to confer oncogenic behavior in several in vitro and in vivo models. Upregulation of TROP2 in solid tumors is associated with increased tumor pathogenicity, metastasis, and an overall decline in survival rates in a large population of difficult-to-treat cancers, making it an attractive target for cancer therapy.

[0003] TROP2-positive cancers have been clinically well targeted with antibody-drug conjugates (ADCs), but are not targeted by naked functional antibodies. ADCs present challenges such as drug toxicity in healthy tissue, off-target effects, and payload delivery. Given the dose-restricted side effects, narrow therapeutic window, and limited efficacy of ADCs, there is an urgent need to improve anti-TROP2 therapies for patients who, even if treatments exist, are not very helpful.

[0004] Camelids and cartilaginous fish naturally produce antibodies composed solely of functional homodimeric heavy chain antibodies (HCAb) (Hamers-Casterman et al., 1993; Muyldermans and Smider, 2016). The heavy chain of HCAb lacks a first constant domain (CH1) and differs from classical antibodies by a few amino acid substitutions that normally participate in light chain pairing (Muyldermans et al., 1994; Vu et al., 1997). These substitutions (Val37Phe / Tyr, Gly44Glu, Leu45Arg, and Trp47Gly) are located in framework region 2 (FR2). The antigen-binding fragment of HCAb is referred to as VHH or nanobody®. VHH has a molecular weight of approximately 15 kDa and is suitable for applications requiring enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic application, the VHH half-life usually needs to be prolonged to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al., Antibodies 8(1), pp. 1-22, 2019). Methods to prolong the VHH half-life, such as pegylation, N-glycosylation, and fusion with HSA or other carrier proteins, have been utilized, but such constructs may introduce immunogenicity or have limited efficacy.

[0005] The variable region of single-domain antibodies is used as a building block for constructing bispecific and polyspecific antibodies. Some studies have shown that bivalent constructs have increased binding affinity or affinity compared to monovalent constructs (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).

[0006] Novel TROP2 conjugates comprising antibodies and their antigen-binding fragments, such as single-domain antibodies and their antigen-binding fragments, are disclosed herein. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Application No. PCT / CA2021 / 050951 [Patent Document 2] WO2022 / 011457 [Patent Document 3] PCT / CA2020 / 051753 [Patent Document 4] WO2021 / 119832A1 [Patent Document 5] WO2022 / 011457 No. A1 [Patent Document 6] U.S. Patent No. 7,405,320 [Patent Document 7] U.S. Patent No. 4,704,692 [Patent Document 8] U.S. Patent No. 4,946,778 [Patent Document 9] U.S. Patent Application Publication No. 20110076275 [Non-patent literature]

[0008] [Non-Patent Document 1] Stepan, J. Histochem and Cytochem, 2011 [Non-licensed Document 2] De Vliegerら, Antibodies 8(1), pages 1~22, 2019 [Non-licensed Document 3] Goldenberg et al., 2018, Ikeda, M. et al., 2015 [Non-licensed Document 4] Kabat, J Immunol., 147: 1709-19(1991) [Non-licensed Document 5] Chothia C, Lesk AM, J Mol Biol. Aug 20;196(4):901-17(1987) [Non-licensed Document 6] Lefranc, M.-P., The Immunologist, 7, 132-136(1999)) [Non-licensed Document 7] Van Der Linden, 1999, Biochim Biophys Act 1431: pages 37~46 [Non-licensed Document 8] Hamsenら, 2007, Appl Microbiol Biotechnol 77: pages 13~22 [Non-licensed Document 9] R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Volume 9: Pages 73~80 (1995) [Non-licensed Document 10] RE Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Volume 9: Pages 132~137 (1991) [Non-licensed Document 11] Tatiana A. Tatusova, Thomas L. Madden(1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250 [Non-licensed Document 12] List of Mutations Antibodies(Basel). 2020 Nov 17;9(4):64 [Non-licensed Document 13] The Canadian Council on Animal Care (CCAC) guidelines [Non-licensed Document 14] The Animal Welfare Committee of UHN recognizes the Animal Use Protocol [Non-licensed Document 15] Kabsch, W. (2010a / b) Acta Cryst D66, pp. 125-132 [Non-licensed Document 16] Evans, PR and Murshudov, GN(2013)Acta Cryst D69, pages 1204~1214 [Non-licensed Document 17] Winn, M.ら, (2011) Acta Cryst D67, pages 235~242 [Non-licensed Document 18] McCoy, (2005) Acta Cryst D 61, pages 458~64 [Non-licensed Document 19] Evansら、(2022)bioRxiv doi: 10.1101 / 2021.10.04.463034 [Non-licensed Document 20] Emsley, P. & Cowtan, K. Coot, (2004) Acta Cryst D60, pp. 2126-32 [Non-licensed Document 21] Murshudov, (1997) Acta Cryst D53, pp. 240-255 [Non-licensed Document 22] Facility Guidelines for Sinclair Research Center, Missouri, USA [Overview of the project] [Means for solving the problem]

[0009] Therefore, the present disclosure generally relates to a binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains specifically binds to trophoblast cell surface antigen-2 (TROP2).

[0010] The binder, or at least one of its one or more binding domains, binds to human TROP2. Therefore, the binders of this disclosure may be used to treat human diseases or disorders associated with the expression or overexpression of TROP2.

[0011] In some embodiments, the binders of this disclosure may be able to bind to the extracellular domain (ECD) of TROP2 (represented by amino acids 27-274 of TROP2 (e.g., SEQ ID NO: 39 or SEQ ID NO: 40), the entire contents of which are incorporated herein by reference; see Goldenberg et al., 2018, Ikeda, M. et al., 2015).

[0012] In some embodiments, one or more antigen-binding domains of the binder include a complementarity-determining region (CDR), heavy chain variable region, or heavy chain of a single-domain antibody (sdAb) as disclosed herein.

[0013] The single-domain antibodies disclosed herein possess unique binding characteristics and activity. For example, the complementarity-determining region 3 (CDR3) of sdAb forms a loop that fits into a groove within the cysteine-rich domain (CRD) of the extracellular domain (ECD) of TROP2. The amino acid residues of CDR3 and framework region 2 (FR2) interact with the amino acid residues of the TROP2 cysteine-rich domain and together form a clip around the CRD as a biparatope antibody. These unique characteristics confer high affinity and slow dissociation rate to the sdAb disclosed herein. Surprisingly, the amino acid residues of complementarity-determining region 1 (CDR1) and complementarity-determining region 2 (CDR2) do not appear to interact with the amino acid residues of the CRD. Therefore, variations in the amino acid sequences of CDR1 and CDR2 are intended, as such variations do not affect binding to TROP2. Accordingly, one or more antigen-binding domains include at least the CDR3 amino acid residue.

[0014] In some cases, one or more antigen-binding domains may include CDR3 amino acid residues and framework region 2 (FR2) amino acid residues.

[0015] In other examples, one or more antigen-binding domains may include CDR3 amino acid residues, framework region 2 (FR2) amino acid residues, and optionally CDR1 and / or CDR2 amino acid residues.

[0016] In further embodiments, one or more antigen-binding domains may include CDR1, CDR2, and CDR3 amino acid residues.

[0017] In further embodiments, one or more antigen-binding domains may include CDR1, FR2, CDR2, and CDR3 amino acid residues.

[0018] In some embodiments, the CDR1, CDR2, CDR3, and FR2 amino acid residues correspond to the amino acid residues of the single-domain antibodies disclosed herein.

[0019] In accordance with this disclosure, CDRs may be identified using the Kabat numbering scheme (e.g., Kabat, J Immunol., 147: 1709-19(1991); Chothia C, Lesk AM, J Mol Biol. Aug 20;196(4):901-17(1987)).

[0020] Alternatively, CDRs can be identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

[0021] In some cases, CDR1, CDR2, CDR3 and / or FR2 correspond to Kabat CDR1, CDR2, CDR3 and / or FR2.

[0022] In other examples, CDR1, CDR2, CDR3 and / or FR2 correspond to IMGT CDR1, CDR2, CDR3 and / or FR2.

[0023] In some embodiments, one or more antigen-binding domains may include amino acid residues in a variable region.

[0024] For example, in some embodiments, one or more antigen-binding domains may include the amino acid sequence of a camelid VH or VHH. In other embodiments, one or more antigen-binding domains may include the amino acid sequence of a mouse VH. In yet another embodiment, one or more antigen-binding domains may include the amino acid sequence of a human VH. In a further embodiment, one or more antigen-binding domains may include the amino acid sequence of a humanized VH or VHH (e.g., humanized camelid VH or VHH, or humanized mouse VH).

[0025] In some cases, this disclosure relates to an anti-TROP2 single-domain antibody capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP). Furthermore, in other cases, this disclosure relates to an anti-TROP2 single-domain antibody capable of internal migration, thereby delivering a drug payload to the tumor microenvironment or tumor cells. In yet other cases, this disclosure relates to an anti-TROP2 single-domain antibody that interferes with / blocks TROP2 function.

[0026] The binders of this disclosure may be active against tumor cells even without conjugation, but this disclosure also encompasses conjugation of the binder with the therapeutic portion. Conjugation may be particularly beneficial when the binder is inactive or has low activity against tumor cells.

[0027] Therefore, this disclosure relates to an unconjugated (naked) single-domain antibody capable of binding to TROP2. This disclosure also relates to an antibody-drug conjugate (ADC) comprising a single-domain antibody capable of binding to TROP2 and a drug payload.

[0028] The binding function of sdAb is conferred by a single polypeptide chain, i.e., a heavy chain variable region. This inherent property provides flexibility in formatting. For example, multiple sdAbs or sdAb antigen-binding domains may be fused to a single polypeptide chain that confers polyvalent and / or polyspecificity. In addition, multiple polypeptide chains may be assembled to increase the diversity of interaction with TROP2 (multi-paratope binding) or avidity. Furthermore, the sdAb antigen-binding domain may be fused to any type of polypeptide chain, including, but not limited to, fusion with the heavy and / or light chains of innate antibodies or their antigen-binding fragments, fusion with protein scaffolds, fusion with immunocytomodulators, etc.

[0029] Therefore, this disclosure relates not only to the single-domain antibodies disclosed herein, but more generally to conjugates containing one or more antigen-binding domains of the single-domain antibodies disclosed herein.

[0030] In addition, this disclosure relates to conjugates that compete with the single-domain antibodies disclosed herein. For example, single-domain antibodies are provided that compete with one or more single-domain antibodies disclosed herein with respect to the binding of at least one epitope containing amino acid residues of the extracellular domain (ECD) of TROP2.

[0031] The binder, or the antigen-binding domain of the binder, can be derived from various sources, including animals capable of producing single-domain antibodies such as camelids, sharks, and transgenic animals, as well as from screening antibody libraries. For example, antibodies can be obtained from immunized hybridomas of rodents (mice, rats, etc.) or from larger mammals such as camelids. In some embodiments, animals may be immunized with the TROP2 antigen.

[0032] In accordance with this disclosure, the binder may comprise two heavy chain variable regions. Each heavy chain variable region may be located on the same or separate polypeptide chain. In some examples, the two heavy chain variable regions are linked to a dimerizing domain that enables the formation of a dimer.

[0033] In some embodiments, the binder comprises a polypeptide chain comprising, in an N-terminal to C-terminal manner, a) an antigen-binding domain of a single-domain antibody, b) optionally a linker, and c) a dimerization domain.

[0034] In some embodiments, the binder comprises a polypeptide chain comprising, in an N-terminal to C-terminal manner, a) a dimerizing domain, b) optionally a linker, and c) an antigen-binding domain for a single-domain antibody.

[0035] In other embodiments, the binder comprises a polypeptide chain comprising, in an N-terminal to C-terminal manner, a) an antigen-binding domain of a single-domain antibody, b) a linker, c) a dimerization domain, and d) a linker and e) an antigen-binding domain of a single-domain antibody, one or more of these.

[0036] In some embodiments, the antigen-binding domain of a) and the antigen-binding domain of e) are different. In some embodiments, the antigen-binding domain of a) and the antigen-binding domain of e) are the same. In some embodiments, the linker of b) and the linker of d) are the same. In some embodiments, the linker of b) and the linker of d) are different.

[0037] In other embodiments, the binder may have the format described in Formulas I, II, III, IIIa and IIIb, IV, V, VI, VII, or VIII disclosed herein.

[0038] In accordance with this disclosure, the binder may include additional amino acid sequences or portions (small molecules, labels, and others) at its N-terminus and / or C-terminus. For example, in accordance with this disclosure, the binder may include an additional antigen-binding domain of a single-domain antibody at its N-terminus or C-terminus. The additional antigen-binding domain may be separated from the core by one or more linkers.

[0039] In accordance with this disclosure, the binder or antigen-binding domain may include the sequences described herein. In accordance with this disclosure, the single domain may include the sequences described herein.

[0040] In some embodiments, the binder, or at least one of one or more antigen-binding domains, is: SEQ ID NOs: SEQ ID NOs: 231, 232, 239, 240, 247, 295, 296, 297, 311, 318, 325, 332, 339, 346, 353, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 3 Includes CDR1, CDR2, CDR3, FR2 or a combination thereof of the heavy chain variable domain described in any one of 82, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 405, SEQ ID NO: 412, SEQ ID NO: 419, SEQ ID NO: 426, SEQ ID NO: 433, SEQ ID NO: 440, SEQ ID NO: 447, SEQ ID NO: 454, SEQ ID NO: 461, SEQ ID NO: 468, SEQ ID NO: 491, SEQ ID NO: 493, SEQ ID NO: 496, SEQ ID NO: 502, or SEQ ID NO: 503.

[0041] In some embodiments, the binder, or at least one of one or more antigen-binding domains, is capable of binding to human trophoblast cell surface antigen 2 (human TROP2), and SEQ ID NOs: SEQ ID NOs: 231, 232, 239, 240, 247, 295, 296, 297, 311, 318, 325, 332, 339, 346, 353, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384 , includes an amino acid sequence that is at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical or identical to the amino acid sequence described in any one of the following: SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 405, SEQ ID NO: 412, SEQ ID NO: 419, SEQ ID NO: 426, SEQ ID NO: 433, SEQ ID NO: 440, SEQ ID NO: 447, SEQ ID NO: 454, SEQ ID NO: 461, SEQ ID NO: 468, SEQ ID NO: 491, SEQ ID NO: 493, SEQ ID NO: 496, SEQ ID NO: 502, or SEQ ID NO: 503.

[0042] In some embodiments, the binder, or at least one of one or more antigen-binding domains, has a sequence described herein (including the original sequence or the humanized sequence) and includes a variable region comprising one to five amino acid substitutions, one to five amino acid deletions, or one to five amino acid additions.

[0043] Specific embodiments of this disclosure include, for example, a conjugate such as a single-domain antibody comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds to trophoblast cell surface antigen 2 (TROP2), and: a) Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 225, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 226, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 320, SEQ ID NO: 392, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 227, or; b) Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 228, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 245, SEQ ID NO: 323, or SEQ ID NO: 395, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 230. Contains a binder.

[0044] In more specific embodiments of this disclosure, at least one of the antigen-binding domains of a binder or one or more antigen-binding domains of a single-domain antibody is a) Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 225, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 226, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 320, SEQ ID NO: 392, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 227, or; b) Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 228, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 245 or SEQ ID NO: 323 or SEQ ID NO: 395, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 230, Amino acid sequences that are at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or identical to the amino acid sequences described in SEQ ID NO: 373, SEQ ID NO: 232, SEQ ID NO: 231, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 372, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 247, SEQ ID NO: 388, SEQ ID NO: 325, SEQ ID NO: 390, SEQ ID NO: 397, or SEQ ID NO: 398 Includes.

[0045] In other specific embodiments, at least one of the antigen-binding domains of the binder or single-domain antibody includes a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 490, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in any one of SEQ ID NO: 493, SEQ ID NO: 496, or SEQ ID NO: 491.

[0046] In other specific embodiments, at least one of the antigen-binding domains of the binder or single-domain antibody includes a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 500, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 501, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in either SEQ ID NO: 503 or SEQ ID NO: 502.

[0047] In a more specific embodiment, at least one of the antigen-binding domains of the binder or single-domain antibody includes a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 308, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 309, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 310, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in either SEQ ID NO: 311 or SEQ ID NO: 389.

[0048] In some cases, at least one of the one or more antigen-binding domains contains the amino acid sequence described in SEQ ID NO: 539.

[0049] In an exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO: 540.

[0050] In other exemplary embodiments, the binder according to any one of the preceding embodiments is wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO: 541.

[0051] In an exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 514 to 516.

[0052] In another exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 517 to 519.

[0053] In another exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 520 to 522.

[0054] In an exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 523 to 525.

[0055] In another exemplary embodiment, the binder according to any one of the preceding embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 526 to 528.

[0056] In exemplary embodiments, the binder or antigen-binding domain of the present disclosure may have ADCP activity against cells expressing human TROP2 and differentiation antigen group 47 (CD47) (Uniprot accession number Q08722-1). Exemplary and non-limiting embodiments of the CD47 antigen are provided in SEQ ID NO: 176.

[0057] Therefore, a binder comprising one or more antigen-binding domains capable of binding to TROP2 and one or more antigen-binding domains capable of binding to CD47 and / or PD-1 is included in this disclosure.

[0058] In some embodiments, the binder is an antibody or its antigen-binding fragment (e.g., a single-domain antibody or its antigen-binding fragment), an antibody-like molecule, a protein scaffold structure, an immunocytomodulator, or similar.

[0059] This disclosure also relates to binders that may compete with one or more binders disclosed herein.

[0060] Other aspects and embodiments of this disclosure relate to compositions or pharmaceutical compositions comprising a binder disclosed herein and a pharmaceutically acceptable carrier.

[0061] Further aspects and embodiments of this disclosure relate to pharmaceutical compositions comprising a conjugated binder and a pharmaceutically acceptable carrier as disclosed herein.

[0062] Other aspects and embodiments of this disclosure relate to nucleic acids or sets of nucleic acids that encode the binders disclosed herein.

[0063] Additional aspects and embodiments of this disclosure relate to vectors comprising nucleic acids disclosed herein, or sets of vectors each comprising nucleic acids disclosed herein.

[0064] Further aspects and embodiments of this disclosure relate to cells expressing the binders disclosed herein.

[0065] In some embodiments, cells are manipulated to express a binder, such as an antibody or its antigen-binding fragment or antibody-like molecule.

[0066] Therefore, this disclosure includes isolated cells expressing an antibody, its antigen-binding fragment, or an antibody-like molecule.

[0067] In other embodiments, cells are manipulated to express a binding agent, such as a chimeric antigen receptor (CAR) construct. Thus, the disclosure includes T cells, NK cells, monocytes, or macrophages expressing a chimeric antigen receptor (CAR) construct.

[0068] Additional aspects and embodiments of this disclosure relate to cells comprising nucleic acids or vectors disclosed herein.

[0069] Further aspects and embodiments of this disclosure relate to kits comprising the binders disclosed herein.

[0070] In some embodiments, the kit may include a binder that binds to TROP2 and a binder that binds to a macrophage marker. For example, the kit may include an antibody or an antigen-binding fragment thereof that specifically binds to TROP2. In some examples, the kit may include the binders described herein. The kit may also include an antibody that binds to a macrophage marker, for example, an antibody or an antigen-binding fragment thereof that binds to CD68 (human CD68), but is not limited to these.

[0071] Further aspects and embodiments of this disclosure relate to kits comprising nucleic acids or vectors disclosed herein.

[0072] Further aspects and embodiments of this disclosure relate to kits comprising the cells disclosed herein.

[0073] In additional aspects and embodiments, the Disclosure relates to methods for treating disorders or diseases, comprising administering a binder, composition or pharmaceutical composition disclosed herein.

[0074] In some embodiments, the TROP2 binders, compositions, or pharmaceutical compositions disclosed herein are administered to subjects having cancer selected from epithelial cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), myeloma, prostate cancer, breast cancer (e.g., triple-negative breast cancer), rectal cancer, pancreatic cancer, glioblastoma, cervical cancer, colorectal cancer, gastric cancer, ovarian cancer, thyroid cancer, bladder cancer, uterine cancer, esophageal cancer, head and neck cancer, and hematological cancers.

[0075] In other embodiments, the TROP2 binders, compositions, or pharmaceutical compositions disclosed herein are administered to subjects having metastatic cancer.

[0076] Another aspect of the present disclosure is a method for treating cancer, comprising administering a binder disclosed herein to a subject having a tumor expressing TROP2 and one or more macrophage markers, such as but not limited to CD68.

[0077] In other aspects and embodiments, the Disclosure relates to methods for producing the conjugates disclosed herein by transforming cells with one or more vectors comprising the nucleic acids disclosed herein, and optionally by conjugating the conjugate to a therapeutic portion, a detectable portion, or a protein or nanoparticle that extends the half-life.

[0078] Further scope, applicability, and merits of this disclosure will become apparent from the non-limiting detailed description set forth below. However, it should be understood that this detailed description, while illustrating exemplary embodiments of this disclosure, is given merely as an example with reference to the accompanying drawings. [Brief explanation of the drawing]

[0079] [Figure 1A-D] This graph illustrates the time course of tumor volume in SCID mice carrying MDA-MB-453 (Figure 1A), MDA-MB-231 (Figure 1B), BxPC-3 (Figure 1C), or T.Tn (Figure 1D) treated with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb, or with PBS. [Figure 1E] This histogram shows the binding of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb to MDA-MB-453, MDA-MB-231, BxPC3, and A375 cells, as analyzed by flow cytometry, compared to the AR47A6.4.2 anti-TROP2 monoclonal antibody. The negative control is sdAb(NC) that binds to HEWL. [Figure 2A] This graph shows in vivo dose / response studies of weekly treatment with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb at doses of 0.5 mg / kg, 2 mg / kg, 8 mg / kg, and 30 mg / kg ip in an MDA-MB-453 transplanted NCG mouse tumor model. PBS was used as the negative control. [Figure 2B] This graph shows the in vivo weekly treatment of large tumors in an MDA-MB-453 transplanted NCG mouse tumor model with KD004, 8 mg / kg, once weekly (QW) in an ip (infusion). The negative control was PBS. Treatment was discontinued at approximately day 70 and replaced with PBS, while negative control treated mice were treated with KD004, 8 mg / kg QW. [Figure 2C]This graph shows the antitumor effects of various treatment regimens using KD004 at a dose of 8 mg / kg at the indicated frequency of ip in an MDA-MB-453 transplanted NCG mouse tumor model. PBS was used as the negative control. [Figure 2D] This graph shows the antitumor effects of various treatment regimens using KD002 or KD005 at a dose of 0.1 mg / kg in an intravenous (IP) approach in an MDA-MB-453 transplanted NCG mouse tumor model. PBS was used as the negative control. [Figure 2E] This figure shows the in vivo pharmacokinetic parameters of anti-TROP2. [Figure 3] This graph shows the time course of tumor volume in MDA-MB-453 transplanted NCG mice treated with KD004 at 8 mg / kg ip at the indicated intervals, with or without human PBMCs. PBS was used as the negative control. [Figure 4A] This histogram shows the cell viability of MDA-MB-453 cells after 12 days of treatment with 1 μM KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb, compared to AR47A6.4.2 anti-TROP2 monoclonal antibody. Negative controls include sdAb (NC) bound to HEWL or untreated cells. [Figure 4B] These are images of MDA-MB-453 cells at 4x resolution 12 days after treatment with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb. Negative controls include sdAb (NC) bound to HEWL or untreated cells. [Figure 5A] This histogram shows the cell viability of MDA-MB-453 cells reflecting the ADCC activity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb. Negative controls include untreated cells (no Ab), no effector cells, no target cells, or sdAb (NC) that binds to HEWL. Comparisons include cells treated with human IgG1 anti-TROP2 antibodies generated using sequences derived from clones hTINA-1 and h7E6. [Figure 5B]This figure shows that sdAb tumor growth inhibitory inhibitory (TGI) was tested in vivo in NCG and SCID mice transplanted with either MDA-MB-231 or MDA-MB-453 cells in sc, with different effector functional backgrounds. Administration of KD002 or KD004 was initiated when the mean tumor volume reached 100 mm3. [Figure 6A-B] These are histograms showing the internal migration of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb (Figure 6A) or AR47A6.4.2 (Figure 6B) after 4 or 24 hours in MDA-MB-453 cells. Negative controls consist of HEWL-binding sdAb (NC) or culture medium only. [Figure 6C] This graph illustrates the internal migration of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb in MDA-MB-453. Internal migration was evaluated by conjugation of HCAb with pH-sensitive probes and monitored by flow cytometry (Em. 560 nm). [Figure 6D] These are confocal microscopy images of MDA-MB-453 cells at 0, 6, and 24 hours, showing antibody binding and internal migration of KD002 and KD005 antibodies. One representative cell is shown for each time point. Cells were stained with anti-human IgG Fc-FITC, and the nuclei were counterstained with DAPI. Images were obtained using a confocal fluorescence microscope. Scale bar, 10 μm. [Figure 7A-D] This figure shows the binding of KD001, KD002, KD003, KD004, KD005, KD006, or KD007 anti-TROP2 sdAb to parental CHO (Figure 7A) or HEK (Figure 7C) cells or to human TROP2-expressing CHO (Figure 7B) or HEK cells (Figure 7D), as evaluated by flow cytometry, compared to a negative control (sdAb (NC) that binds to HEWL) or comparative monoclonal antibodies AR47A6.4.2, hTINA-1, h7E6, and hRS7. [Figure 7E]This bar graph illustrates the binding of KD001, KD002, KD003, KD004, KD005, KD006, or KD007 anti-TROP2 sdAb to recombinant human EpCAM (rhEpCAM) and recombinant human IGFBP-1 (rhIGFB-1) protein. [Figure 8] This graph illustrates competitive assays performed on human recombinant TROP2 using one of the His-tagged conjugates KD001 (Figure 8A), KD002 (Figure 8B), KD003 (Figure 8C), KD004 (Figure 8D), or KD005 (Figure 8E) against each of the KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb strains. [Figure 9A] This graph illustrates a competitive assay for the binding of human recombinant TROP2 between the His-tagged conjugate KD004 and hRS7 or AR47A6.4.2 antibodies. [Figure 9B] This graph illustrates a competitive assay between His-tagged KD006 and KD007 anti-TROP2 sdAb regarding the binding of human recombinant TROP2. [Figure 10A] This is a schematic diagram showing various constructs used to indicate the location of the binding domain of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb. [Figure 10B] This figure shows images of immunoblots performed on the human TROP2 extracellular domain (ECD) and human TROP2 cysteine-poor domain (CPD) using KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb, or AR47A6.4.2 and hRS7. The negative control includes sdAb(NC) that binds to HEWL. [Figure 11A-E] This figure shows images of immunoblots performed on various forms of recombinant human TROP2 (undenatured, linear, reduced, and deglycosylated using PNGase F) using KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb. [Figures 12A-12D]This graph shows the binding of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb to recombinant cynomolgus monkey / rhesus monkey TROP2 protein (Figure 12A), recombinant human TROP2 protein (Figure 12B), mouse TROP2 protein (Figure 12C), or rat TROP2 protein (Figure 12D). Negative controls include sdAb that bind to HEWL. Positive controls include anti-TROP2 polyclonal antibodies. [Figure 12E] This table illustrates the affinity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb to human TROP2 and to cynomolgus monkey TROP2, based on SPR. [Figure 12F] This table illustrates the affinity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb to human TROP2, using the indicated ka, kd, and KD. [Figures 13A-13B] This figure shows a histogram illustrating the binding of the indicated TROP2 antibody at 100 nM to HEK cells transfected with TROP2 constructs having various cysteine-rich domain shortenings. [Figure 13C] This graph shows tumor growth in a xenograft model of T.Tn in SCID mice treated with an anti-TROP2 antibody, 8 mg / kg ip, once per week. Treatment with KD006 resulted in a 48.6% inhibition of tumor growth in the T.Tn xenograft model. [Figure 14A] This graph shows the antibody-mediated cytophagous activity of the IgG1 or IgG4 versions of KD002 and KD004 against MDA-MB-453 tumors. [Figure 14B]This graph shows the expression levels of CD47 and TROP2 in MDA-MB-453, MDA-MB-231, and NCI-H292 cells, measured by staining with anti-CD47 (Biolegend, catalog number 323124) and anti-TROP2 (Biolegend, catalog number 363804) antibodies at a 1 / 100 dilution. The data are shown as a multiplier change in CD47 and TROP2 levels compared to isotype control staining. [Figure 14C-E] This graph shows the dose-response ADCP activity of KD002 and KD004 TROP2 antibodies against MDA-MB-453 (Figure 14C), MDA-MB-231 (Figure 14D), and NCI-H292 (Figure 14E) cells. The ADCP assay was performed using the Promega ADCP assay kit (Promega, catalog number CS314906). The test antibodies were serially diluted from 1.88 nM to 1 / 2.2 dilutions. TROP2 antibodies KD002 and KD004 showed high levels of ADCP activity against MDA-MB-453 cells, while showing relatively low ADCP activity against MDA-MB-231 and NCI-H292 cells. [Figure 14F] This graph illustrates the ADCP activity of KD002 and KD004 and their corresponding IgG4 versions; KD017 and KD018 against MDA-MB-453, MDA-MB-231, and NCI-H292 cells. The assays were performed at an antibody concentration of 1 nM. [Figure 15A] This graph illustrates the CD47 expression levels in MDA-MB-231 and NCI-H292 cells after CD47 knockdown using siRNA. CD47 siRNA and control siRNA were used in the knockdown assays (Thermofisher CD47 silencer select pre-designed siRNA (catalog number 4392421) and silencer negative control siRNA (catalog number AM4635)). [Figure 15B-C]This graph shows the dose-response ADCP activity of KD002 and KD005 TROP2 antibodies against MDA-MB-231 (Figure 15B) and NCI-H292 cells (Figure 15C) in CD47 knockdown. The test antibodies were sequentially diluted from 2.1 nM to 1 / 2.5 dilutions. [Figure 15D] This graph illustrates the ADCP activity of KD002 and KD005, as well as their corresponding IgG4 versions, KD017 and KD019, in MDA-MB-453, MDA-MB-231, and NCI-H292 cells with or without siRNA (siCD47) inhibition of CD47 expression. The assays were performed at an antibody concentration of 1 nM. [Figure 15E] This graph shows the in vivo efficacy of TROP2-CD47 bispecific antibodies in a xenograft model of MDA-MB-231 in SCID mice. Mice were treated once a week with KD004 at 8 mg / kg, and KD065 and KD066 were administered intravenously at the same molar concentration (11 mg / kg). [Figure 16A] This figure shows plots of THP-1 differentiated M1 and M2 macrophages after treatment with PMA and cytokines. THP-1 cells showed upregulation of CD68 after PMA treatment. M1 cells showed upregulation of CD38 and CD86 after treatment with IFN-γ and LPS. M2 macrophages showed upregulation of CD206 and downregulation of CD38 after treatment with IL-4 and IL-13. [Figure 16B] This bar graph shows the percentage of M1 or M2 macrophages phagocytosed by treatment with KD002, KD004, or KD005 at 10 nM, as exemplified by gating with pHrodo-positive and Celltrace Violet-positive cells. [Figure 17A] This bar graph illustrates the binding of a 25 nM TROP2 antibody to the TROP2 recombinant protein using ELISA with an anti-IgG4 Fc secondary antibody. [Figure 17B]This graph shows the antitumor effects of KD002 and KD017 at a dose of 8 mg / kg IV in an MDA-MB-453 transplanted NCG mouse tumor model. PBS was used as the negative control. [Figure 18A] This graph shows the dose-response ADCP activity of KD002 and KD005 TROP2 antibodies against MDA-MB-453 (top panel), MDA-MB-468 (center panel), and T.Tn cells (bottom panel). The ADCP assay was performed using the Promega ADCP assay kit (Promega, catalog number CS314906). [Figure 18B] This graph shows the antitumor effects of KD002 and KD005 in xenograft tumor models of MDA-MB-453 (top panel), MDA-MB-468 (center panel), and TTn (bottom panel). The test antibodies were administered once a week at a dose of 8 mg / kg ip. [Figure 19A] These are micrographs of IHC staining of mouse macrophages using F4 / 80 antibody in xenograft tumors recovered from COLO205, CAPAN1, MDA-MB-231, TTn, and MDA-MB-453 xenograft models treated with TROP2 antibodies KD002 and KD005 at a dose level of 8 mg / kg. The tumors were recovered at the end of the study. [Figure 19B] This figure shows a microarray of human colon adenocarcinoma tumor tissue using KD067, KD068, or isotype-controlled KD069 antibody. The human multiple tumor tissue microarray was purchased from Novus Biologics (catalog number NBP2-42056) and stained with 2 μg / ml antibody. [Figure 19C] This figure shows a microarray of human invasive ductal carcinoma tumor tissue using KD067, KD068, or isotype-controlled KD069 antibody. The human multiple tumor tissue microarray was purchased from Novus Biologics (catalog number NBP2-42052) and stained with 2 μg / ml antibody. [Figure 19D]This figure shows a human head and neck tumor tissue microarray using KD067, KD068, or isotype-controlled KD069 antibody (H&N squamous cell carcinoma). The human multiple tumor tissue microarray was purchased from Novus Biologics (catalog number NBP2-42056) and stained with 2 μg / ml antibody. [Figure 19E] This figure shows a human renal cell carcinoma microarray using KD067, KD068, or isotype-controlled KD069 antibody. The human multiple tumor tissue microarray was purchased from Novus Biologics (catalog number NBP2-42052) and stained with 2 μg / ml antibody. [Figure 20A] This figure shows the X-ray crystal structure of the extracellular domain of the TROP2 protein using translucent surface rendering. The relative positions of the cysteine-rich domain (CRD), thyroglobulin domain (TY-1), and cysteine-poor domain (CPD) are shown. [Figure 20B] This figure shows an X-ray cocrystal (shown by surface rendering) of KD012 and human TROP2. The contact region in TROP2 is shown in dark gray on the surface of TROP2. Contact between KD012 and TROP2 mainly occurs in the CRD of TROP2 and the CDR3 and framework 2 of KD012. The CDR and framework 2 are indicated by arrows. [Figure 20C] This figure shows the X-ray cocrystal structure of KD012 and human TROP2. KD102 is shown as a ribbon diagram. Interacting residues on TROP2 are shown in dark gray, and selected residues on KD012 are shown. [Figure 20D] This figure shows the amino acid residues involved in the interaction between KD012 and human TROP2 (within 4.5 Å; CDRs are annotated according to IMGT numbering). [Figures 21A-21B] This figure shows the dose-response ELISA binding of KD002 and its humanized variant to the human TROP2 recombinant protein. The TROP2 antibody was sequentially diluted from 200 nM to 0.00256 nM by a 1 / 5 dilution. [Figures 22A-22B] Figure showing the dose-response ELISA binding of KD005 and humanized variants to human TROP2 recombinant protein. The TROP2 antibodies were serially diluted 1 / 5 from 200 nM to 0.00256 nM. [Figures 23A-23B] Bar graph illustrating the relative luminescence of the KD002 humanized variant at 0.5 nM (Figure 23A) or 0.1 nM (Figure 23B) compared to KD002, measured using the Promega ADCP reporter assay. [Figures 24A-24B] Bar graph showing the relative luminescence of KD005 and KD005 humanized variants at 0.5 nM (Figure 24A) and 0.1 nM (Figure 24B), measured using the Promega ADCP reporter assay. [Figure 24C-24I] Figure showing the dose-response ADCP assay performed in MDA-MB-453 cells using KD045 (Figure 24C), KD047 (Figure 24D), KD048 (Figure 24E), KD049 (Figure 24F), KD053 (Figure 24G), KD059 (Figure 24H), and KD063 (Figure 24I). [Figures 25A-25B] Bar graph showing the relative luminescence of the KD002 humanized variant at 0.5 nM (Figure 25A) or 0.1 nM (Figure 25B) compared to KD002, measured using the Promega ADCC reporter assay. [Figures 26A-26B] Bar graph showing the relative luminescence of KD005 and KD005 humanized variants at 0.5 nM (Figure 26A) and 0.1 nM (Figure 26B), measured using the Promega ADCC reporter assay. [Figure 27] Figure showing the alignment between the variable regions of KD001, KD, 002, KD003, KD004, and KD005. [Figure 28] Figure showing the alignment and consensus sequence of the KD002 variant. [Figure 29] Figure showing the alignment of selected KD002 variants for ADCC and ADCP activities. [Figure 30]This figure shows the alignment and consensus sequences of the KD005 variant. [Figure 31] This figure shows the alignment of the selected KD005 variants based on their binding activity. [Figure 32] This figure shows the alignment of the selected KD005 variants in terms of ADCP activity. [Figure 33] This figure shows the alignment of the selected KD005 variants regarding ADCC activity. [Figure 34] This figure shows the alignment of the KD005 variant selected based on all its features. [Figure 35] This figure shows that KD005 induced complete tumor regression with a long response period in a large Colo201 colorectal cancer xenograft model in SCID mice. [Figure 36] This figure shows that the KD005 treatment inhibited tumor growth in an NCI-H2228 non-small cell lung cancer xenograft model in SCID mice. [Figure 37] This figure shows that the KD005 treatment inhibited tumor growth in a Cal27 head and neck cancer xenograft model in SCID mice. [Figure 38] This figure shows that KD005 treatment induced regression in a Colo201 colorectal cancer xenograft model in SCID mice. Treatment with a KD005 effector-inactive LALAPG Fc mutant version did not affect tumor growth. [Figure 39] This figure shows that KD005 treatment induced regression in the MDA-MB-453 breast cancer xenograft model in NCG mice. KD005 treatment did not alter tumor growth in the MDA-MB-453 xenograft model transplanted into mice lacking all Fcg receptors (FcgRko). [Figure 40A] This table shows the EC50 values ​​(nM) of KD005, KD001, KD132, and KD134 binding to human TROP2 recombinant protein. [Figure 40B] This table shows the EC50 values ​​(nM) of KD001, KD132, and KD134 binding to MDA-MB-453 and COLO 201 cell lines. [Figure 40C] This table shows the EC50 values ​​(nM) of KD001, KD132, and KD134 ADCP activity in MDA-MB-453 and COLO 201 cells. [Figure 41A-D] This figure shows that treatment with KD133, KD135, and KD136 induces tumor regression in MDA-MB-453 (A-B) and COLO 201 (C-D) xenografts compared to the positive control KD005 and negative isotype controls specific to chicken egg white lysozyme (HEWL). [Figure 42A] This table shows the EC50 values ​​(nM) of KD005, KD137, KD138, and KD139 binding to human TROP2 recombinant protein. [Figure 42B] This table shows the EC50 values ​​(nM) of KD005, KD138, and KD139 binding to MDA-MB-453 and COLO 201 cell lines. [Figure 42C] This table shows the EC50 values ​​(nM) of KD005, KD138, and KD139 ADCP activity in MDA-MB-453 and COLO 201 cells. [Figure 43A-D] This figure shows that treatment with KD137, KD138, and KD139 induces tumor regression in MDA-MB-453 (A-B) and COLO 201 (C-D) xenografts compared to the positive control KD005 and negative isotype controls specific to chicken egg white lysozyme (HEWL). [Figure 44A-B] This figure shows the binding of KD142 and KD143 to human TROP2 recombinant protein (A), and to MDA-MB-453 cells and NCI-H292 cells (B). [Figure 45] This figure shows the alignment of the heavy chain variable region in Table 15E (Table 24) and Table 15F (Table 25). [Modes for carrying out the invention]

[0080] Detailed description Definition The use of the terms "a", "an", and "the" and similar designators is to be construed to cover both the singular and the plural forms in the context of describing embodiments (especially in the context of the claims), unless otherwise indicated herein or clearly contradicted by the content.

[0081] Unless explicitly stated or obvious from the content, as used herein, the term "or" is to be understood as inclusive and to cover both "or" and "and".

[0082] As used herein, the term "and / or" is to be understood as a clear disclosure of each of the specified features or components, including or not including the others.

[0083] The terms "comprising", "having", "including", and "containing" are to be construed as open-form terms (i.e., meaning "including but not limited to") unless otherwise specified. The term "consisting of" is to be construed as a closed form.

[0084] As used herein, the term "TR0P2" refers to the TROP2 protein or a polypeptide containing the TROP2 amino acid sequence. The term "TROP2" encompasses the human TROP2 protein and any protein having at least 80% identity to the human TROP2 protein.

[0085] The term "anti-TROP2 antibody" refers to any form of antibody that binds to the TROP2 protein, such as the TROP2 antigen, or a part thereof, and includes, without limitation, "anti-TROP2 single domain antibody".

[0086] The term "binding agent" refers to a molecule that contains a part of a polypeptide (such as a polypeptide chain) and is capable of specifically binding to an antigen, or a molecule that contains a domain such as an antigen-binding domain that is capable of specifically binding to an antigen.

[0087] The term "antibody" is used in a broad sense and encompasses a wide range of antibody forms and structures, including any immunoglobulin, monoclonal antibody, polyclonal antibody, bivalent antibody, monovalent antibody, bispecific antibody, polyspecific antibody, conventional antibody, single-domain antibody, single-chain antibody, heavy-chain-only antibody, nanobody, full-length antibody, humanized antibody, chimeric antibody that binds to a specific antigen, and any antigen-binding fragment exhibiting desirable antigen-binding activity. Antibodies may be naturally occurring (natural) or the result of recombinant technology.

[0088] As used herein, the expressions “single-domain antibody” and “heavy-chain-only antibody” are interchangeable. The term “single-domain antibody” includes naturally occurring single-domain antibodies or heavy-chain-only antibodies, as well as single-domain antibodies in which the naturally occurring constant region is replaced by variants such as dimerized domains and humanized or chimeric single-domain antibodies disclosed herein. If a single-domain antibody has a dimerized domain, it generally consists of two polypeptide chains.

[0089] "Naturally occurring single-domain antibodies" include antibodies produced by camelids (camelid antibodies) or sharks. "Naturally occurring single-domain antibodies" also include antibodies produced by transgenic animals modified to express only heavy-chain antibodies. Exemplary embodiments of transgenic animals are provided in International Application PCT / CA2021 / 050951, filed July 21, 2021, and WO2022 / 011457, published January 20, 2022, whose entire contents are incorporated herein by reference.

[0090] In this specification, the terms "KD001", "KD002", "KD003", "KD004", "KD005", "KD006", "KD007", "KD008", "KD009", "KD010", "KD011", "KD012", "KD013", "KD014", "KD015", "KD016", "KD017", "KD018", "KD019", "KD020", "KD021", "KD022", "KD023", "KD024", "KD025", "KD026", "KD027", "KD028", "KD029", and "KD030" are used. , "KD031", "KD032", "KD033", "KD034", "KD035", "KD036", "KD037", "KD03" 8”, “KD039”, “KD040”, “KD041”, “KD042”, “KD043”, “KD044”, “KD045”, “KD0 46'', ``KD047'', ``KD048'', ``KD049'', ``KD050'', ``KD051'', ``KD052'', ``KD053'', ``K D054", "KD055", "KD056", "KD057", "KD058", "KD059", "KD060", "KD061", " KD062", "KD063", "KD064", "KD067", "KD068", "KD069", "KD071", "KD072" , "KD073", "KD074", "KD075", "KD076", "KD077", "KD078", "KD079", KD080" , "KD081", "KD082", "KD083", "KD084", "KD085", "KD086", "KD087", "KD088" ", "KD089", "KD090", "KD091", "KD092", "KD093", "KD094", "KD095", "KD09 6”, “KD097”, “KD098”, “KD099”, “KD100”, KD101”, “KD102”, “KD103”, “KD1 04", "KD105", "KD106", "KD107", "KD108", "KD109", "KD110", "KD111", "KD 112'', ``KD113'', ``KD114'', ``KD115'', ``KD116'', ``KD117'', ``KD118'', ``KD119'', `` DK120'', ``KD121'', ``KD122'', ``KD123'', ``KD124'', ``KD125'', ``KD126'', ``KD127'',It should be understood that "KD128," "KD129," "KD130," "KD131," "KD132," "KD133," "KD134," "KD135," "KD136," "KD137," "KD138," "KD139," "KD140," or "KD141" each refer to single-domain antibodies containing two heavy chains, each having either the corresponding heavy chain amino acid sequence described in Table 15A (Table 20) to Table 15K (Table 30), Table 16 (Table 31), Table 18 (Table 34), or Table 19 (Table 35), or each having the corresponding variable region amino acid sequence described in Table 15A (Table 20) to Table 15K (Table 30), Table 16 (Table 31), Table 18 (Table 34), or Table 19 (Table 35).

[0091] The terms "KD065" or "KD066" refer to a binder containing two chains, each having the corresponding amino acid sequence of the polypeptide chain described in SEQ ID NO: 150 or SEQ ID NO: 151, respectively, or each having the corresponding variable region amino acid sequence of the polypeptide chain.

[0092] In some embodiments, either KD065 or KD066 may have a corresponding variable region amino acid sequence of KD065 or KD66, as well as a hinge and / or dimerization domain (e.g., CH2 and / or CH3) that is different from KD065 or KD66.

[0093] As used herein, the term “subjects requiring treatment” refers to subjects having or suspected to have a disorder or disease involving TROP2, TROP2 expression (e.g., expression in tissue, cells, or serum), or TROP2 overexpression. The term “subjects requiring treatment” also refers to subjects having or suspected to have a disorder or disease that may benefit from treatment with a TROP2-targeting conjugate. “Subjects requiring treatment” includes subjects having or suspected to have cancer.

[0094] For the purposes of this disclosure, the term “treatment” refers to both therapeutic treatments and preventive or tumor-preventive measures. Subjects requiring treatment include those who already have a disability, those prone to developing a disability, or those whose disability is being prevented. Accordingly, subjects requiring treatment also include those who already have cancer, those prone to developing cancer, or those whose cancer is being managed.

[0095] When used in the context of sequences, the term "natural" refers to sequences that exist naturally.

[0096] With respect to a given value, the terms “about” or “approximately” mean that variation in the value is expected. In some embodiments, the terms “about” or “approximately” generally mean a range of + / - 10 percent, + / - 5 percent, + / - 4 percent, + / - 3 percent, + / - 2 percent, or + / - 1 percent of a given value or range.

[0097] It is understood herein that expressions relating to a range of values ​​in the form of "A to B" include individual values ​​and any partial ranges that are included in and contain such ranges. For example, the expression "1 to 10" includes, non-restrictively, partial ranges such as "2 to 10", "2 to 9", "3 to 6", "5 to 7", and any individual values ​​that are included in between, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0098] With respect to a given value, the term "at least" is understood herein to include that value and any greater value. For example, the term "at least 80%" includes "at least 80%", "at least 81%", "at least 82%", "at least 83%", "at least 84%", "at least 85%", "at least 86%", "at least 87%", "at least 88%", "at least 89%", "at least 90%", "at least 91%", "at least 92%", "at least 93%", "at least 94%", "at least 95%", "at least 96%", "at least 97%", "at least 98%", "at least 99%", "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%.

[0099] As used herein, the term “humanized” means that the binder, such as an antibody or antigen-binding fragment, comprises a CDR or CDR amino acid residue derived from a non-human animal antibody, an FR region derived from a human antibody, and, where applicable, a constant region derived from a human antibody.

[0100] As used herein, the expression “fully humanized framework region” means that the amino acid sequence of all framework regions of a given binder, such as an antibody or antigen-binding fragment, is identical to that of a human antibody variable region (e.g., human germline antibody variable region sequence).

[0101] As used herein, the expression “partially humanized” means that the amino acid sequence of one or more, or all, framework regions of a binder such as an antibody or antigen-binding fragment is not entirely identical to that of a human antibody variable region, for example, a germline human antibody variable region (e.g., a human germline antibody variable region sequence).

[0102] In some cases, a humanized, fully humanized or partially humanized binding agent, such as an antibody or an antigen-binding fragment, may contain the CDRs or CDR amino acid residues of a human antibody variable region (e.g., a human germline antibody variable region sequence).

[0103] Thus, a "partially humanized framework region" includes, by way of example and not limitation, a human framework region containing a reverse mutation in which the original amino acid is reintroduced at the original position. A "partially humanized framework region" also includes, by way of example and not limitation, a human framework region in which one or more amino acid residues have been replaced, added or deleted.

[0104] As used herein, the term "affinity" refers to the strength of the non-covalent interaction between a binding agent or its antigen-binding domain and an antigen. "Affinity" is, for example, when the binding between an antigen and a binding agent (e.g., an antibody such as a single domain antibody) reaches equilibrium, for example, the K D value, i.e., the ratio of the association rate to the dissociation rate (k オフ / k オン ). Affinity can be determined by using any conventional method well known in the art, including but not limited to surface plasmon resonance, microscale thermophoresis, HPLC-MS, and flow cytometry (such as FACS). Antibodies generally have a K -6 value of ≤ 10 -7 M (e.g., ≤ 5x10 -7 M, ≤ 2x10 -7 M, ≤ 10 -8 M, ≤ 5x10 -8 M, ≤ 10 -8 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M or ≤ 10 -9 M and ≤ 10 -9 of any value). Preferably, the antibody has a K D value below the nanomolar range (e.g., ≤ 9x10-9 M, ≤ 8x10 -9 M, ≤ 7x10 -9 M, ≤ 6x10 -9 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M, ≤ 1x10 -9 M or less, for example, ≤ 1 x 10 -10 M, ≤ 1x10 -11 M, ≤ 1 x 10⁻ 12 M)'s K D The antibody has a value. More preferably, the antibody is less than or equal to the picomolar range (e.g., ≤9x10). -12 M, ≤ 8x10 -12 M, ≤ 7x10 -12 M, ≤ 6x10 -12 M, ≤ 5x10 -12 M, ≤ 4x10 -12 M, ≤ 3x10 -12 M, ≤ 2x10 -12 M, ≤ 1x10 -12 K (M or less) D It has a value.

[0105] As used herein, the term “specific binding” or “specifically binding” refers to a non-random binding reaction between two molecules, such as between an antibody and an antigen. Specific binding is characterized by binding affinity, ≤ 10 -6 M (for example, ≤ 5x10) -7 M, ≤ 2x10 -7 M, ≤10 -7 M, ≤ 5x10 -8 M, ≤ 2x10 -8 M, ≤10 -8 M, ≤ 5x10 -9 M, ≤ 4x10 -9 M, ≤ 3x10 -9 M, ≤ 2x10 -9 M or ≤ 10 -9 M and ≤10 -9 K (any value of) D The value may indicate specific binding between the binder (e.g., an antibody such as a single-domain antibody) and TROP2 (e.g., human TROP2).

[0106] As used herein, the terms “compete for binding,” “compete for binding,” “compete with,” or “compete with” refer to the ability of a first antibody or its antigen-binding fragment (e.g., a single-domain antibody or its antigen-binding fragment) to inhibit the binding interaction between TROP2 and a second anti-TROP2 antibody (e.g., a single-domain antibody or its antigen-binding fragment) to any detectable extent.

[0107] As used herein, the term “epitope” means, as used herein, a specific group of atoms or amino acid residues on an antigen to which an antibody (e.g., a single-domain antibody) binds. Two antibodies may bind to the same or closely related epitopes within an antigen if they exhibit competitive binding to the antigen. Epitopes may be linear or conformal (i.e., containing spaced amino acid residues). For example, if an antibody or antigen-binding fragment blocks at least 85%, at least 90%, or at least 95% of the binding of a reference antibody (e.g., a single-domain antibody) to an antigen, then the antibody or antigen-binding fragment is considered to bind to the same / closely related epitope as the reference antibody. For example, monoclonal, chimeric, human, or humanized antibodies or their antigen-binding fragments may compete with the single-domain antibodies of this disclosure for binding to human TROP2.

[0108] As used herein, the term “sequence identity” in this invention refers to the degree of identity between two nucleic acid sequences or two amino acid sequences when best compared and when appropriate mutations such as substitutions, insertions, or deletions are permitted. Sequence identity can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0109] When used in the expressions "antibody or its antigen-binding fragment" or "single-domain antibody or its antigen-binding fragment," the term "antigen-binding fragment" refers to an antibody or a fragment of a single antibody that contains an antigen-binding domain and may or may not incorporate other parts of the antibody or single-domain antibody, such as amino acid residues in the hinge region, amino acid residues in the constant region, or a portion of the Fc region. Regardless of its structure, the antigen-binding fragment binds to the same antigen recognized by the complete antibody (e.g., a single-domain antibody).

[0110] The antigen-binding fragment of a single-domain antibody may include, for example, a sequence that encompasses the CDR and the entire variable region or a portion thereof, or the entire heavy chain or a portion thereof, and may or may not include other parts of the antibody.

[0111] The term "antigen-binding domain" refers to a portion of an antibody that is involved in antigen binding and includes, for example, one or more CDRs, one or more framework regions (FRs), or an entire variable region. In the context of a single-domain antibody, the term "antigen-binding domain" refers to a portion of a single-domain antibody that is involved in antigen binding and includes, for example, one or more CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more framework regions FR1, FR2, FR3, FR4, or an entire variable region (VH or VHH). In the context of a native antibody, the term "antigen-binding domain" refers to a portion of a native antibody that is involved in antigen binding and includes, for example, one or more CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light chain or heavy chain framework regions FR1, FR2, FR3, FR4, or an entire variable region (heavy chain variable region (VH) and / or light chain variable region (VL)).

[0112] As used herein, the terms "CDRH1," "CDRH2," and "CDRH3" refer to the CDR1, CDR2, or CDR3 antibody heavy chains, respectively.

[0113] TROP2 antigen The antibodies or antigen-binding fragments of this disclosure can be obtained, for example, by immunizing animals with the TROP2 antigen.

[0114] Alternatively, the antibody or antigen-binding fragment of this disclosure may be obtained from an antibody library.

[0115] Binding agents that conjugate TROP2 can be identified by computer, in vitro, and / or in vivo methods. Some in vitro methods rely on the binding of a candidate antibody or its antigen-binding fragment to the antigen. Such anti-TROP2 antibodies or their antigen-binding fragments can be identified by methods relating to binding to the TROP2 antigen disclosed herein and / or by assays disclosed herein.

[0116] In one embodiment, the TROP2 antigen includes the human TROP2 protein (e.g., SEQ ID NO: 39), or a TROP2 homolog, or a TROP2 variant, homolog, or variant having at least 80% identity with the human TROP2 protein or a portion of the human TROP2 protein (e.g., the extracellular domain of human TROP2: SEQ ID NO: 164). In some embodiments, the TROP2 homolog is a primate TROP2 sequence or a portion thereof.

[0117] In one embodiment, the TROP2 antigen is a TROP2 variant having a sequence that is at least 80% identical to human TROP2, a TROP2 homolog, or a fragment thereof.

[0118] In exemplary embodiments, the TROP2 homolog is the cynomolgus monkey TROP2.

[0119] In another exemplary embodiment, the TROP2 homolog is rhesus macaque TROP2.

[0120] In some embodiments, the TROP2 antigen includes the extracellular domain of TROP2 (e.g., human TROP2, a TROP2 homolog, or a variant thereof).

[0121] In some embodiments, the TROP2 antigen includes the extracellular domain (ECD) of TROP2 (represented by amino acids 27-274 of TROP2 (e.g., SEQ ID NO: 39 or SEQ ID NO: 40), the full content of which is incorporated herein by reference; see Goldenberg et al., 2018, Ikeda, M. et al., 2015).

[0122] In some embodiments, the TROP2 antigen includes an epitope common to human TROP2 and cynomolgus monkey TROP2.

[0123] In some embodiments, the TROP2 antigen includes epitopes similar to human TROP2 and cynomolgus monkey TROP2.

[0124] In some embodiments, the TROP2 antigen includes amino acid residues 27-274 of human TROP2 or a TROP2 homolog, or a fragment thereof. Therefore, the TROP2 antigen may include a polypeptide or fragment thereof containing the human TROP2 amino acid sequence consisting of amino acid residues 27-274.

[0125] In other embodiments, the TROP2 antigen comprises amino acid residues 27-146 of human TROP2 or a TROP2 homolog, or a fragment thereof. Therefore, the TROP2 antigen may comprise a polypeptide or fragment thereof containing the human TROP2 amino acid sequence consisting of amino acid residues 27-146.

[0126] In yet another embodiment, the TROP2 antigen comprises amino acid residues 27-73 of human TROP2 or a TROP2 homolog, or a fragment thereof. Therefore, the TROP2 antigen may comprise a polypeptide or fragment thereof containing the human TROP2 amino acid sequence consisting of amino acid residues 27-73.

[0127] This disclosure indicates that the TROP2 antigen may contain at least 10 amino acid residues of human TROP2.

[0128] More specifically, the TROP2 antigen is a fragment of at least 10 amino acid residues from the human TROP2 amino acid sequence, which consists of amino acid residues 27-146.

[0129] This disclosure describes the TROP2 antigen as a human TROP2 amino acid sequence consisting of 27 to 146 amino acid residues, for example, amino acid residues between 10 and 100, between 10 and 90, between 10 and 80, between 10 and 70, between 10 and 60, between 10 and 50, between 10 and 40, between 10 and 30, between 10 and 29, between 10 and 28, between 10 and 27, between 10 and 26, and between 10 and 25 amino acid residues. Examples include groups with 10 to 24 amino acid residues, groups with 10 to 23 amino acid residues, groups with 10 to 22 amino acid residues, groups with 10 to 21 amino acid residues, groups with 10 to 20 amino acid residues, groups with 10 to 19 amino acid residues, groups with 10 to 18 amino acid residues, groups with 10 to 17 amino acid residues, groups with 10 to 16 amino acid residues, groups with 10 to 15 amino acid residues, groups with 10 to 14 amino acid residues, groups with 10 to 13 amino acid residues, groups with 10 to 12 amino acid residues, groups with 10 to 11 amino acid residues, and groups with 10 to 110 amino acid residues.

[0130] For example, the TROP2 antigen contains at least 10 amino acid residues from the human TROP2 amino acid sequence, which consists of amino acid residues 27 to 73.

[0131] This disclosure describes the TROP2 antigen as a human TROP2 amino acid sequence consisting of amino acid residues 27 to 73, for example, amino acid residues between 10 and 40, between 10 and 35, between 10 and 30, between 10 and 29, between 10 and 28, between 10 and 27, between 10 and 26, between 10 and 25, between 10 and 24, between 10 and 23, and between 10 and 22 Examples include amino acid residues between 1, 10 to 21, 10 to 20, 10 to 19, 10 to 18, 10 to 17, 10 to 16, 10 to 15, 10 to 14, 10 to 13, 10 to 12, and 10 to 11, and also include amino acid residues between 10 and 45.

[0132] In some cases, the TROP2 antigen contains a human TROP2 amino acid sequence consisting of amino acid residues 27-56 of human TROP2, or a fragment thereof.

[0133] In other cases, the TROP2 antigen contains a human TROP2 amino acid sequence consisting of amino acid residues 27-66 of human TROP2, or a fragment thereof.

[0134] In other cases, the TROP2 antigen contains a human TROP2 amino acid sequence or a fragment thereof, consisting of amino acid residues 47-56 of human TROP2.

[0135] In other cases, the TROP2 antigen contains a human TROP2 amino acid sequence consisting of 60-66 amino acid residues of human TROP2, or a fragment thereof.

[0136] In other cases, the TROP2 antigen contains a human TROP2 amino acid sequence or a fragment thereof, consisting of amino acid residues 47-66 of human TROP2.

[0137] For example, the TROP2 antigen contains a human TROP2 amino acid sequence consisting of amino acid superscripts 27-56, 27-66, 28-66, 29-66, 30-66, 31-66, 32-66, 33-66, 34-66, 35-66, 36-66, 37-66, 38-66, 39-66, 40-66, 41-66, 42-66, 43-66, 44-66, 45-66, 46-66, or 47-66.

[0138] In some cases, the TROP2 antigen may exclude the cysteine-poor (CPD) domain of TROP2.

[0139] Exemplary and non-limiting embodiments of the TROP2 antigen are provided in SEQ ID NO: 164.

[0140] Therefore, in some embodiments, the binder of this disclosure, or the antigen-binding domain of the binder, is capable of binding to human TROP2.

[0141] In some embodiments, the binder or the antigen-binding domain of the binder of this disclosure can bind to human TROP2 and cynomolgus monkey TROP2.

[0142] In some embodiments, the binder or the antigen-binding domain of the binder of this disclosure preferentially binds to human TROP2 compared to mouse TROP2, or has a good affinity for human TROP2 compared to mouse TROP2.

[0143] In some embodiments, the binder or the antigen-binding domain of the binder of this disclosure preferentially binds to human TROP2 compared to rat TROP2, or has a good affinity for human TROP2 compared to rat TROP2.

[0144] In some embodiments, the binder or the antigen-binding domain of the binder of this disclosure binds to an amino acid residue common to the extracellular domain of human TROP2 and the extracellular domain of cynomolgus monkey TROP2.

[0145] In some embodiments, the binder or the antigen-binding domain of the binder of this disclosure binds to an amino acid residue that is not present in the extracellular domain of mouse TROP2.

[0146] In some embodiments, the antigen-binding domain of the binder or the binder of this disclosure binds to an amino acid residue that is not present in the extracellular domain of rat TROP2.

[0147] In some embodiments, the antigen-binding domain of the binder or the binder of this disclosure binds to amino acid residues present in the extracellular domains of human TROP2, cynomolgus monkey TROP2, mouse TROP2, and rat TROP2.

[0148] Therefore, the binder or the antigen-binding domain of the binder may be capable of binding to human TROP2 and cynomolgus monkey TROP2.

[0149] In exemplary embodiments, the binder or the antigen-binding domain of the binder may be capable of binding to a denatured form of human TROP2.

[0150] In another exemplary embodiment, the binder or the antigen-binding domain of the binder may be capable of binding to the reduced form of human TROP2.

[0151] In other exemplary embodiments, the binder or the antigen-binding domain of the binder may be capable of binding to the deglycosylated form of human TROP2.

[0152] Binder Exemplary embodiments of the binders of this disclosure include polypeptide chains disclosed herein.

[0153] Exemplary embodiments of the binders of this disclosure include an antibody or an antigen-binding fragment thereof.

[0154] Accordingly, exemplary embodiments of the binder include, but are not limited to, a natural antibody or its antigen-binding fragment, or a single-domain antibody or its antigen-binding fragment, or a variant thereof, such as a humanized, chimeric, or human antibody or its antigen-binding fragment, or a humanized or chimeric single-domain antibody or its antigen-binding fragment.

[0155] The single-domain antibodies or antigen-binding fragments thereof disclosed herein will be specifically investigated, and binders containing one or more antigen-binding domains of one or more single-domain antibodies disclosed herein will also be investigated.

[0156] This disclosure also includes single-domain antibodies that compete for binding to the TROP2 antigen with one or more single-domain antibodies (having the amino acid sequences disclosed herein).

[0157] Other exemplary embodiments of the binders of this disclosure include binders comprising antigen-binding domains such as antibody-like molecules (including single-chain antibodies), protein scaffold molecules, and immunocytomodulators.

[0158] Accordingly, exemplary embodiments of the binders of this disclosure include antibody-like molecules comprising one or more antigen-binding domains of one or more monoantibodies disclosed herein.

[0159] Additional exemplary embodiments of the binders of this disclosure include protein scaffold molecules comprising one or more antigen-binding domains of one or more monoantibodies disclosed herein.

[0160] Further additional exemplary embodiments of the binders of this disclosure include immunocytomodulators comprising one or more antigen-binding domains of one or more monoantibodies disclosed herein.

[0161] As an example, and not limited to, antibodies or antigen-binding fragments thereof, and binders comprising single-domain antibodies or antigen-binding fragments thereof, which compete with single-domain antibodies having the amino acid sequences disclosed herein, are also included in this disclosure.

[0162] This disclosure relates to a binder comprising one or more antigen-binding domains, wherein at least one of the antigen-binding domains comprises an antigen-binding fragment capable of binding to trophoblast cell surface antigen-2 (TROP2).

[0163] In some embodiments, the binders of this disclosure can bind to the extracellular domain (ECD) of TROP2.

[0164] In some embodiments, the binders of this disclosure can bind to the cysteine-rich domain and / or thyroglobulin type 1 domain of TROP2.

[0165] In some embodiments, the binders of this disclosure can bind to an epitope containing amino acids in the extracellular domain of TROP2.

[0166] In some embodiments, the binders of the present disclosure can bind to epitopes containing amino acids of the cysteine-rich domain and / or thyroglobulin type 1 domain of TROP2.

[0167] In some embodiments, the binders of the present disclosure can bind to epitopes or fragments thereof that include amino acid residues contained in amino acid sequence 27-274 of TROP2.

[0168] In some embodiments, the binders of this disclosure are not capable of binding to the cysteine ​​poor domain (CPD) of TROP2.

[0169] In some embodiments, the binders of this disclosure are not capable of binding to amino acids 146 to 274 of TROP2.

[0170] In some embodiments, the binder includes the antigen-binding domain of a single-domain antibody capable of binding to TROP2.

[0171] In some embodiments, the binder includes a single-domain antibody.

[0172] In other embodiments, the binder is a single-domain antibody.

[0173] In some embodiments, the antigen-binding domain of the binder includes a CDR of a single-domain antibody.

[0174] In some embodiments, the antigen-binding domain of the binder is the variable region of a single-domain antibody. In some examples, the variable region may be a camelid VH or VHH. In some examples, the variable region includes, for example, a camelid CDR and framework regions such as a camelid framework region, a humanized framework region, a human framework region, and a mouse framework region.

[0175] In some embodiments, the binder includes a variable region comprising a CDR of camelid origin and a humanized framework region.

[0176] In other embodiments, the binder includes a variable region comprising a CDR of camelid origin and a human framework region.

[0177] In some embodiments, the binder comprises two heavy chain variable regions of a single-domain antibody, each containing a dimerizing domain, thereby forming a dimer.

[0178] In some embodiments, the level of humanization of a single-domain antibody may be calculated based on sequence identity with closely related human VH germline genes. Therefore, the percentage of the humanization score is based on framework regions 1-3 (FR1, FR2, FR3) and CDR1 and CDR2. According to one embodiment, the sequences of CDR3 and FR4 are not considered for evaluating the level of single-domain humanization disclosed herein.

[0179] In exemplary embodiments, the humanization level of the antigen-binding domain is at least 80%.

[0180] In other exemplary embodiments, the humanization level of the variable region of the single-domain antibody of this disclosure is at least 80%.

[0181] In some embodiments, the binders of the present disclosure each independently have an amino acid sequence of formula I in an N-terminal to C-terminal manner: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y (wherein m is 0, 1, 2 or an integer greater than 2; n is an integer greater than 2, or 0, 1, 2; m and n are not both 0 at the same time; Ab a Ab d Each of these represents an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3); X or Y may exist independently or not exist, and may include an amino acid sequence; L b , L c Each of these independently includes one or more linkers; and (DD stands for dimerization domain) It may contain one or more polypeptide chains, including the following.

[0182] In some embodiments, the binder comprises one or more polypeptide chains, at least one of which is of formula II: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Equation II) is included.

[0183] In some embodiments, the binder comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula III: X-(Ab a1 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula III).

[0184] In some embodiments, the binder comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula IV: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-Y (Formula IV).

[0185] In some embodiments, the binder comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula V: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-Y (Formula V).

[0186] In some embodiments, the binder comprises one or more polypeptide chains, and at least one of the polypeptide chains has the formula VI: X-(Ab a1 )-(L b2 )-(Ab a2 )-(L b1 )-(DD)-(L c1 )-(Ab d1 )-(L c2 )-(Ab d2 )-(L c3 )-(Ab d3 )-Y (Formula VI).

[0187] In some embodiments, the binder comprises one or more polypeptide chains, at least one of which is of 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 (Equation VII) is included.

[0188] In some embodiments, the binder comprises one or more polypeptide chains, at least one of which is of 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 (Equation VIII) is included.

[0189] In some embodiments, Ab a1、 Ab a2 Ab a3、 Ab d1 Ab d2 , or Ab d3 Each of these independently contains an antigen-binding domain.

[0190] In some embodiments, L b1This includes a linker or a plurality of linkers and / or hinge regions of the antibody or its antigen-binding fragment. In some embodiments, the hinge region is a naturally occurring hinge region of an antibody. In exemplary embodiments, the hinge may include the amino acid sequence or a portion thereof described in SEQ ID NO: 36, SEQ ID NO: 42, and SEQ ID NO: 481. In other embodiments, the hinge region is a mutated or synthetic hinge region disclosed herein. In some embodiments, the mutated or synthetic hinge may have a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 87%, at least 98%, and at least 99% identical to the naturally occurring antibody hinge. In exemplary embodiments, the mutated or synthetic hinge may have the sequence or a portion thereof described in SEQ ID NO: 481.

[0191] In some embodiments, the hinge region may be replaced with a linker or spacer. In some embodiments, the hinge region, linker, or spacer are optional.

[0192] In some embodiments, L b2 , L b3 L c1 , L c2 , and L c3 Each of these independently includes one or more linkers disclosed herein.

[0193] Dimerization domain and linker In accordance with this disclosure, the binder may consist of two polypeptide chains, each comprising one or more antigen-binding domains and dimerization domains. The one or more antigen-binding domains may be separated by a linker.

[0194] Exemplary embodiments of the dimerization domain and linker are provided in PCT / CA2020 / 051753, filed on 18 December 2020 and published on 24 June 2021 as WO2021 / 119832A1, the entire contents of which are incorporated herein by reference.

[0195] In some embodiments, the dimerization domain enables the formation of homodimers.

[0196] In some embodiments, the dimerizing domain enables the formation of a heterodimer.

[0197] In some embodiments, the dimerization domain includes an amino acid sequence that is continuous with the amino acid sequence of the antigen-binding domain.

[0198] In some embodiments, the dimerization domain includes an immunoglobulin dimerization domain. Other dimerization domains known to those skilled in the art, including leucine zippers, are intended herein.

[0199] In some embodiments, the dimerization domain includes an IgG, IgM, IgA, IgD, or IgE dimerization domain (derived from human or animal IgGss, IgM, IgA, IgD, or IgE).

[0200] In some embodiments, the dimerization domain includes a constant region or a portion thereof.

[0201] In some embodiments, the constant region or a portion thereof is derived from an IgG1, IgG2, IgG3, or IgG4 heavy chain, or a combination thereof. For example, the constant region or a portion thereof includes the amino acid sequence of an IgG1, IgG2, IgG3, or IgG4 heavy chain, or a combination thereof. In some examples, the amino acid sequence of the constant region or a portion thereof (e.g., the CH3 domain) is at least 80% identical to the sequence of a naturally occurring IgG1, IgG2, IgG3, or IgG4 heavy chain.

[0202] In some embodiments, the constant region or a portion thereof is derived from a single human IgG chain.

[0203] In some embodiments, the constant region or a portion thereof is derived from a human IgG double chain.

[0204] In some embodiments, the constant region or a portion thereof is derived from a human IgG triple chain.

[0205] In some embodiments, the constant region or a portion thereof is derived from a human IgG quadruple chain.

[0206] In some embodiments, the binder includes the Fc region of the antibody or a portion thereof.

[0207] In some embodiments, the binder includes an Fc region or a portion thereof that includes or does not include Fc modification.

[0208] In exemplary embodiments, the binder may include Fc or a portion thereof that is capable of binding to Fc receptors on monocytes and / or macrophages.

[0209] In another exemplary embodiment, the binder may include Fc or a portion thereof that is capable of binding to an Fc receptor on a CD68+ macrophage.

[0210] In other embodiments, the binder includes a modified or mutated Fc region or portion thereof. For example, the Fc region or portion thereof may be modified or mutated to alter one or more characteristics of the binder. In exemplary embodiments, the Fc region or portion thereof is non-glycosylated. In other exemplary embodiments, the Fc region or portion thereof includes one or more Fc modifications from Table A (Table 4).

[0211] In other exemplary embodiments, the Fc region or a portion thereof is modified or mutated to increase ADCC activity. For example, afucosylation (e.g., N297 according to the EU numbering system) can result in increased FcRgII binding in NK cells and potently increase ADCC. Therefore, in some embodiments, the Fc region or a portion thereof may have a reduced number of fucose residues. In other embodiments, the binder includes an afucosylated Fc region or a portion thereof. In other embodiments, the Fc region or a portion thereof of the binder lacks fucose residues. In additional embodiments, the Fc region or a portion thereof of the binder lacks core fucose residues. In further embodiments, the Fc region or a portion thereof of the binder completely lacks core fucose residues.

[0212] In some embodiments, the dimerization domain includes a CH3 domain.

[0213] In some embodiments, the CH3 domain is either a natural CH3 domain or a mutated CH3 domain.

[0214] In some embodiments, the dimerization domain also includes a CH2 domain.

[0215] In some embodiments, the dimerization domain includes the CH3 domain of the antibody. The dimerization domain may also include the CH2 domain of the antibody.

[0216] In some embodiments, the CH2 domain is either a natural CH2 domain or a mutated CH2 domain.

[0217] In some embodiments, the dimerized domain includes a natural CH2 domain and a natural CH3 domain.

[0218] In some embodiments, the dimerized domain includes a native CH2 domain and a mutated CH3 domain.

[0219] In some embodiments, the dimerized domain includes a mutated CH2 domain and a native CH3 domain.

[0220] In some embodiments, the dimerization domain includes a mutated CH2 domain and a mutated CH3 domain.

[0221] In some embodiments, the CH3 domain may have the sequence described in Sequence ID No. 38, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0222] In other embodiments, the CH3 domain may have the sequence described in Sequence ID No. 43, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0223] In some embodiments, the CH2 domain may have the sequence described in Sequence ID No. 37, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0224] In some embodiments, the CH2 domain may have the sequence described in Sequence ID No. 42, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0225] In some embodiments, the dimerization domain may include a CH3 domain having the sequence described in Sequence ID No. 38, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical thereto, and a CH2 domain having the sequence described in Sequence ID No. 37 or Sequence ID No. 42, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical thereto.

[0226] In some embodiments, the dimerization domain may include a CH3 domain having the sequence described in Sequence ID No. 43, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical thereto, and a CH2 domain having the sequence described in Sequence ID No. 37 or Sequence ID No. 42, or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical thereto.

[0227] In some embodiments, the dimerization domain may include the sequence described in Sequence ID No. 212, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0228] In some embodiments, the dimerization domain may include the sequence described in Sequence ID No. 506, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0229] In some embodiments, the dimerization domain may include the sequence described in Sequence ID No. 214, or a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical thereto.

[0230] In some embodiments, the dimerization domain includes a hinge region such as a naturally occurring antibody or its antigen-binding fragment, or a mutated or synthetic hinge region.

[0231] In some embodiments, the dimerization domain includes a peptide linker.

[0232] In some embodiments, the linker is an amino acid sequence with a length of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues.

[0233] In some embodiments, the linker is incutable.

[0234] In some embodiments, the linker includes one or more units of GGGGS (Sequence ID 206).

[0235] In some embodiments, the linker is of the formula (X(PAPAP)) n The linker may include KA (sequence number 207) (wherein n is an integer selected from 1 to 10, X is either present or absent, and A). In some embodiments, the linker may include the sequence described in sequence number 163.

[0236] In some embodiments, the linker is an antibody hinge or a part thereof. For example, in some embodiments, the linker may include a human IgG1 hinge sequence or a part thereof. In some embodiments, the linker may include a human IgG2 hinge sequence or a part thereof. In some embodiments, the linker may include a human IgG3 hinge sequence or a part thereof. In other embodiments, the linker may include a human IgG4 hinge sequence or a part thereof.

[0237] Antibody and antigen-binding fragments In some embodiments, the binder is an anti-TROP2 antibody or an antigen-binding fragment thereof. It is understood herein that the following are exemplary and non-limiting embodiments of the antibody or its antigen-binding fragment.

[0238] Antibodies or their antigen-binding fragments (or sequences thereof) can be obtained from immunization of animals (including camels, llamas, dromedary camels, alpacas, and transgenic animals) with the TROP2 antigen disclosed herein. For example, single-domain antibodies or their antigen-binding fragments (or sequences thereof) can be obtained by immunizing animals, such as dromedary camels, camels, llamas, alpacas, and rabbits, with the TROP2 antigen (e.g., ECD or fragments thereof). Alternatively, single-domain antibodies or sequences of their antigen-binding domains can be obtained by immunizing transgenic animals capable of expressing single-domain antibodies (see WO2022 / 011457 A1, international application PCT / CA2021 / 050951, filed July 12, 2021, and published January 20, 2022, whose entire contents are incorporated herein by reference).

[0239] In some cases, antibodies or their antigen-binding fragments (or sequences thereof) are obtained from the serum or tissues of immunized animals, such as bone marrow, spleen, and / or lymph nodes.

[0240] Fusion cells can be produced from the fusion of immunized animal cells with malignant and immortal plasma cells, such as myeloma cells lacking hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), an enzyme that enables cell proliferation in HAT medium (hypoxanthine-aminopterin-thymidine). Splenocytes are particularly useful for generating fusion cells. Antibodies or their antigen-binding fragments can be isolated from the fusion cells.

[0241] The nucleic acid sequence of an antibody or its antigen-binding fragment can be obtained from cells or tissues capable of expressing the antibody or its antigen-binding fragment by PCR amplification and sequencing. Amino acid sequence information is obtained from the nucleic acid sequence. Due to the inherent degeneracy of the genetic code, nucleic acid molecules may be generated that encode the same amino acid sequence but have a different nucleic acid sequence compared to the original nucleic acid sequence.

[0242] Once the nucleic acid sequence encoding the amino acid sequence of the antibody is obtained, recombinant expression of the antibody can be enabled by transfection of one or more expression vectors into an immortalized mammalian cell line.

[0243] Alternatively, the sequence of an antibody or its antigen-binding fragment can be obtained by screening an established antibody library for sequences that possess desirable characteristics, such as homology or identity with a reference antibody.

[0244] The antibodies of this disclosure include natural or intact antibodies that specifically bind to TROP2. Natural or intact antibodies include recombinantly expressed antibodies, antibodies isolated from immunized animals, or antibodies isolated from fusion cells.

[0245] Exemplary embodiments of the binders of this disclosure include an antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing amino acids of the extracellular domain (ECD) of TROP2. Other exemplary embodiments of the binders of this disclosure include an antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing amino acids of the cysteine-rich domain of TROP2. In some embodiments, the antibody or the antigen-binding fragment thereof binds to the TROP2 antigen disclosed herein.

[0246] In one embodiment, the antibody of the Disclosure specifically binds to human TROP2. In another embodiment, the antibody of the Disclosure specifically binds to cynomolgus monkey TROP2. In yet another embodiment, the antibody of the Disclosure specifically binds to rhesus monkey TROP2.

[0247] More specifically, the antibodies of this disclosure specifically bind to the ECD or CRD of human TROP2.

[0248] Natural or intact antibodies consist of two heavy chains (H) and two light chains (L). Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each consisting of a variable region (VH) and a first, second, third, and optionally a fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are classified as λ or κ, each consisting of a variable region (VL) and a constant region. Antibodies have a "Y" shape, where the stem of Y consists of a second and third constant region to which two heavy chains are linked via disulfide bonds. Each arm of Y contains a variable region and a first constant region of a single heavy chain bound to a variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs containing CDRL1, CDRL2, and CDRL3; heavy chain CDRs containing CDRH1, CDRH2, and CDRH3). The CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by the conventions of Kabat, IMGT, Chothia, or A1-Lazikani. The three CDRs are inserted between adjacent stretches known as framework regions (FRs) (light chain FRs containing LFR1, LFR2, LFR3, and LFR4; heavy chain FRs containing HFR1, HFR2, HFR3, and HFR4), which are even more conserved than the CDRs and form a scaffold for supporting the highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit various effector functions. Antibodies are designated into classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes and isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of alpha, delta, epsilon, gamma, and muon heavy chains. Several major antibody classes are further classified into subclasses such as IgG1 (gamma single-chain), IgG2 (gamma double-chain), IgG3 (gamma triple-chain), IgG4 (gamma quadruple-chain), IgA1 (alpha single-chain), or IgA2 (alpha double-chain).

[0249] The antibodies disclosed herein include chimeric antibodies that specifically bind to TROP2. Chimeric antibodies are recombinant proteins in which a variable region containing a complementarity-determining region (CDR) of an antibody derived from one or more species, such as a rodent antibody, is present, while the constant domain of the antibody molecule is derived from another species, such as a human antibody.

[0250] The antibodies of this disclosure include humanized antibodies that specifically bind to TROP2. A humanized antibody is a recombinant protein in which a species-derived antibody; for example, a rodent antibody, a CDR, or CDR residues derived from the rodent antibody are translocated from the heavy and light variable chains of the rodent antibody to the human heavy and light variable regions (e.g., framework region sequences). The constant domain of the antibody molecule is derived from that of the human antibody. In certain embodiments, a limited number of framework region amino acid residues derived from the parent (rodent) antibody may be substituted with the human antibody framework region sequence. In other embodiments, simply translocating mouse CDRs to human FRs often results in reduced or even lost antibody affinity, so additional modifications may be necessary to restore the original affinity of the murin antibody. This can be achieved by replacing one or more partial human residues in the FR region with their murin counterparts to obtain an antibody with good binding affinity to its epitope. In some embodiments, the humanized antibody is a humanized single-domain antibody. In some embodiments, humanized antibodies that compete with the single-domain antibodies disclosed herein are also included in this disclosure.

[0251] The antibodies of this disclosure include human antibodies that specifically bind to TROP2. Fully human antibodies can be obtained from transgenic non-human animals. Methods for producing fully human antibodies using either a combinatorial approach or transgenic animals transformed using human immunoglobulin gene sites are well known in the art. In one alternative method, phage display technology may be used to generate human antibodies. Fully human antibodies are expected to exhibit fewer side effects than chimeric or humanized antibodies and to function in vivo as essentially endogenous human antibodies. In some embodiments, human antibodies that compete with the single-domain antibodies disclosed herein are also included in this disclosure.

[0252] The antibodies of this disclosure include single-domain antibodies (sdAb) that specifically bind to TROP2. As used herein, the expressions “single-domain antibody” and “heavy-chain-only antibody” are interchangeable. The term “single-domain antibody” is used in its broadest sense and includes naturally occurring single-domain antibodies or their variants, e.g., humanized single-domain antibodies or chimeric single-domain antibodies, but not limited to these. A single-domain antibody is called a chimeric antibody if it possesses a constant region of another species. In one embodiment, a single-domain antibody is a chimeric single-domain antibody. In some embodiments, a chimeric single-domain antibody includes a humanized variable region and a human constant region. In other embodiments, a chimeric single-domain antibody includes the variable region of a naturally occurring single-domain antibody. The term “single-domain antibody” also encompasses mutated versions of naturally occurring single-domain antibodies, mutated versions of humanized single-domain antibodies, and mutated versions of chimeric single-domain antibodies, whose amino acid sequences include amino acid substitutions, deletions, and / or additions.

[0253] Single-domain antibodies (sdAbs) are typically composed of two heavy chains. They are approximately 12–15 kDa in size (about 110 amino acids in length). Like full-size antibodies, sdAbs can selectively bind to target antigens and have similar affinity for them. However, due to their smaller size, they may be able to penetrate solid tumors more effectively. The smaller size also contributes to the stability of sdAbs, making them more tolerant to extreme pH and temperature conditions than full-size antibodies (Van Der Linden et al., 1999, Biochim Biophys Act 1431: pp. 37–46). Single-domain antibodies were initially developed following the discovery that camelids (camels, alpacas, llamas) possess fully functional antibodies without light chains (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77: pp. 13–22). Naturally occurring single-domain antibodies consist of a single variable region (VH or VHH) and two constant domains (CH2 and CH3). The variable region of a naturally occurring single-domain antibody can be cloned and fused with the constant region of a naturally occurring antibody heavy chain. In some embodiments, the Fc domain of a naturally occurring single-domain antibody is replaced with the Fc or its Fc portion of a human IgG antibody. In some embodiments, the heavy chain constant region of a naturally occurring single-domain antibody is replaced with the human IgG heavy chain constant region containing the CH1, CH2, and CH3 domains, and optionally the CH4 domain, and optionally a hinge region. In some embodiments, the heavy chain constant region of a naturally occurring single-domain antibody is replaced with the CH3 or CH2-CH3 domain of a human IgG heavy chain, and optionally a hinge region.

[0254] The antigen-binding properties of a single-domain antibody are conferred by the heavy chain variable region. Therefore, a single heavy chain or a further single heavy chain variable region (VH or VHH) may be sufficient for specific binding to an antigen. However, single-domain antibodies are typically composed of two heavy chains. These two heavy chains can be assembled via a dimerization domain, such as the constant region of a class G human immunoglobulin. The two heavy chains can also be expressed as a single polypeptide chain.

[0255] Therefore, in some embodiments, the binder includes, for example and not limited to, single-domain antibodies derived from camelids or sharks, human antibodies containing IgG (including human IgG1, human IgG2, human IgG3, and human IgG4), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD, animal antibodies, for example, antibodies and antigen-binding fragments thereof, including IgG (IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE, and IgD, and variants having a similar form (e.g., chimeric, humanized, or human).

[0256] In other embodiments, the binders of the Disclosure include, for example and not limited to, antigen-binding fragments such as Fab, Fab', F(ab')2, complementarity-determining regions, VH, VHH, VL, and variable regions.

[0257] Antigen-binding fragments can be prepared by known techniques, such as proteolytic hydrolysis of full-length antibodies or by expression of the DNA encoding the fragment in Escherichia coli (E. coli) or another host. Antigen-binding fragments can also be obtained by pepsin or papain digestion of full-length antibodies by conventional methods. For example, an antigen-binding fragment can be produced by enzymatic cleavage of an antibody using pepsin, yielding an approximately 100 kD fragment denoted as F(ab')2. The F(ab')2 fragment can be further cleaved using a thiol reducing agent and optionally a protecting group for the sulfhydryl group resulting from the cleavage of the disulfide bond, to produce an approximately 50 kD monovalent Fab' fragment. Alternatively, enzymatic cleavage using papain directly produces two monovalent Fab and Fc fragments.

[0258] In some embodiments, the antibody that binds to TROP2 may be bispecific or polyspecific. A "polyspecific antibody" is an antibody that can simultaneously bind to at least two targets that have different structures, for example, two or more different antigens, the same antigen or hapten and / or two or more different epitopes on an antigen or epitope. A "polyvalent antibody" is an antibody that can simultaneously bind to at least two targets that have the same or different structures. The titer indicates how many antigen-binding domains the antibody has; i.e., monovalent, bivalent, trivalent, or polyvalent. Polyvalentity gives the antibody the advantage of multiple interactions in binding to the antigen, thereby increasing the binding strength to the antigen. Specificity indicates how many antigens or epitopes the antibody can bind to; i.e., monospecific, bispecific, trispecific, or polyspecific. Using these definitions, a natural antibody (e.g., IgG) is bivalent because it has two antigen-binding domains (each consisting of amino acid residues from the light and heavy chain variable regions), but monospecific because each antigen-binding domain binds to the same epitope. A polyspecific, multivalent antibody includes antibodies that have more than one antigen-binding domain of different specificities. A "bispecific antibody" is an antibody that can bind simultaneously to two targets of different structures. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) may have at least one antigen-binding domain that specifically binds to TROP2.

[0259] In addition to recombinant techniques, numerous methods for producing bispecific or polyspecific antibodies are well known, as disclosed, for example, in U.S. Patent No. 7,405,320, the Examples section of which is incorporated herein by reference. Bispecific antibodies can be produced by a quadroma, which involves the fusion of two different fusion cells, each producing a monoclonal antibody that recognizes a different antigen site. Another method for producing bispecific antibodies involves using a heterobifunctional crosslinking agent to chemically link two different monoclonal antibodies.

[0260] Single-domain antibody An exemplary embodiment of the binders of this disclosure is a single-domain antibody. It is understood herein that the following are exemplary and non-limiting embodiments of a single-domain antibody or its antigen-binding fragment.

[0261] Single-domain antibodies possess unique binding characteristics. For example, the complementarity-determining region 3 (CDR3) of the sdAb disclosed herein forms a loop that fits into a groove within the cysteine-rich domain (CRD) of the extracellular domain (ECD) of TROP2. The amino acid residues of CDR3 and framework region 2 (FR2) interact with the amino acid residues of the TROP2 cysteine-rich domain, and together form a clip around the CRD. These unique characteristics confer high affinity and slow dissociation rate of sdAb to TROP2. Surprisingly, the amino acid residues of complementarity-determining region 1 (CDR1) and complementarity-determining region 2 (CDR2) do not appear to interact with the amino acid residues of the CRD.

[0262] The single-domain antibodies disclosed herein also possess anticancer activity, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) activity. The single-domain antibodies disclosed herein may also be conjugated for therapeutic or detection use.

[0263] Exemplary embodiments of the binders of this disclosure include a single-domain antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing amino acids of the extracellular domain (ECD) of TROP2. Other exemplary embodiments of the binders of this disclosure include a single-domain antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing amino acids of the cysteine-rich domain of TROP2. In some embodiments, the single-domain antibody or the antigen-binding fragment thereof binds to the TROP2 antigen disclosed herein.

[0264] In one embodiment, the single-domain antibody of the Disclosure specifically binds to human TROP2. In another embodiment, the single-domain antibody of the Disclosure specifically binds to cynomolgus monkey TROP2. In yet another embodiment, the single-domain antibody of the Disclosure specifically binds to rhesus monkey TROP2.

[0265] More specifically, the single-domain antibody of this disclosure specifically binds to the ECD or CRD of human TROP2.

[0266] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 54, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 52 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 53.

[0267] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 47.

[0268] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CD3 described in SEQ ID NO: 220, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 218 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 219.

[0269] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 220, the amino acid sequence of FR2 described in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 218 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 219.

[0270] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 490, the amino acid sequence of FR2 described in SEQ ID NO: 505, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 488 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 489.

[0271] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 501, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 500 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 489.

[0272] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 230, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 228 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 229.

[0273] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 230, the amino acid sequence of FR2 described in SEQ ID NO: 361, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 228 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 229.

[0274] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 238, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 236 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 237.

[0275] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 246, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 244 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 245.

[0276] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 303, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 301 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 302.

[0277] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 310 and the amino acid sequence of FR2 described in SEQ ID NO: 360, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 308 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 309.

[0278] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 317, the amino acid sequence of FR2 described in SEQ ID NO: 354, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 315 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 316.

[0279] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 324, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 322 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 323.

[0280] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 331, the amino acid sequence of FR2 described in SEQ ID NO: 355, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 329 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 330.

[0281] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 338, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 336 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 337.

[0282] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 345, the amino acid sequence of FR2 described in SEQ ID NO: 356, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 343 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 344.

[0283] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 352, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 350 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 351.

[0284] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 396, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 394 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 395.

[0285] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 404, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 402 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 403.

[0286] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 411, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 409 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 410.

[0287] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 418, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 416, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 416.

[0288] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 425, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 423 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 424.

[0289] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 432, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 430 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 431.

[0290] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 439, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 437 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 438.

[0291] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 446, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 444 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 445.

[0292] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 453, the amino acid sequence of FR2 described in SEQ ID NO: 360, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 451 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 452.

[0293] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 460, the amino acid sequence of FR2 described in SEQ ID NO: 360, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 458 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 459.

[0294] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 467, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 465 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 466.

[0295] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 58, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 56 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 57.

[0296] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 61, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 59 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 60.

[0297] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 51, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 50.

[0298] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 47.

[0299] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 47.

[0300] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 118, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 47.

[0301] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 119, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 47.

[0302] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 124.

[0303] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 48, the amino acid sequence of FR2 described in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 123.

[0304] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 51, the amino acid sequence of FR2 described in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 50.

[0305] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 51, the amino acid sequence of FR2 described in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 50.

[0306] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 51, the amino acid sequence of FR2 described in SEQ ID NO: 118, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 50.

[0307] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 51, the amino acid sequence of FR2 described in SEQ ID NO: 120, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 50.

[0308] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 5.

[0309] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 130.

[0310] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 131.

[0311] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 133.

[0312] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 131.

[0313] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 132.

[0314] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 133.

[0315] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 172, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 133.

[0316] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 134.

[0317] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 4 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 135.

[0318] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 6, the amino acid sequence of FR2 described in SEQ ID NO: 174, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 127 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 129.

[0319] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 14.

[0320] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 137.

[0321] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 138.

[0322] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 217, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 215 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 216.

[0323] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 227, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 225 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 226.

[0324] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 235, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 233 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 234.

[0325] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 243, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 241 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 242.

[0326] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 300, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 298 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 299.

[0327] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 307, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 305 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 306.

[0328] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 314, the amino acid sequence of FR2 described in SEQ ID NO: 357, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 312 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 313.

[0329] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 321, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 319 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 320.

[0330] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 328, the amino acid sequence of FR2 described in SEQ ID NO: 358, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 326 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 327.

[0331] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 335, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 333, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 334.

[0332] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 342, the amino acid sequence of FR2 described in SEQ ID NO: 359, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 340 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 341.

[0333] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 349, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 347 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 348.

[0334] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 393, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 391 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 392.

[0335] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 401, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 399 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 400.

[0336] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 408, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 406 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 407.

[0337] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 415, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 413 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 414.

[0338] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 422, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 420 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 421.

[0339] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 429, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 427 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 428.

[0340] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 436, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 434 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 435.

[0341] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 443, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 441 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 442.

[0342] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 450, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 448 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 449.

[0343] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 457, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 455 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 456.

[0344] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 464, the amino acid sequence of FR2 described in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 462 and / or the amino acid sequence of CDR2 described in SEQ ID NO: 463.

[0345] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 136.

[0346] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 137.

[0347] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 138.

[0348] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 171, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 139.

[0349] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 described in SEQ ID NO: 15, the amino acid sequence of FR2 described in SEQ ID NO: 173, and optionally the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 137.

[0350] In some embodiments, the single-domain antibody or its antigen-binding fragment comprises three CDRs of the single-domain antibody disclosed herein.

[0351] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes a CDR1 having the amino acid sequence described in SEQ ID NO: 1, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 2, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 3.

[0352] In exemplary embodiments, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 4, CDRH2 having the amino acid sequence described in SEQ ID NO: 5, and CDRH3 having the amino acid sequence described in SEQ ID NO: 6.

[0353] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 7, CDRH2 having the amino acid sequence described in SEQ ID NO: 8, and CDRH3 having the amino acid sequence described in SEQ ID NO: 9.

[0354] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 10, CDRH2 having the amino acid sequence described in SEQ ID NO: 11, and CDRH3 having the amino acid sequence described in SEQ ID NO: 12.

[0355] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 13, CDRH2 having the amino acid sequence described in SEQ ID NO: 14, and CDRH3 having the amino acid sequence described in SEQ ID NO: 15.

[0356] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 16, CDRH2 having the amino acid sequence described in SEQ ID NO: 17, and CDRH3 having the amino acid sequence described in SEQ ID NO: 18.

[0357] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 19, CDRH2 having the amino acid sequence described in SEQ ID NO: 20, and CDRH3 having the amino acid sequence described in SEQ ID NO: 21.

[0358] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 46, CDRH2 having the amino acid sequence described in SEQ ID NO: 47, and CDRH3 having the amino acid sequence described in SEQ ID NO: 48.

[0359] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 55, CDRH2 having the amino acid sequence described in SEQ ID NO: 47, and CDRH3 having the amino acid sequence described in SEQ ID NO: 48.

[0360] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 49, CDRH2 having the amino acid sequence described in SEQ ID NO: 50, and CDRH3 having the amino acid sequence described in SEQ ID NO: 51.

[0361] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 62, CDRH2 having the amino acid sequence described in SEQ ID NO: 50, and CDRH3 having the amino acid sequence described in SEQ ID NO: 51.

[0362] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 52, CDRH2 having the amino acid sequence described in SEQ ID NO: 53, and CDRH3 having the amino acid sequence described in SEQ ID NO: 54.

[0363] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 56, CDRH2 having the amino acid sequence described in SEQ ID NO: 57, and CDRH3 having the amino acid sequence described in SEQ ID NO: 58.

[0364] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 59, CDRH2 having the amino acid sequence described in SEQ ID NO: 60, and CDRH3 having the amino acid sequence described in SEQ ID NO: 61.

[0365] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 63, CDRH2 having the amino acid sequence described in SEQ ID NO: 64, and CDRH3 having the amino acid sequence described in SEQ ID NO: 65.

[0366] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence described in SEQ ID NO: 66, CDRH2 having the amino acid sequence described in SEQ ID NO: 67, and CDRH3 having the amino acid sequence described in SEQ ID NO: 68.

[0367] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 123, and the amino acid sequence of CDR3 described in SEQ ID NO: 48.

[0368] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 124, and the amino acid sequence of CDR3 described in SEQ ID NO: 48.

[0369] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 130, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0370] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 131, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0371] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 132, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0372] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 133, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0373] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 134, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0374] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 135, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0375] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 127, the amino acid sequence of CDR2 described in SEQ ID NO: 129, and the amino acid sequence of CDR3 described in SEQ ID NO: 6.

[0376] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 14, and the amino acid sequence of CDR3 described in SEQ ID NO: 15.

[0377] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 136, and the amino acid sequence of CDR3 described in SEQ ID NO: 15.

[0378] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 137, and the amino acid sequence of CDR3 described in SEQ ID NO: 15.

[0379] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 138, and the amino acid sequence of CDR3 described in SEQ ID NO: 15.

[0380] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 139, and the amino acid sequence of CDR3 described in SEQ ID NO: 15.

[0381] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 215, the amino acid sequence of CDR2 described in SEQ ID NO: 216, and the amino acid sequence of CDR3 described in SEQ ID NO: 217.

[0382] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 218, the amino acid sequence of CDR2 described in SEQ ID NO: 219, and the amino acid sequence of CDR3 described in SEQ ID NO: 220.

[0383] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 225, the amino acid sequence of CDR2 described in SEQ ID NO: 226, and the amino acid sequence of CDR3 described in SEQ ID NO: 227.

[0384] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 228, the amino acid sequence of CDR2 described in SEQ ID NO: 229, and the amino acid sequence of CDR3 described in SEQ ID NO: 230.

[0385] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 233, the amino acid sequence of CDR2 described in SEQ ID NO: 234, and the amino acid sequence of CDR3 described in SEQ ID NO: 235.

[0386] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 236, the amino acid sequence of CDR2 described in SEQ ID NO: 237, and the amino acid sequence of CDR3 described in SEQ ID NO: 238.

[0387] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 241, the amino acid sequence of CDR2 described in SEQ ID NO: 242, and the amino acid sequence of CDR3 described in SEQ ID NO: 243.

[0388] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 244, the amino acid sequence of CDR2 described in SEQ ID NO: 245, and the amino acid sequence of CDR3 described in SEQ ID NO: 246.

[0389] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 298, the amino acid sequence of CDR2 described in SEQ ID NO: 299, and the amino acid sequence of CDR3 described in SEQ ID NO: 300.

[0390] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 301, the amino acid sequence of CDR2 described in SEQ ID NO: 302, and the amino acid sequence of CDR3 described in SEQ ID NO: 303.

[0391] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 305, the amino acid sequence of CDR2 described in SEQ ID NO: 306, and the amino acid sequence of CDR3 described in SEQ ID NO: 307.

[0392] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 308, the amino acid sequence of CDR2 described in SEQ ID NO: 309, and the amino acid sequence of CDR3 described in SEQ ID NO: 310.

[0393] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 312, the amino acid sequence of CDR2 described in SEQ ID NO: 313, and the amino acid sequence of CDR3 described in SEQ ID NO: 314.

[0394] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 315, the amino acid sequence of CDR2 described in SEQ ID NO: 316, and the amino acid sequence of CDR3 described in SEQ ID NO: 317.

[0395] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 319, the amino acid sequence of CDR2 described in SEQ ID NO: 320, and the amino acid sequence of CDR3 described in SEQ ID NO: 321.

[0396] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 322, the amino acid sequence of CDR2 described in SEQ ID NO: 323, and the amino acid sequence of CDR3 described in SEQ ID NO: 324.

[0397] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 326, the amino acid sequence of CDR2 described in SEQ ID NO: 327, and the amino acid sequence of CDR3 described in SEQ ID NO: 328.

[0398] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 329, the amino acid sequence of CDR2 described in SEQ ID NO: 330, and the amino acid sequence of CDR3 described in SEQ ID NO: 331.

[0399] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 333, the amino acid sequence of CDR2 described in SEQ ID NO: 334, and the amino acid sequence of CDR3 described in SEQ ID NO: 335.

[0400] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 336, the amino acid sequence of CDR2 described in SEQ ID NO: 337, and the amino acid sequence of CDR3 described in SEQ ID NO: 338.

[0401] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 340, the amino acid sequence of CDR2 described in SEQ ID NO: 341, and the amino acid sequence of CDR3 described in SEQ ID NO: 342.

[0402] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 343, the amino acid sequence of CDR2 described in SEQ ID NO: 344, and the amino acid sequence of CDR3 described in SEQ ID NO: 345.

[0403] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 347, the amino acid sequence of CDR2 described in SEQ ID NO: 348, and the amino acid sequence of CDR3 described in SEQ ID NO: 349.

[0404] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 350, the amino acid sequence of CDR2 described in SEQ ID NO: 351, and the amino acid sequence of CDR3 described in SEQ ID NO: 352.

[0405] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 391, the amino acid sequence of CDR2 described in SEQ ID NO: 392, and the amino acid sequence of CDR3 described in SEQ ID NO: 393.

[0406] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 394, the amino acid sequence of CDR2 described in SEQ ID NO: 395, and the amino acid sequence of CDR3 described in SEQ ID NO: 396.

[0407] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 399, the amino acid sequence of CDR2 described in SEQ ID NO: 400, and the amino acid sequence of CDR3 described in SEQ ID NO: 401.

[0408] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 402, the amino acid sequence of CDR2 described in SEQ ID NO: 403, and the amino acid sequence of CDR3 described in SEQ ID NO: 404.

[0409] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 406, the amino acid sequence of CDR2 described in SEQ ID NO: 407, and the amino acid sequence of CDR3 described in SEQ ID NO: 408.

[0410] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 409, the amino acid sequence of CDR2 described in SEQ ID NO: 410, and the amino acid sequence of CDR3 described in SEQ ID NO: 411.

[0411] In exemplary embodiments, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 413, the amino acid sequence of CDR2 described in SEQ ID NO: 414, and the amino acid sequence of CDR3 described in SEQ ID NO: 415.

[0412] In exemplary embodiments, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 416, the amino acid sequence of CDR2 described in SEQ ID NO: 417, and the amino acid sequence of CDR3 described in SEQ ID NO: 418.

[0413] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 420, the amino acid sequence of CDR2 described in SEQ ID NO: 421, and the amino acid sequence of CDR3 described in SEQ ID NO: 422.

[0414] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 423, the amino acid sequence of CDR2 described in SEQ ID NO: 424, and the amino acid sequence of CDR3 described in SEQ ID NO: 425.

[0415] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 427, the amino acid sequence of CDR2 described in SEQ ID NO: 428, and the amino acid sequence of CDR3 described in SEQ ID NO: 429.

[0416] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 430, the amino acid sequence of CDR2 described in SEQ ID NO: 431, and the amino acid sequence of CDR3 described in SEQ ID NO: 432.

[0417] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 434, the amino acid sequence of CDR2 described in SEQ ID NO: 435, and the amino acid sequence of CDR3 described in SEQ ID NO: 436.

[0418] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 437, the amino acid sequence of CDR2 described in SEQ ID NO: 438, and the amino acid sequence of CDR3 described in SEQ ID NO: 439.

[0419] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 441, the amino acid sequence of CDR2 described in SEQ ID NO: 442, and the amino acid sequence of CDR3 described in SEQ ID NO: 443.

[0420] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 444, the amino acid sequence of CDR2 described in SEQ ID NO: 445, and the amino acid sequence of CDR3 described in SEQ ID NO: 446.

[0421] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 448, the amino acid sequence of CDR2 described in SEQ ID NO: 449, and the amino acid sequence of CDR3 described in SEQ ID NO: 450.

[0422] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 451, the amino acid sequence of CDR2 described in SEQ ID NO: 452, and the amino acid sequence of CDR3 described in SEQ ID NO: 453.

[0423] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 455, the amino acid sequence of CDR2 described in SEQ ID NO: 456, and the amino acid sequence of CDR3 described in SEQ ID NO: 457.

[0424] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 458, the amino acid sequence of CDR2 described in SEQ ID NO: 459, and the amino acid sequence of CDR3 described in SEQ ID NO: 460.

[0425] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 462, the amino acid sequence of CDR2 described in SEQ ID NO: 463, and the amino acid sequence of CDR3 described in SEQ ID NO: 464.

[0426] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 465, the amino acid sequence of CDR2 described in SEQ ID NO: 466, and the amino acid sequence of CDR3 described in SEQ ID NO: 467.

[0427] In exemplary embodiments, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 485, the amino acid sequence of CDR2 described in SEQ ID NO: 486, and the amino acid sequence of CDR3 described in SEQ ID NO: 487.

[0428] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 488, the amino acid sequence of CDR2 described in SEQ ID NO: 489, and the amino acid sequence of CDR3 described in SEQ ID NO: 490.

[0429] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 485, the amino acid sequence of CDR2 described in SEQ ID NO: 492, and the amino acid sequence of CDR3 described in SEQ ID NO: 487.

[0430] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 485, the amino acid sequence of CDR2 described in SEQ ID NO: 495, and the amino acid sequence of CDR3 described in SEQ ID NO: 487.

[0431] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 485, the amino acid sequence of CDR2 described in SEQ ID NO: 498, and the amino acid sequence of CDR3 described in SEQ ID NO: 499.

[0432] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 500, the amino acid sequence of CDR2 described in SEQ ID NO: 489, and the amino acid sequence of CDR3 described in SEQ ID NO: 501.

[0433] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 485, the amino acid sequence of CDR2 described in SEQ ID NO: 486, and the amino acid sequence of CDR3 described in SEQ ID NO: 499.

[0434] In some embodiments, the single-domain antibody or antigen-binding fragment thereof of the Disclosure comprises the CDR1, CDR2, CDR3, and FR2 amino acid sequences of the single-domain antibody disclosed herein.

[0435] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 52, the amino acid sequence of CDR2 described in SEQ ID NO: 53, the amino acid sequence of CDR3 described in SEQ ID NO: 54, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0436] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 47, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0437] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 56, the amino acid sequence of CDR2 described in SEQ ID NO: 57, the amino acid sequence of CDR3 described in SEQ ID NO: 58, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0438] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 59, the amino acid sequence of CDR2 described in SEQ ID NO: 60, the amino acid sequence of CDR3 described in SEQ ID NO: 61, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0439] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 62, the amino acid sequence of CDR2 described in SEQ ID NO: 50, the amino acid sequence of CDR3 described in SEQ ID NO: 51, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0440] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 47, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0441] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 218, the amino acid sequence of CDR2 described in SEQ ID NO: 219, the amino acid sequence of CDR3 described in SEQ ID NO: 220, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0442] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 218, the amino acid sequence of CDR2 described in SEQ ID NO: 219, the amino acid sequence of CDR3 described in SEQ ID NO: 220, and the amino acid sequence of FR2 described in SEQ ID NO: 117.

[0443] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 228, the amino acid sequence of CDR2 described in SEQ ID NO: 229, the amino acid sequence of CDR3 described in SEQ ID NO: 230, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0444] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 236, the amino acid sequence of CDR2 described in SEQ ID NO: 237, the amino acid sequence of CDR3 described in SEQ ID NO: 238, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0445] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 244, the amino acid sequence of CDR2 described in SEQ ID NO: 245, the amino acid sequence of CDR3 described in SEQ ID NO: 246, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0446] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 301, the amino acid sequence of CDR2 described in SEQ ID NO: 302, the amino acid sequence of CDR3 described in SEQ ID NO: 303, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0447] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 308, the amino acid sequence of CDR2 described in SEQ ID NO: 309, the amino acid sequence of CDR3 described in SEQ ID NO: 310, and the amino acid sequence of FR2 described in SEQ ID NO: 360.

[0448] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 315, the amino acid sequence of CDR2 described in SEQ ID NO: 316, the amino acid sequence of CDR3 described in SEQ ID NO: 317, and the amino acid sequence of FR2 described in SEQ ID NO: 354.

[0449] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 322, the amino acid sequence of CDR2 described in SEQ ID NO: 323, the amino acid sequence of CDR3 described in SEQ ID NO: 324, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0450] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 329, the amino acid sequence of CDR2 described in SEQ ID NO: 330, the amino acid sequence of CDR3 described in SEQ ID NO: 331, and the amino acid sequence of FR2 described in SEQ ID NO: 355.

[0451] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 336, the amino acid sequence of CDR2 described in SEQ ID NO: 337, the amino acid sequence of CDR3 described in SEQ ID NO: 338, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0452] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 343, the amino acid sequence of CDR2 described in SEQ ID NO: 344, the amino acid sequence of CDR3 described in SEQ ID NO: 345, and the amino acid sequence of FR2 described in SEQ ID NO: 356.

[0453] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 350, the amino acid sequence of CDR2 described in SEQ ID NO: 351, the amino acid sequence of CDR3 described in SEQ ID NO: 352, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0454] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 394, the amino acid sequence of CDR2 described in SEQ ID NO: 395, the amino acid sequence of CDR3 described in SEQ ID NO: 396, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0455] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 402, the amino acid sequence of CDR2 described in SEQ ID NO: 403, the amino acid sequence of CDR3 described in SEQ ID NO: 404, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0456] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 409, the amino acid sequence of CDR2 described in SEQ ID NO: 410, the amino acid sequence of CDR3 described in SEQ ID NO: 411, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0457] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 416, the amino acid sequence of CDR2 described in SEQ ID NO: 417, the amino acid sequence of CDR3 described in SEQ ID NO: 418, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0458] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 423, the amino acid sequence of CDR2 described in SEQ ID NO: 424, the amino acid sequence of CDR3 described in SEQ ID NO: 425, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0459] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 430, the amino acid sequence of CDR2 described in SEQ ID NO: 431, the amino acid sequence of CDR3 described in SEQ ID NO: 432, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0460] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 437, the amino acid sequence of CDR2 described in SEQ ID NO: 438, the amino acid sequence of CDR3 described in SEQ ID NO: 439, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0461] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 444, the amino acid sequence of CDR2 described in SEQ ID NO: 445, the amino acid sequence of CDR3 described in SEQ ID NO: 446, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0462] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 451, the amino acid sequence of CDR2 described in SEQ ID NO: 452, the amino acid sequence of CDR3 described in SEQ ID NO: 453, and the amino acid sequence of FR2 described in SEQ ID NO: 360.

[0463] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 458, the amino acid sequence of CDR2 described in SEQ ID NO: 459, the amino acid sequence of CDR3 described in SEQ ID NO: 460, and the amino acid sequence of FR2 described in SEQ ID NO: 360.

[0464] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 465, the amino acid sequence of CDR2 described in SEQ ID NO: 466, the amino acid sequence of CDR3 described in SEQ ID NO: 467, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0465] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 488, the amino acid sequence of CDR2 described in SEQ ID NO: 489, the amino acid sequence of CDR3 described in SEQ ID NO: 490, and the amino acid sequence of FR2 described in SEQ ID NO: 505.

[0466] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 500, the amino acid sequence of CDR2 described in SEQ ID NO: 489, the amino acid sequence of CDR3 described in SEQ ID NO: 501, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0467] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 47, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 117.

[0468] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 47, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 118.

[0469] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 47, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 119.

[0470] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 124, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 117.

[0471] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 55, the amino acid sequence of CDR2 described in SEQ ID NO: 123, the amino acid sequence of CDR3 described in SEQ ID NO: 48, and the amino acid sequence of FR2 described in SEQ ID NO: 117.

[0472] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 62, the amino acid sequence of CDR2 described in SEQ ID NO: 50, the amino acid sequence of CDR3 described in SEQ ID NO: 51, and the amino acid sequence of FR2 described in SEQ ID NO: 116.

[0473] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 62, the amino acid sequence of CDR2 described in SEQ ID NO: 50, the amino acid sequence of CDR3 described in SEQ ID NO: 51, and the amino acid sequence of FR2 described in SEQ ID NO: 117.

[0474] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 62, the amino acid sequence of CDR2 described in SEQ ID NO: 50, the amino acid sequence of CDR3 described in SEQ ID NO: 51, and the amino acid sequence of FR2 described in SEQ ID NO: 118.

[0475] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 62, the amino acid sequence of CDR2 described in SEQ ID NO: 50, the amino acid sequence of CDR3 described in SEQ ID NO: 51, and the amino acid sequence of FR2 described in SEQ ID NO: 120.

[0476] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 5, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0477] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 130, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0478] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 131, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0479] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 133, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0480] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 131, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0481] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 132, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0482] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 133, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0483] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 133, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 172.

[0484] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 134, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0485] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 4, the amino acid sequence of CDR2 described in SEQ ID NO: 135, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0486] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 127, the amino acid sequence of CDR2 described in SEQ ID NO: 129, the amino acid sequence of CDR3 described in SEQ ID NO: 6, and the amino acid sequence of FR2 described in SEQ ID NO: 174.

[0487] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 14, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0488] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 137, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0489] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 138, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0490] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 215, the amino acid sequence of CDR2 described in SEQ ID NO: 216, the amino acid sequence of CDR3 described in SEQ ID NO: 217, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0491] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 225, the amino acid sequence of CDR2 described in SEQ ID NO: 226, the amino acid sequence of CDR3 described in SEQ ID NO: 227, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0492] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 233, the amino acid sequence of CDR2 described in SEQ ID NO: 234, the amino acid sequence of CDR3 described in SEQ ID NO: 235, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0493] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 241, the amino acid sequence of CDR2 described in SEQ ID NO: 242, the amino acid sequence of CDR3 described in SEQ ID NO: 243, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0494] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 298, the amino acid sequence of CDR2 described in SEQ ID NO: 299, the amino acid sequence of CDR3 described in SEQ ID NO: 300, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0495] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 305, the amino acid sequence of CDR2 described in SEQ ID NO: 306, the amino acid sequence of CDR3 described in SEQ ID NO: 307, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0496] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 312, the amino acid sequence of CDR2 described in SEQ ID NO: 313, the amino acid sequence of CDR3 described in SEQ ID NO: 314, and the amino acid sequence of FR2 described in SEQ ID NO: 357.

[0497] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 319, the amino acid sequence of CDR2 described in SEQ ID NO: 320, the amino acid sequence of CDR3 described in SEQ ID NO: 321, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0498] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 326, the amino acid sequence of CDR2 described in SEQ ID NO: 327, the amino acid sequence of CDR3 described in SEQ ID NO: 328, and the amino acid sequence of FR2 described in SEQ ID NO: 358.

[0499] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 333, the amino acid sequence of CDR2 described in SEQ ID NO: 334, the amino acid sequence of CDR3 described in SEQ ID NO: 335, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0500] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 340, the amino acid sequence of CDR2 described in SEQ ID NO: 341, the amino acid sequence of CDR3 described in SEQ ID NO: 342, and the amino acid sequence of FR2 described in SEQ ID NO: 359.

[0501] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 347, the amino acid sequence of CDR2 described in SEQ ID NO: 348, the amino acid sequence of CDR3 described in SEQ ID NO: 349, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0502] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 391, the amino acid sequence of CDR2 described in SEQ ID NO: 392, the amino acid sequence of CDR3 described in SEQ ID NO: 393, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0503] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 399, the amino acid sequence of CDR2 described in SEQ ID NO: 400, the amino acid sequence of CDR3 described in SEQ ID NO: 401, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0504] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 406, the amino acid sequence of CDR2 described in SEQ ID NO: 407, the amino acid sequence of CDR3 described in SEQ ID NO: 408, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0505] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 413, the amino acid sequence of CDR2 described in SEQ ID NO: 414, the amino acid sequence of CDR3 described in SEQ ID NO: 415, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0506] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 420, the amino acid sequence of CDR2 described in SEQ ID NO: 421, the amino acid sequence of CDR3 described in SEQ ID NO: 422, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0507] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 427, the amino acid sequence of CDR2 described in SEQ ID NO: 428, the amino acid sequence of CDR3 described in SEQ ID NO: 429, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0508] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 434, the amino acid sequence of CDR2 described in SEQ ID NO: 435, the amino acid sequence of CDR3 described in SEQ ID NO: 436, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0509] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 441, the amino acid sequence of CDR2 described in SEQ ID NO: 442, the amino acid sequence of CDR3 described in SEQ ID NO: 443, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0510] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 448, the amino acid sequence of CDR2 described in SEQ ID NO: 449, the amino acid sequence of CDR3 described in SEQ ID NO: 450, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0511] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 455, the amino acid sequence of CDR2 described in SEQ ID NO: 456, the amino acid sequence of CDR3 described in SEQ ID NO: 457, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0512] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment includes the amino acid sequence of CDR1 described in SEQ ID NO: 462, the amino acid sequence of CDR2 described in SEQ ID NO: 463, the amino acid sequence of CDR3 described in SEQ ID NO: 464, and the amino acid sequence of FR2 described in SEQ ID NO: 170.

[0513] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 136, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0514] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, and / or the amino acid sequence of CDR2 described in SEQ ID NO: 137, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0515] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 138, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0516] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 139, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 171.

[0517] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 described in SEQ ID NO: 13, the amino acid sequence of CDR2 described in SEQ ID NO: 137, the amino acid sequence of CDR3 described in SEQ ID NO: 15, and the amino acid sequence of FR2 described in SEQ ID NO: 173.

[0518] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises a variable region of a single-domain antibody disclosed herein.

[0519] In a further exemplary embodiment, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 22.

[0520] In other exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 23.

[0521] In a further exemplary embodiment, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 24.

[0522] In an additional exemplary embodiment, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 25.

[0523] In other exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 26.

[0524] In a further exemplary embodiment, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 27.

[0525] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 28.

[0526] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 221.

[0527] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 222.

[0528] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 223.

[0529] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 224.

[0530] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 231.

[0531] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 232.

[0532] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 239.

[0533] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 240.

[0534] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 247.

[0535] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 304.

[0536] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 311.

[0537] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 318.

[0538] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 325.

[0539] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 332.

[0540] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 339.

[0541] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 346.

[0542] In further exemplary embodiments, the single-domain antibody includes a variable region having the amino acid sequence described in SEQ ID NO: 353.

[0543] In some embodiments, the single-domain antibody may contain a sequence derived from a combination of CDRH1, CDRH2, and CDRH3 and a heavy chain variable region skeleton as described in Table 23 (Table 45) or Table 24 (Table 46).

[0544] Therefore, in some embodiments, the single-domain antibody may contain the sequence shown in Table 1A.

[0545] [Table 1A]

[0546] [Table 1B]

[0547] [Table 1C]

[0548] [Table 1D]

[0549] [Table 1E]

[0550] [Table 1F]

[0551] The positions of the residues referenced in Table 23 (Table 45) and Table 24 (Table 46) should be understood to refer to the positions of the amino acid residues resulting from Sequence ID No. 145 in Table 1C (Table 5), taking into account the insertion of the complete amino acid sequences of IMGT CDR1, CDR2, and CDR3 into the humanized heavy chain variable region backbone. Therefore, when inserting the complete amino acid sequences of IMGT CDR1, CDR2, and CDR3, IMGT framework 1 generally extends to positions 1-25, IMGT framework 2 generally extends to positions 34-50, IMGT framework 3 generally extends to positions 58-95, and IMGT framework 4 generally extends to positions 110-120. Since the length of IMGT CDR may vary slightly, the arrangement of the frameworks may vary accordingly. However, those skilled in the art can determine the number of positions of a given variable region by alignment with Sequence ID No. 145 in Table 1C (Table 5).

[0552] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 29.

[0553] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 71.

[0554] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 72.

[0555] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 73.

[0556] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 74.

[0557] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 75.

[0558] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 76.

[0559] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 77.

[0560] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 78.

[0561] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 79.

[0562] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 80.

[0563] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 81.

[0564] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 82.

[0565] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 83.

[0566] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 84.

[0567] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 85.

[0568] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 86.

[0569] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 87.

[0570] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 88.

[0571] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 89.

[0572] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 90.

[0573] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 91.

[0574] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 92.

[0575] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 93.

[0576] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 94.

[0577] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 95.

[0578] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 96.

[0579] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 97.

[0580] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 98.

[0581] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 99.

[0582] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 100.

[0583] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 101.

[0584] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 012.

[0585] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 103.

[0586] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 104.

[0587] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 105.

[0588] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 106.

[0589] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 107.

[0590] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 108.

[0591] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 109.

[0592] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 110.

[0593] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 111.

[0594] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 112.

[0595] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 113.

[0596] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 114.

[0597] In an additional exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 115.

[0598] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 221.

[0599] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 222.

[0600] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 223.

[0601] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 224.

[0602] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 231.

[0603] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 232.

[0604] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 239.

[0605] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 240.

[0606] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 247.

[0607] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 295.

[0608] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 296.

[0609] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 297.

[0610] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 304.

[0611] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 311.

[0612] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 318.

[0613] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 325.

[0614] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 332.

[0615] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 339.

[0616] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 346.

[0617] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 353.

[0618] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 362.

[0619] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 363.

[0620] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 364.

[0621] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 365.

[0622] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 366.

[0623] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 367.

[0624] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 368.

[0625] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 369.

[0626] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 370.

[0627] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 371.

[0628] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 372.

[0629] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 373.

[0630] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 374.

[0631] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 375.

[0632] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 376.

[0633] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 377.

[0634] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 378.

[0635] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 379.

[0636] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 380.

[0637] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 381.

[0638] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 382.

[0639] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 383.

[0640] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 384.

[0641] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 385.

[0642] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 386.

[0643] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 387.

[0644] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 388.

[0645] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 389.

[0646] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 390.

[0647] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 397.

[0648] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 398.

[0649] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 405.

[0650] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 412.

[0651] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 419.

[0652] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 426.

[0653] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 433.

[0654] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 440.

[0655] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 447.

[0656] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 454.

[0657] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 461.

[0658] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 468.

[0659] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 469.

[0660] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 470.

[0661] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 471.

[0662] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 472.

[0663] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 473.

[0664] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 491.

[0665] In additional exemplary embodiments, a single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 493.

[0666] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 496.

[0667] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 502.

[0668] In additional exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 503.

[0669] In an exemplary embodiment, Sequence IDs 221, 222, 223, 224, 231, 232, 239, 240, 247, 325, 332, 339, 346, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 296, 297, 372, 373, 374, 375, 376, 377, 378, 37 Single-domain antibodies containing the CDR or heavy chain variable region or a humanized version thereof as described in 9, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 397, 398, 405, 412, 419, 426, 433, 440, 447, 454, 461, 468, and 318 are particularly selected.

[0670] In another exemplary embodiment, a single-domain antibody or its antigen-binding fragment comprises the heavy chain of a single-domain antibody disclosed herein.

[0671] In an exemplary embodiment, the single-domain antibody contains the amino acid sequence described in SEQ ID NO: 29.

[0672] In exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 30.

[0673] In yet another exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 31.

[0674] In a further exemplary embodiment, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 32.

[0675] In further exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 33.

[0676] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 34.

[0677] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 35.

[0678] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 44.

[0679] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 45.

[0680] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 482.

[0681] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 483.

[0682] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 484.

[0683] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 494.

[0684] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 497.

[0685] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence described in SEQ ID NO: 504.

[0686] In some cases, the binders of this disclosure do not contain any of the amino acid sequences described in SEQ ID NOs. 22-28, 71-115, or 143-146.

[0687] In some cases, the binders of this disclosure do not contain the amino acid sequence described in SEQ ID NO: 295.

[0688] In some cases, the binders of this disclosure do not contain the amino acid sequence described in SEQ ID NO: 304.

[0689] In some cases, the binders of this disclosure do not contain the amino acid sequence described in SEQ ID NO: 311.

[0690] In some cases, the binders of this disclosure do not contain the amino acid sequence described in SEQ ID NO: 353.

[0691] In some cases, the binders of this disclosure do not contain the amino acid sequence described in SEQ ID NO: 362.

[0692] In some examples, the binders of the present disclosure may include any one of the amino acid sequences described in SEQ ID NOs. 22-28, 71-115, and 143-146, however, the binders of the present disclosure may also include SEQ ID NOs. 221-224, 231, 232, 239, 240, 247, 304, 295, 296, 297, 311, 318, 325, 332, 339, 346, 353, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, and 37 5. Also includes the amino acid sequences described in any one of the following: SEQ ID NOs: 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 397, 398, 405, 412, 419, 426, 433, 440, 447, 454, 461, 468, 469, 470, 471, 472, 473, 491, 493, 496, 502, or 503.

[0693] In accordance with this disclosure, single-domain antibodies may include the amino acid sequence combinations and dimerization domains shown in Table 1A.

[0694] In some embodiments, the dimerization domain may include SEQ ID NO: 38 or a variant thereof. In other embodiments, the dimerization domain may include SEQ ID NO: 43 or a variant thereof. The dimerization domain may also include SEQ ID NO: 37 or SEQ ID NO: 179 or a variant thereof in combination with either SEQ ID NO: 36 or SEQ ID NO: 42 or a variant thereof.

[0695] In further embodiments, the dimerizing domain may include SEQ ID NO: 212 or a variant thereof. In further embodiments, the dimerizing domain may include SEQ ID NO: 506 or a variant thereof. In even further embodiments, the dimerizing domain may include SEQ ID NO: 214 or a variant thereof.

[0696] Therefore, in the exemplary embodiments, the single-domain antibody may contain the amino acid sequence shown in Table 1B (Table 2).

[0697] [Table 2A]

[0698] [Table 2B]

[0699] [Table 2C]

[0700] [Table 2D]

[0701] [Table 2E]

[0702] [Table 2F]

[0703] [Table 2G]

[0704] [Table 2H]

[0705] [Table 2I]

[0706] Binding agent containing the antigen-binding domain of sdAb The binders of this disclosure comprise one or more antigen-binding domains of an antibody or its antigen-binding fragment that binds to TROP2. For example, the binders of this disclosure comprise one or more antigen-binding domains of a single-domain antibody disclosed herein. For example, the binder may comprise three CDRs of a single domain disclosed herein. In another example, the binder may comprise a variable region or a portion thereof of a single domain disclosed herein.

[0707] It is understood herein that the following represent exemplary and non-limiting embodiments of a binder containing the antigen-binding domain of a single-domain antibody.

[0708] In some embodiments, the binders of the Disclosure include one or more antigen-binding domains that specifically bind to trophoblast cell surface antigen-2 (TROP2). In some embodiments, the binders of the Disclosure include one or more antigen-binding domains that bind to a target other than TROP2.

[0709] Exemplary embodiments of the binders of this disclosure include those comprising one or more antigen-binding domains that specifically bind to at least one epitope containing amino acids of the extracellular domain (ECD) of TROP2. Other exemplary embodiments of the binders of this disclosure include those comprising one or more antigen-binding domains that specifically bind to at least one epitope containing amino acids of the cysteine-rich domain of TROP2. In some embodiments, the binder comprises one or more antigen-binding domains that specifically bind to the TROP2 antigen disclosed herein.

[0710] In one embodiment, the binder of the Disclosure comprises one or more antigen-binding domains that specifically bind to human TROP2. In another embodiment, the binder of the Disclosure comprises one or more antigen-binding domains that specifically bind to cynomolgus monkey TROP2. In yet another embodiment, the binder of the Disclosure comprises one or more antigen-binding domains that specifically bind to rhesus monkey TROP2.

[0711] More specifically, the binder of this disclosure comprises one or more antigen-binding domains that specifically bind to the ECD or CRD of human TROP2.

[0712] In other embodiments, the binder of the present disclosure comprises one or more antigen-binding domains of a native antibody (CDR and / or VL and VH) that binds to TROP2.

[0713] The CDR or variable regions (VL and VH) of antibodies and their antigen-binding fragments can be expressed as a single polypeptide chain to generate single-chain Fv(scFV) molecules and variants. Numerous forms are considered, as shown below.

[0714] Similarly, the CDRs or variable regions of multiple single-domain antibodies may be expressed as a single polypeptide chain, thereby conferring polyvalence and / or polyspecificity to the binder. Furthermore, multiple polypeptide chains may be assembled to increase the diversity of interaction with TROP2 or the binding affinity. Numerous configurations shown below are considered.

[0715] Therefore, the antigen-binding fragments or antigen-binding domains of single-domain antibodies disclosed herein can be used to generate conjugates that are monospecific, polyspecific, monovalent, or polyvalent.

[0716] For example, the variable region or CDR of a single-domain antibody can be fused with the antibody's Fc, CH3, or CH2-CH3 domain to enable homodimerization, thereby generating a polyvalent conjugate.

[0717] Alternatively, multiple copies of the variable region or CDR of a single-domain antibody may be fused in tandem with spacers or linkers necessary to generate a multivalent and monospecific binder.

[0718] For example, the variable regions or CDRs of two single-domain antibodies can be fused with Fc, CH3, or CH2-CH3 domains to enable heterodimerization, thereby generating a multivalent and multispecific binder.

[0719] Alternatively, the variable regions or CDRs of two or more single-domain antibodies may be fused in tandem with spacers or linkers necessary to generate a multivalent and multispecific binder.

[0720] The binders of this disclosure may take the form of antibodies or antigen-binding fragments thereof, antibody-like molecules (single-domain antibodies or natural antibodies fused with antigen-binding domains such as sdAb, Fc-, CH3 fusions), fusions with protein scaffolds, immunocytomodulators, and the like.

[0721] The binders also include immunocytomodulators such as biaffinity retargeting molecules (DARTs), chimeric antigen receptor (CAR) constructs, bispecific T cell engager constructs (BiTEs), bispecific killer cell engagers (BiKEs), and trispecific killer cell engagers (TriKEs) containing scFv or VHH.

[0722] Examples of binders include ankyrin repeat protein, the Z domain of Staphylococcus protein A, fibronetin type III, and Nottin, and also include fusion products with protein scaffolds. Exemplary embodiments of the binder include monospecific antibodies, dispecific antibodies (symmetric or asymmetric), trispecific antibodies or polyspecific antibodies, and monovalent antibodies, divalent antibodies, trivalent antibodies or polyvalent antibodies, single-chain FV (scFV), and derivatives such as diabody, triabody, tetrabody, tandem di-scFvs, tandem tri-scFvs, scFV-Fc, minibody (scFV-CH3), tandem diabody, didiabody, and bibody, VH or VHH, and derivatives such as tandem dispecific or polyspecific VHH, divalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, divalent CH3 fusions, VHH pentabody, and decabody.

[0723] A single-chain Fv molecule (scFv) contains a VL domain and a VH domain. The VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L). The scFv molecule is denoted as VL-L-VH if the VL domain is the N-terminal portion of the scFv molecule, or as VH-L-VL if the VH domain is the N-terminal portion of the scFv molecule. Methods for constructing scFv molecules and designing suitable peptide linkers are described in U.S. Patents 4,704,692 and 4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs,” FASEB Vol. 9: pp. 73-80 (1995), and RE Bird and BW Walker, “Single Chain Antibody Variable Regions,” TIBTECH Vol. 9: pp. 132-137 (1991).

[0724] In some embodiments, the binders of this disclosure may have the forms of formulas Ia, Ib, Ic, II, III, IV, V, VI, VII, or VIII, etc., disclosed herein, or formulas I, II, III, IIIa and IIIb, IV, V, VI, VII, or VIII, etc., whose entire contents are incorporated herein by reference (PCT / CA2020 / 051753 filed on 18 December 2020 and WO2021 / 119832A1 published on 24 June 2021).

[0725] The binders of this disclosure may be formed by the assembly of two polypeptide chains having the same (or different) configuration (having the same or different amino acid sequences) or different configurations, where the same or different configurations include those described in Formulas I, II, III, IIIa and IIIb, IV, V, VI, VII or VIII disclosed herein.

[0726] In some embodiments, the binders of the Disclosure are monospecific. In some embodiments, the binders of the Disclosure are polyspecific.

[0727] The binders of this disclosure (including antibodies or antigen-binding fragments thereof) may be monospecific, polyspecific, monovalent, or polyvalent.

[0728] A binder containing two or more antigen-binding domains of a single-domain antibody is considered polyvalent. A polyvalent single-domain antibody can be monospecific (all antigen-binding domains bind to exactly the same epitope) or polyspecific (antigen-binding domains bind to different epitopes on the same target, or to different epitopes or targets). A binder containing only one antigen-binding domain is considered monovalent.

[0729] The production of binders, such as antibodies or antigen-binding fragments, may involve cloning procedures and recombinant expression. Antigen-binding domain sequences (e.g., the sequences of the variable light and heavy chains of an intact antibody, or the sequence of the variable heavy chain of a single-domain antibody) can be obtained by various molecular cloning procedures, such as RT-PCR, 5'-RACE, and cDNA library screening. The sequence of interest can be cloned into an expression vector, the expression vector can be transfected into cells, and the binder can be isolated and / or purified. In the case of an intact antibody, the light and heavy chains can be cloned into a single expression vector or into two separate expression vectors. Simultaneous transfection of separate expression vectors is necessary for the intact antibody to be correctly assembled. In the case of a single-domain antibody, a single expression vector (expressing the heavy chain) is sufficient.

[0730] Monospecific binders include binders that are specific to a single epitope of a given antigen.

[0731] An exemplary embodiment of a monospecific binder includes a binder containing one antigen-binding fragment of a single-domain antibody. Another exemplary embodiment of a monospecific binder includes a binder containing more than one antigen-binding fragment of a single-domain antibody, but the antigen-binding fragments have the same CDR and framework region. Yet another exemplary embodiment of a binder includes a binder containing more than one antigen-binding fragment of a single-domain antibody, but the variable region has the same CDR and different framework region. A further exemplary embodiment of a monospecific binder includes a binder containing antigen-binding fragments of a single-domain antibody that differ in one or more amino acid sequences of their CDRs (e.g., conserved substitutions in one or more CDRs) without affecting their ability to bind to the same antigen or epitope.

[0732] A multispecific binder includes a binder that is specific to one or more epitopes (of the same or different antigens) or to one or more antigens. Thus, for example, a multispecific polypeptide chain or binder may have one or more variable regions of a single-domain antibody, at least two of which bind to different antigens or epitopes.

[0733] Accordingly, the binders of this disclosure may be bispecific, trispecific, tetraspecific, vspecific, heptaspecific, etc. In some embodiments, each variable region may be specific to a given antigen. In some embodiments, two or more antigen-binding fragments of a given binder may be specific to the same or different antigens. In some embodiments, three or more antigen-binding fragments of a given binder may be specific to the same or different antigens. In some embodiments, four or more antigen-binding fragments of a given binder may be specific to the same or different antigens. In some embodiments, five or more antigen-binding fragments of a given binder may be specific to the same or different antigens. In some embodiments, six or more antigen-binding fragments of a given binder may be specific to the same or different antigens. Specificity may depend on the number of antigen-binding fragments present in a given binder.

[0734] Other exemplary, non-limiting embodiments of the multispecific binder include those having two antigen-binding fragments of different specificities. Still other exemplary, non-limiting embodiments of the multispecific binder include those having more than two antigen-binding fragments that bind to two different antigens, proteins, or two different epitopes on the same antigen or protein.

[0735] In some embodiments, the binder of this disclosure is monovalent.

[0736] In some embodiments, the binder of this disclosure is polyvalent.

[0737] Exemplary, non-limiting embodiments of a polyvalent binder include a binder composed of a polyvalent polypeptide chain. Other non-limiting, exemplary embodiments of a polyvalent binder include a binder composed of one or more monovalent polypeptide chains.

[0738] This disclosure allows the dispecificity binder to be divalent or polyvalent depending on the number of variable regions it contains. Exemplary, non-limiting embodiments of the dispecificity binder include those comprising two identical dispecificity polypeptide chains that form a dimer.

[0739] Exemplary and non-limiting embodiments of two specific binders are provided herein.

[0740] In some embodiments, the bispecificity binder binds to TROP2 and includes an antigen-binding domain comprising formula II (see PCT / CA2020 / 051753 filed on 18 December 2020 and WO2021 / 119832A1 published on 24 June 2021, which incorporate the entire content by reference).

[0741] In other embodiments, the binder comprises a polypeptide chain comprising, in an N-terminal to C-terminal manner, a) an antigen-binding domain of a single-domain antibody, b) a linker, c) a dimerization domain, d) another linker, and e) an antigen-binding domain of a single-domain antibody, wherein at least one of the antigen-binding domains of a) or e) is an antigen-binding domain that binds to TROP2.

[0742] In some embodiments, the antigen-binding domain of a) and the antigen-binding domain of e) are different.

[0743] In some embodiments, the antigen-binding domain in a) and the antigen-binding domain in e) are the same.

[0744] In some embodiments, linker b) and linker d) are the same.

[0745] In some embodiments, linker b) and linker d) are different.

[0746] In an exemplary embodiment, the antigen-binding domain of a) includes the antigen-binding domain of a single-domain antibody that specifically binds to human TROP2.

[0747] In another exemplary embodiment, the antigen-binding domain of a) includes, for example, the antigen-binding domain of a single-domain antibody that specifically binds to CD47, such as human CD47.

[0748] In an exemplary embodiment, the antigen-binding domain of e) includes the antigen-binding domain of a single-domain antibody that specifically binds to human TROP2.

[0749] In an exemplary embodiment, the antigen-binding domain of e) includes, for example, the antigen-binding domain of a single-domain antibody that specifically binds to CD47, such as human CD47.

[0750] In some embodiments, the bispecificity binder comprises two polypeptide chains. The two polypeptide chains are non-covalently assembled by amino acid residues of the CH2 and / or CH3 domains contained therein to obtain a bispecificity and tetravalent binder.

[0751] In exemplary embodiments, the binders or antigen-binding domains of the present disclosure can inhibit the proliferation of tumor cells expressing human TROP2 and CD47.

[0752] In exemplary embodiments, the binder comprises one or more antigen-binding domains capable of binding to CD47.

[0753] In another exemplary embodiment, the binder includes one or more antigen-binding domains capable of binding to TROP2 and one or more antigen-binding domains capable of binding to CD47.

[0754] In exemplary embodiments, the antigen-binding domain capable of binding to CD47 is a. CDRH1 having the amino acid sequence described in SEQ ID NO: 156, CDRH2 having the amino acid sequence described in SEQ ID NO: 157, and CDRH3 having the amino acid sequence described in SEQ ID NO: 158, or b. CDRH1 having the amino acid sequence described in SEQ ID NO: 159, CDRH2 having the amino acid sequence described in SEQ ID NO: 160, and CDRH3 having the amino acid sequence described in SEQ ID NO: 161 Includes.

[0755] In exemplary embodiments, the antigen-binding domain capable of binding to CD47 includes the amino acid sequence described in SEQ ID NO: 155.

[0756] In exemplary embodiments, the binder is capable of binding to CD47 and TROP2 and comprises the amino acid sequence described in SEQ ID NO: 155 and the amino acid sequence of at least one antigen-binding domain of at least one single-domain antibody disclosed herein.

[0757] In other exemplary embodiments, the binder may include at least one antigen-binding domain capable of binding to CD47 and at least one antigen-binding domain capable of binding to TROP2, as well as SEQ ID NOs: 22-26, 73-114, 221-224, 231, 232, 239, 240, 247, 304, 295, 296, 297, 311, 318, 325, 332, 339, 346, 353, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, SEQ ID NOs: The sequence includes the amino acid sequence of the antigen-binding domain of at least one single-domain antibody described in any one of the following: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 405, SEQ ID NO: 412, SEQ ID NO: 419, SEQ ID NO: 426, SEQ ID NO: 433, SEQ ID NO: 440, SEQ ID NO: 447, SEQ ID NO: 454, SEQ ID NO: 461, SEQ ID NO: 468, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472, SEQ ID NO: 473, SEQ ID NO: 491, SEQ ID NO: 493, SEQ ID NO: 496, SEQ ID NO: 502, or SEQ ID NO: 503.

[0758] In other exemplary embodiments, the binder may include at least one antigen-binding domain capable of binding to CD47 and at least one antigen-binding domain capable of binding to TROP2, and also include the amino acid sequence of a humanized version of a single-domain antibody disclosed herein.

[0759] In other exemplary embodiments, the binder may include at least one antigen-binding domain capable of binding to PD-1 and at least one antigen-binding domain capable of binding to TROP2.

[0760] In other exemplary embodiments, the binder may include at least one antigen-binding domain capable of binding to TROP2, at least one antigen-binding domain capable of binding to CD47, and at least one antigen-binding domain capable of binding to PD-1.

[0761] In an exemplary embodiment, the antigen-binding domain capable of binding to PD-1 is: a. CDRH1 having the amino acid sequence described in SEQ ID NO: 532, CDRH2 having the amino acid sequence described in SEQ ID NO: 535, and CDRH3 having the amino acid sequence described in SEQ ID NO: 534, or b. CDRH1 having the amino acid sequence described in SEQ ID NO: 535, CDRH2 having the amino acid sequence described in SEQ ID NO: 536, and CDRH3 having the amino acid sequence described in SEQ ID NO: 537 Includes.

[0762] In an exemplary embodiment, the antigen-binding domain capable of binding to PD-1 includes the amino acid sequence described in SEQ ID NO: 538.

[0763] In exemplary embodiments, the binder is capable of binding to PD-1 and TROP2 and includes the amino acid sequence described in SEQ ID NO: 538 and the amino acid sequence of at least one antigen-binding domain of at least one single-domain antibody disclosed herein.

[0764] In some cases, the humanized version has amino acid residues in its framework region replaced with amino acid residues in the corresponding human or humanized framework region. In some cases, the humanized version has its framework region replaced with the region described in Sequence ID No. 211. In other cases, the humanized version has its framework region replaced with the region described in Sequence ID No. 213.

[0765] In some embodiments, the humanized version replaces its framework region with the region described in any one of sequence numbers 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, or 271.

[0766] In some embodiments, the humanized version replaces its framework region with the region described in any one of the sequence numbers: 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, or 294.

[0767] In another exemplary embodiment, the binder can bind to CD47 and TROP2 and comprises the amino acid sequence described in SEQ ID NO: 150 or SEQ ID NO: 151.

[0768] variant Variants of the sequences disclosed herein are also included by this disclosure.

[0769] Therefore, the binders of this disclosure include antibody variants (e.g., single-domain antibody variants), variants of antibody-like molecules, variants of protein scaffolds, and variants of immunocytomodulators. In addition, the binders of this disclosure may include antigen-binding fragments or antigen-binding domains of antibody variants, for example, antigen-binding fragments or antigen-binding domains of single-domain antibody variants.

[0770] More specifically, this disclosure includes variants of the single-domain antibodies disclosed herein, and conjugates comprising one or more antigen-binding domains of the single-domain antibody variants. The conjugate variants disclosed herein may have the same format as the conjugates disclosed herein. Therefore, the conjugate variants encompassed by this disclosure may be polyspecific and / or polyvalent.

[0771] The variants encompassed by this disclosure include variants that may include the insertion of one or more amino acid residues at one or more positions, the deletion of one or more amino acid residues at one or more positions, or the substitution (conservative or non-conservative substitution) of one or more amino acid residues at one or more positions.

[0772] Variations can occur in the antigen-binding domain, or in other elements of the binder such as the dimerization domain, linker, or spacer.

[0773] For example, naturally occurring residues are classified into groups based on common side-chain properties. Conservative substitutions can be made by replacing an amino acid from one of the groups (groups 1-6) listed below with another amino acid from the same group. Non-conservative substitutions involve replacing one member of one group with 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 the direction of the chain: glycine (Gly), proline (Pro); and (Group 6) Aromatics: Tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0774] Other exemplary embodiments of conservative substitutions are shown in Table 2 (Table 3) under the heading “Preferred Substitutions”. If such substitutions result in undesirable properties, more substantial changes may be introduced, which are labeled “Exemplary Substitutions” in Table 2 (Table 3) or described in detail below with reference to amino acid classes, and the product may be screened.

[0775] One skilled in the art recognizes that a particular amino acid is less positively charged, neutral, negatively charged, or has a reduced charge compared to other amino acids. Amino acids can be classified based on their net charge as indicated by the isoelectric point of the amino acid. The isoelectric point is the pH at which the average net charge of an amino acid molecule is zero. When pH > pI, the amino acid has a net negative charge, and when pH < pI, the amino acid has a net positive charge. In some embodiments, the pI value measured for an antibody is between about 3 - 9 (e.g., 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, and 9), and any value therebetween. In some embodiments, the pI value measured for an antibody is between 4 - 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0), and any value therebetween. Exemplary isoelectric points of amino acids are shown in Table 2 (Table 3) below. Generally, amino acids with positively charged side chains include, for example, arginine (R), histidine (H), and lysine (K). Amino acids with negatively charged side chains include, for example, aspartic acid (D) and glutamic acid (E). Amino acids with polar characteristics include, for example, serine (S), threonine (T), asparagine (N), glutamine (Q), and cysteine (C), tyrosine (Y) and tryptophan (W). Non-polar amino acids include, for example, alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), glycine (G), and proline (P).

[0776] In some embodiments, the isoelectric point of an antibody is modified via amino acid substitution. See, for example, U.S. Patent Application Publication No. 20110076275. In some embodiments, modifying the isoelectric point of the polypeptide constituting an antibody results in a change in the half-life of the antibody.

[0777]

Table 3

[0778] Generally, the degree of similarity and identity between variable strands is determined using the Blast2 sequence program (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) as described herein, with default settings, namely the blastp program, BLOSUM62 matrix (open gap 11 and extension gap penalty 1; gapx drop-off 50, expected value 10.0, word size 3) and activation filter.

[0779] Therefore, percentage identity indicates amino acids that are identical to the original peptide and can occupy the same or similar positions.

[0780] Percent similarity indicates amino acids that are identical to the original peptide at the same or similar positions, and amino acids that have been replaced by conservative amino acid substitutions.

[0781] Accordingly, the variants disclosed herein may include sequences that are at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the original sequence or reference sequence, or a portion of the original sequence.

[0782] In exemplary embodiments, mutations in one or more CDRs, variable regions, or constant regions may be made by amino acid substitutions. Exemplary embodiments of substitutions include conservative and non-conservative amino acid substitutions. Exemplary substitutions are provided in Table 2 (Table 3). Other exemplary substitutions are shown in Table 13A (Table 18). Further exemplary substitutions are shown in Table 13B (Table 18).

[0783] In some embodiments, polypeptide chains having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or less than 100% identical to a predetermined amino acid sequence may generally have amino acid substitutions, additions, or deletions located outside the complementarity-determining region.

[0784] In some embodiments, the variant may have at least 80% sequence identity with the sequence disclosed herein. In other embodiments, the variant may have at least 85% sequence identity with the sequence disclosed herein. In further embodiments, the variant may have at least 90% sequence identity with the sequence disclosed herein. In even further embodiments, the variant may have at least 95% sequence identity with the sequence disclosed herein. In other embodiments, the variant may have at least 99% sequence identity with the sequence disclosed herein.

[0785] In some embodiments, variations may occur in one or more CDRs. Mutations may occur, for example, in CDR amino acid residues not involved in interaction with TROP2. In exemplary embodiments, amino acid residues of CDR1 and / or CDR2 may be mutated. In other exemplary embodiments, amino acid residues of CDR3 may be mutated. CDR mutations that improve affinity for TROP2 are particularly intended.

[0786] In other embodiments, variations may occur in the framework region. Mutations may occur, for example, in framework amino acid residues not involved in interaction with TROP2. In exemplary embodiments, amino acid residues of FR1, FR3, and / or FR4 may be mutated. In other exemplary embodiments, amino acid residues of FR2 may be mutated. FR2 mutations that improve affinity for TROP2 are particularly intended.

[0787] In other embodiments, variations may occur in the steady region. In an exemplary embodiment, the steady region may be mutated to modulate ADCC activity. In another exemplary embodiment, the steady region may be mutated to modulate ADCP activity. In yet another exemplary embodiment, the steady region may be mutated to improve ADCP activity. In yet another exemplary embodiment, the steady region may be mutated to improve internal translocation. In yet another exemplary embodiment, the steady region may be mutated to improve stability.

[0788] Exemplary, non-limiting embodiments of mutations in a steady-state region (e.g., the Fc region) that improve one or more effector functions are encompassed by this disclosure and are provided in Table A (Table 4), the full contents of which are incorporated herein by reference, in List of Mutations Antibodies (Basel). 2020 Nov 17;9(4):64.

[0789] [Table 4]

[0790] Exemplary and non-limiting embodiments of single-domain antibody variants are provided herein. Binding agents comprising one or more antigen-binding domains of one or more single-domain antibody variants are also included herein.

[0791] Exemplary variants of this disclosure may include polypeptides comprising the variable region sequences or heavy chain sequences of Table 1A (Table 1), Table 1B (Table 2), Table 15A (Table 20) to Table 15K (Table 30), Table 16 (Table 31), Table 18 (Table 34), or Table 19 (Table 35), having one or more variations selected from amino acid substitutions, deletions, and / or additions. Variations may include one to five amino acid substitutions, one to five amino acid deletions, and / or one to five amino acid insertions. In exemplary embodiments, the deletion or addition is conserved. In another exemplary embodiment, the deletion or addition is non-conserved. In yet another exemplary embodiment, the deletion or addition is at the N-terminus. In yet another exemplary embodiment, the deletion or addition is at the C-terminus. In yet another exemplary embodiment, the deletion or addition is outside the IMGT CDR sequence. In yet another exemplary embodiment, the deletion or addition is outside the IMGT CDR3 sequence and / or the IMGT FR2 sequence. In further exemplary embodiments, the deletion or addition is located outside the Kabat CDR sequence. In further exemplary embodiments, the deletion or addition is located outside the Kabat CDR3 sequence and / or the Kabat FR2 sequence.

[0792] As used herein, the term "1 to 5" includes any individual values ​​such as "1," "2," "3," "4," and "5," as well as any ranges between them such as "1 to 2," "1 to 3," "1 to 4," "1 to 5," "2 to 3," "2 to 4," "2 to 5," "3 to 4," "3 to 5," and "4 to 5."

[0793] A variant of this disclosure may include at least one amino acid substitution compared to the sequence disclosed herein. Alternatively, a variant of this disclosure may include at least one amino acid deletion compared to the sequence disclosed herein. Alternatively, a variant of this disclosure may include at least one amino acid addition compared to the sequence disclosed herein.

[0794] In some embodiments, variants of the disclosure may include at least one amino acid substitution and at least one amino acid deletion compared to the sequences disclosed herein. In some embodiments, variants of the disclosure may include at least one amino acid substitution and at least one amino acid addition compared to the sequences disclosed herein. In some embodiments, variants of the disclosure may include at least one amino acid addition and at least one amino acid deletion compared to the sequences disclosed herein. In some embodiments, variants of the disclosure may include at least one amino acid substitution, at least one amino acid addition and at least one amino acid deletion compared to the sequences disclosed herein.

[0795] In some embodiments, the amino acid substitution, deletion, or addition is outside the amino acid sequence of CDR3 (IMGT or Kabat). In some embodiments, the amino acid substitution, deletion, or addition is outside the amino acid sequence of FR2 (IMGT or Kabat). In some embodiments, the amino acid substitution, deletion, or addition is outside the amino acid sequence of CDR1 (IMGT or Kabat). In some embodiments, the amino acid substitution, deletion, or addition is outside the amino acid sequence of CDR2 (IMGT or Kabat).

[0796] In aspects of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NOs. 143-146.

[0797] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 143.

[0798] Sequence ID 143 (KD002 consensus)

[0799] [ka]

[0800] In an exemplary embodiment, X 1a ~X 1rEach of these is, independently, any (e.g., naturally occurring) amino acid residue.

[0801] In another exemplary embodiment, X 1a ~X 1r Each of these is, independently, an amino acid residue found at the corresponding position in either SEQ ID NO: 23 or SEQ ID NOs. 73-93 (SEQ ID NO: 196), or a conserved or non-conserved amino acid substitution thereof.

[0802] In another exemplary embodiment, X 1a ~X 1r Each of these is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 22, 23, 24, 25, or 26 (SEQ ID NO: 195), or a conserved or non-conserved amino acid substitution thereof.

[0803] In a further exemplary embodiment, X 1a ~X 1r Each of these is, independently, the amino acid residue most frequently found at the corresponding position in either SEQ ID NO: 23 or SEQ ID NOs: 73-93.

[0804] In another exemplary embodiment, X 1a ~X 1r Each of these is, independently, the amino acid residue most frequently found at the corresponding position in any one of SEQ ID NOs: 22, 23, 24, or 25-26.

[0805] In another exemplary embodiment, X 1a ~X 1r Each of these is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 73, 75, 76, 77, 78, 81, 82, 83, 85, 89, or 91 (SEQ ID NO: 208), or a conserved or non-conserved amino acid substitution thereof.

[0806] In further exemplary embodiments, any X 1a ~X 1r Each of these is independently the amino acid residue most frequently found at its corresponding position in any one of sequence numbers 73, 75, 76, 77, 78, 81, 82, 83, 85, 89, or 91.

[0807] In a further exemplary embodiment, X 1a is Q or E; X 1b is Q, V, or L; X 1c is M or L; X 1d is V or P; X 1e is G or Q; X 1f It is P, L, or R; X 1g is Y or V; X 1h D, N, E; X 1iは、 P or A; X 1j is S or T; X 1k is A or S; X 1l is L or Q; X 1m is N or S; X 1n is either K or R; X 1o is P or A; X 1p is L or V; X 1q is R or Y; and / or X 1r This is either Q or M (Sequence ID 196).

[0808] In another exemplary embodiment, X 1a is Q or E; X 1b is Q, V, or L; X 1c is M or L; X 1d is V or P; X 1e is G or Q; X 1f is P, L, or R; X 1g Y is X 1h D, X 1iは、 P or A; X 1j is S or T; X 1k is A or S; X1l is L or Q; X 1m is N or S; X 1n is either K or R; X 1o is P or A; X 1p is L or V; X 1q is R or Y; and / or X 1r This is either Q or M (Sequence ID 197).

[0809] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 211.

[0810] [ka]

[0811] In some embodiments, X 1a ~X 1r Any of these is as described herein (e.g., Sequence IDs 477, 478).

[0812] In other embodiments, X 1a ~X 1r One of these is the corresponding amino acid residue in the variable region of the antibody heavy chain.

[0813] In other embodiments, X 1a ~X 1r One of these is the corresponding amino acid residue in the sequence described in any one of sequence numbers 248-271.

[0814] In other embodiments, X 1a ~X 1r One of these is the corresponding amino acid residue of a human antibody heavy chain, for example, a residue of a germline human antibody heavy chain.

[0815] In accordance with this disclosure, [CDRH1], [CDRH2], and [CDRH3] in SEQ ID NO: 211 correspond to the amino acid sequences of IMGT CDR1, CDR2, and CDR3 of predetermined variable region sequences disclosed herein, particularly in Tables 15A (Table 20) to 15K (Table 30), Table 16 (Table 31), Table 18 (Table 34), or Table 19 (Table 35).

[0816] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 144.

[0817] Sequence ID 144 (KD005 consensus)

[0818] [ka]

[0819] In an exemplary embodiment, X 2a ~X 2p Each of these is, independently, any (e.g., naturally occurring) amino acid residue.

[0820] In another exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in either SEQ ID NO: 26 or SEQ ID NOs. 94-114 (SEQ ID NO: 199), or a conserved or non-conserved amino acid substitution thereof.

[0821] In another exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 22, 23, 24, 25, or 26 (SEQ ID NO: 198), or a conserved or non-conserved amino acid substitution thereof.

[0822] In another exemplary embodiment, any X 2a ~X2p Each of these is independently the amino acid residue most frequently found at its corresponding position in either SEQ ID NO: 26 or SEQ ID NOs: 94-114.

[0823] In further exemplary embodiments, any X 2a ~X 2p Each of these is independently the amino acid residue most frequently found at its corresponding position in one of the following sequence numbers: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NOs: 25-26.

[0824] In a further exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107, or 108 (sequence number 209), or a conserved or non-conserved amino acid substitution thereof.

[0825] In additional exemplary embodiments, any X 2a ~X 2p Each of these is independently the amino acid residue most frequently found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107, or 108.

[0826] In another exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 100, 102, 103, 104, 105, 106, 108, 112, or 114 (sequence number 210), or a conserved or non-conserved amino acid substitution thereof.

[0827] In further exemplary embodiments, any X 2a ~X 2pEach of these is independently the amino acid residue most frequently found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 100, 102, 103, 104, 105, 106, 108, 112, or 114.

[0828] In a further exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 105, 112, 113, or 114 (sequence number 210), or a conserved or non-conserved amino acid substitution thereof.

[0829] In another exemplary embodiment, any X 2a ~X 2p Each of these is independently the amino acid residue most frequently found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 105, 112, 113, or 114.

[0830] In another exemplary embodiment, X 2a ~X 2p Each of these is, independently, an amino acid residue found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 102, or 105 (sequence number 210), or a conserved or non-conserved amino acid substitution thereof.

[0831] In additional exemplary embodiments, any X 2a ~X 2p Each of these is independently the amino acid residue most frequently found at the corresponding position in any one of sequence numbers 94, 95, 96, 97, 102, or 105.

[0832] In another exemplary embodiment, X 2a is Q or E; X 2b is Q, L, or V; X 2c is V or I; X 2dis E or Q; X 2e is A or P; X 2f is P or L; X 2g is Y, A, or V; X 2h is P or A; X 2i is S or T; X 2j is A or S; X 2k is either K or R; X 2l is P or A; X 2m is either D or E; X 2n is L or V; X 2o is R or Y, and / or X 2p This is either Q or L (Sequence ID 199).

[0833] In another exemplary embodiment, X 2a is Q or E; X 2b is Q or L; X 2c is V or I; X 2d is E or Q; X 2e is A or P; X 2f is P or L; X 2g is either Y or A; X 2h is P or A; X 2i is S or T; X 2j is A or S; X 2k is either K or R; X 2l is P or A; X 2m is either D or E; X 2n is L or V; X 2o is R or Y, and / or X 2p This is either Q or L (Sequence ID 200).

[0834] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 213.

[0835] [ka]

[0836] In some embodiments, X 2a ~X 2p Any of the following is as described herein, X 2q L is X 2r is A, and / or X 2s It is W.

[0837] In other embodiments, X 2a ~X 2s One of these is the corresponding amino acid residue in the variable region of the antibody heavy chain.

[0838] In other embodiments, X 2a ~X 2s One of these is the corresponding amino acid residue in the sequence described in any one of sequence numbers 272-294.

[0839] In other embodiments, X 2a ~X 2s One of these is the corresponding amino acid residue of a human antibody heavy chain, for example, a residue of a germline human antibody heavy chain.

[0840] In some embodiments, X 2a is Q or E; X 2b is Q, L, or V; X 2c is V or I; X 2d is E, Q, D, or H; X 2e is A or P; X 2f is P or L; X 2g is Y, A, or V; X 2h is P or A; X 2i is S or T; X 2j is A or S; X 2k is K, R, or T; X 2l is P or A; X 2m is either D or E; X 2n is L or V; X 2o is R or Y; X 2pis Q or L; X 2q L is X 2r is A and / or X 2s This is W (sequence number 479).

[0841] In some embodiments, X 2a is Q or E; X 2b is Q, L, or V; X 2c is V or I; X 2d is E, Q, D, or H; X 2e is A or P; X 2f is P or L; X 2g is Y, A, V, or I; X 2h is P or A; X 2i is S or T; X 2j is A or S; X 2k is K, R, or T; X 2l is P or A; X 2m is either D or E; X 2n is L or V; X 2o is R or Y; X 2p is Q or L; X 2q is L or M; X 2rは、 A or V and / or X 2s This is either W or Q (Sequence ID 480).

[0842] In accordance with this disclosure, [CDRH1], [CDRH2], and [CDRH3] in SEQ ID NO: 213 correspond to the amino acid sequences of IMGT CDR1, CDR2, and CDR3 of predetermined variable region sequences disclosed herein, particularly in Tables 15A (Table 20) to 15K (Table 30), Table 16 (Table 31), Table 18 (Table 34), or Table 19 (Table 35).

[0843] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 145.

[0844] Sequence ID 145 (Consensus from KD001~KD005 heavy chain)

[0845] [ka]

[0846] In an exemplary embodiment, X 3a ~X 3y Each of these is, independently, any (e.g., naturally occurring) amino acid residue.

[0847] In another exemplary embodiment, X 3a ~X 3y Each of these is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 22, 23, 24, 25, or 26 (SEQ ID NO: 201), or a conserved or non-conserved amino acid substitution thereof.

[0848] In another exemplary embodiment, X 3a ~X 3y Each of these is, independently, the most frequently found amino acid residue in the group consisting of SEQ ID NOs: 22, 23, 24, 25, and 26.

[0849] In a further exemplary embodiment, X 3a is M or L; X 3b is Q, H, or E; X 3c is V, P, or A; X 3d is G or R; X 3e is P or T; X 3f is S, N, or G; X 3g is A, Y, or S; X 3h is N or T; X 3i is A, S, or G; X 3j is D, G, or S; X 3k is either G or does not exist; X 3l is S or G; X3m is K, N, or T; X 3n is either Y or D; X 3o is I or T; X 3p is T or M; X 3q is L or Q; X 3r is N or S; X 3s is K or T; X 3t is E or D; X 3u is R or K; X 3v is A or S; X 3w is either K or R; X 3x is T or S; and / or X 3y is either Y or A (Sequence ID 202).

[0850] In a further exemplary embodiment, X 3a is M or L; X 3b is Q, H, or E; X 3c is V, P, or A; X3d is G or R; X 3e is P or T; X3f is S, N or G; X 3g is A, Y, or S; X 3h is N or T; X 3i is A, S, or G; X 3j is D, G, or S; X 3k is either G or does not exist; X 3l is S or G; X 3m is K, N, or T; X 3n Y is X 3o is I or T; X 3p is T or M; X 3q is L or Q; X 3r is N or S; X 3s is K or T; X 3t is E or D; X 3u is R or K; X 3v is A or S; X 3w R is X3x is S; and / or X 3y This is A (sequence number 203).

[0851] Generally, the positions of amino acid residues are as described in Sequence ID No. 145 and as illustrated in Table 1C (Table 5).

[0852] [Table 5]

[0853] In accordance with this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence described in SEQ ID NO: 146.

[0854] Sequence ID 146 (Consensus of KD001-KD005 and selected variant heavy chains)

[0855] [ka]

[0856] In an exemplary embodiment, X 4a ~X 5n Each of these is, independently, any (e.g., naturally occurring) amino acid residue.

[0857] In another exemplary embodiment, X 4a ~X 5n Each of these is an amino acid residue (sequence number 205) found independently at the corresponding position in any one of sequence numbers 22-26 or 73-114, or a conserved or non-conserved amino acid substitution thereof.

[0858] In another exemplary embodiment, X 4a ~X 5nEach of these is an amino acid residue (SEQ ID NO: 204) found independently at the corresponding position in any one of SEQ ID NOs: 22, 23, 24, 25, or 26, or a conserved or non-conserved amino acid substitution thereof.

[0859] In another exemplary embodiment, X 4a ~X 5n Each of these is, independently, the amino acid residue that is most frequently disrupted at the corresponding position in one of the sequence numbers 22-26 or 73-114.

[0860] In another exemplary embodiment, X 4a ~X 5n Each of these is, independently, the amino acid residue most frequently found at the corresponding position in one of the following sequence numbers: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NOs. 25-26.

[0861] In a further exemplary embodiment, X 4a is Q or E, and X 4b is Q, L, or V, and X 4c It is M or L, X 4d is V or I, and X 4e is Q, H, or E, and X 4f is P, A, or V, and X 4g is G or R, X 4h is P or T, and X 4i is G, S, or N, and X 4j is A or S, and X 4k is S or H, and X 4l is V or Y, and X 4m is G or Q, and X 4n is P, L, or R, and X 4o is E or V, and X 4p is Y, A, or R, and X 4q is either T or N, and X 4r is A, S, or G, and X 4sis G, S, D, N, or E, and X 4t It either does not exist or is S, X 4u is either K or T, and X 4v is Y, D, or S, and X 4w is P or A, and X 4x is S or T, X 4y is I or T, X 4z is S or A, X 5a is T or M, X 5b is Q or L, and X 5c is either S or N, and X 5d is K, R, or T, and X 5e is P or A, and X 5f is E or D, and X 5g is V or L, and X 5h is either Y or R, and X 5i is R or K, and X 5j is A or S, and X 5k is either K or R, and X 5l is S or T, X 5m is Y or A, and / or X 5n This is Q, L, or M (Sequence ID 205).

[0862] All antibodies in Table 15B (Table 21) and Table 15C (Table 22) have the same CDR1, CDR2, and CDR3 as KD001 (KD008) CDR.

[0863] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 507, SEQ ID NO: 508, and SEQ ID NO: 509 (e.g., Table 15B (Table 21) and Table 15C (Table 22)).

[0864] Sequence IDs 507, 508, and 509 (consensus of KD008 and KD008 variants)

[0865] [ka]

[0866] In an exemplary embodiment, X at positions 1 to 115 of Sequence ID No. 507 is each independently any (e.g., naturally occurring) amino acid residue.

[0867] In other exemplary embodiments, X at position 1 is E, Q, N, K, or D; X at position 5 is any amino acid residue; X at position 11 is L, M, I, or V; X at position 12 is I, V, L, or M; X at position 13 is Q, H, or D; X at position 14 is any amino acid residue; X at position 16 is G, D, S, or N; X at position 23 is A, V, or T; X at position 45 is any amino acid residue; X at position 60 is any amino acid residue; X at position 62 is S, T, or is A; X at position 74 is A, S, or T; X at position 75 is K, N, E, Q, D, or H; X at position 77 is any amino acid residue; X at position 86 is any amino acid residue; X at position 87 is any amino acid residue; X at position 88 is D, E, N, or Q; X at position 92 is L, V, M, or I; X at position 93 is any amino acid residue; X at position 110 is any amino acid residue, and / or X at position 115 is any amino acid residue (SEQ ID NO: 508).

[0868] In some embodiments, the 1st position X is E or Q; the 5th position X is Q or L; the 11th position X is L or M; the 12th position X is I or V; the 13th position X is Q, H or D; the 14th position X is P or A; the 16th position X is G or D; the 23rd position X is A or V; the 45th position X is P or L; the 60th position X is P or A; the 62nd position X is S or The X at position 74 is A or S; the X at position 75 is K or N; the X at position 77 is T or M; the X at position 86 is R, K or T; the X at position 87 is P or A; the X at position 88 is D or E; the X at position 92 is L or V; the X at position 93 is Y or D; the X at position 110 is W or Q, and / or the X at position 115 is L or Q (Sequence ID 509).

[0869] In some embodiments, the 1st position X is E or Q; the 5th position X is Q or L; the 11th position X is L or M; the 12th position X is I or V; the 13th position X is Q, H or D; the 14th position X is P or A; the 16th position X is D; the 23rd position X is A or V; the 45th position X is P or L; the 60th position X is P or A; the 62nd position X is The X at position 74 is S or T; the X at position 75 is A or S; the X at position 75 is N; the X at position 77 is M; the X at position 86 is R, K or T; the X at position 87 is P or A; the X at position 88 is D or E; the X at position 92 is L or V; the X at position 93 is D; the X at position 110 is W or Q, and / or the X at position 115 is L or Q (Sequence ID 509).

[0870] In some embodiments, the 11th position X is M or L; the 13th position X is H, D or Q; the 14th position X is P or A; the 16th position X is G or D; the 23rd position X is A or V; the 75th position X is K or N; the 77th position X is T or M; the 86th position X is K or T; the 88th position X is D or E; the 93rd position X is Y or D, and / or the 110th position X is W or Q (Sequence ID 510).

[0871] In some embodiments, the X at position 1 is E or Q; the X at position 5 is Q or L; the X at position 11 is L or M; the X at position 12 is I or V; the X at position 13 is Q or H; the X at position 45 is P or L; the X at position 60 is P or A; the X at position 62 is S or T; the X at position 74 is A or S; the X at position 86 is R or T; the X at position 87 is A or P; the X at position 88 is D or E; the X at position 92 is L or V, and / or the X at position 115 is L or Q (Sequence ID 511).

[0872] In other exemplary embodiments, any one of the X at positions 1 to 115 of SEQ ID NO: 507 is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 221, 222, 223, 224, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 469, 470, 471, 472, or 473, or a conserved or non-conserved amino acid substitution thereof.

[0873] In other exemplary embodiments, any one of the X at positions 1 to 115 of SEQ ID NO: 507 is, independently, the amino acid residue most frequently found at the corresponding position in any one of SEQ ID NOs: 221, 222, 223, 224, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 469, 470, 471, 472, or 473.

[0874] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 512 or SEQ ID NO: 513 (e.g., Table 15D (Table 23)).

[0875] [ka]

[0876] In exemplary embodiments, X at positions 16, 69, or 77 of SEQ ID NO: 512 is independently any (e.g., naturally occurring) amino acid residue.

[0877] In some embodiments, the X at position 16 is R or G; the X at position 69 is T or I; and / or the X at position 77 is M or T (Sequence ID 513).

[0878] In other embodiments, either X at position 16 or 69 of SEQ ID NO: 512 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in SEQ ID NO: 25 or SEQ ID NO: 304.

[0879] The antibodies in Table 15E (Table 24), Table 15F (Table 25), Table 15G (Table 26), Table 15H (Table 27), and Table 15I (Table 28) all have the same CDR1 and CDR3, and variations within CDR2 (Figures 45A-45B).

[0880] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 539, SEQ ID NO: 540, or SEQ ID NO: 541 (for example, Tables 15E (Table 24) to Table F (Table 25)).

[0881] [ka]

[0882] In exemplary embodiments, X at positions 11, 13, 14, 23, 48, 75, 77, 85, 88, 92, or 110 is any amino acid residue, and X at position 53 is A or G (SEQ ID NO: 539).

[0883] In other embodiments, X at positions 11, 13, 14, 23, 48, 75, 77, 85, 88, 92, or 110 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in any one of SEQ ID NOs: 231, 232, 295, 296, 297, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​239, 240, 383, 384, 385, 386, or 387, and X at position 53 is A or G (SEQ ID NO: 539).

[0884] In some embodiments, X at position 11 is M, L, I, or V; X at position 13 is H, Q, N, K, or R; X at position 14 is any amino acid residue; X at position 23 is A, V, or T; X at position 48 is any amino acid residue; X at position 53 is A or G, X at position 75 is K, N, E, Q, or H; X at position 77 is any amino acid residue; X at position 85 is any amino acid residue; X at position 88 is E, D, N, or Q; X at position 92 is L, V, M, or I; and / or X at position 110 is any amino acid residue.

[0885] In other embodiments, the 11th position X is M or L; the 13th position X is H or Q; the 14th position X is P or A; the 23rd position X is A or V; the 48th position X is V or G; the 53rd position X is A or G, the 75th position X is K or N; the 77th position X is T or M; the 85th position X is R or L; the 88th position X is E or D; the 92nd position X is L or V; and / or the 110th position X is W or Q.

[0886] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 514, SEQ ID NO: 515, or SEQ ID NO: 516 (for example, Table 15E (Table 24)).

[0887] [ka]

[0888] In an exemplary embodiment, X at positions 11-110 of SEQ ID NO: 514 is each independently any (e.g., naturally occurring) amino acid residue.

[0889] In some embodiments, X at position 11 is M, L, I, or V; X at position 13 is H, Q, N, K, or R; X at position 14 is any amino acid residue; X at position 23 is A, V, or T; X at position 48 is any amino acid residue; X at position 75 is K, N, E, Q, or H; X at position 77 is any amino acid residue; X at position 85 is any amino acid residue; X at position 88 is E, D, N, or Q; X at position 92 is L, V, M, or I, and / or X at position 110 is any amino acid residue (SEQ ID NO: 515).

[0890] In some embodiments, the 11th position X is M or L; the 13th position X is H or Q; the 14th position X is P or A; the 23rd position X is A or V; the 48th position X is V or G; the 75th position X is K or N; the 77th position X is T or M; the 85th position X is R or L; the 88th position X is E or D; the 92nd position X is L or V, and / or the 110th position X is W or Q (Sequence ID 516).

[0891] In other exemplary embodiments, any one of the X at positions 11 to 110 of SEQ ID NO: 514 is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 372, SEQ ID NO: 373, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, or SEQ ID NO: 382, ​​or a conserved or non-conserved amino acid substitution thereof.

[0892] In other exemplary embodiments, any one of the X at positions 11 to 110 of SEQ ID NO: 514 is, independently, the amino acid residue most frequently found at the corresponding position in any one of SEQ ID NOs: 231, 232, 295, 296, 297, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, or 382.

[0893] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 517, SEQ ID NO: 518, or SEQ ID NO: 519 (for example, Table 15F (Table 25)).

[0894] [ka]

[0895] In an exemplary embodiment, X at positions 11-110 of SEQ ID NO: 517 is each independently any (e.g., naturally occurring) amino acid residue.

[0896] In some embodiments, X at position 11 is L, M, I, or V; X at position 13 is Q, H, N, or K; X at position 14 is any amino acid; X at position 23 is A, V, or T, and / or X at position 110 is any amino acid (SEQ ID NO: 518).

[0897] In some embodiments, the 11th position X is L or M; the 13th position X is Q or H; the 14th position X is A or P; the 23rd position X is A or V, and / or the 110th position X is W or Q (Sequence ID 519).

[0898] In other exemplary embodiments, any one of the X at positions 11-110 of SEQ ID NO: 517 is, independently, an amino acid residue found at the corresponding position in any one of SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, or SEQ ID NO: 387, or a conserved or non-conserved amino acid substitution thereof.

[0899] In other exemplary embodiments, any one of the X at positions 11-110 of SEQ ID NO: 517 is, independently, the amino acid residue most frequently found at the corresponding position in any one of SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, or SEQ ID NO: 387.

[0900] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 520, SEQ ID NO: 521, or SEQ ID NO: 522 (for example, Table 15G (Table 26)).

[0901] [ka]

[0902] In an exemplary embodiment, X at positions 11 and 14 of Sequence ID No. 520 is independently any (e.g., naturally occurring) amino acid residue.

[0903] In some embodiments, the 11th position X is M, L, I, or V, and / or the 14th position X is H, Q, N, or K (Sequence ID 521).

[0904] In some embodiments, the 11th position X is M or L, and / or the 14th position X is H or Q (Sequence ID 522).

[0905] In other exemplary embodiments, either X at positions 11 and 14 of SEQ ID NO: 520 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in SEQ ID NO: 247 or SEQ ID NO: 388.

[0906] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 523, SEQ ID NO: 524, or SEQ ID NO: 525 (for example, Table 15H (Table 27)).

[0907] [ka]

[0908] In an exemplary embodiment, X at positions 73 and 77 in Sequence ID No. 523 is independently any (e.g., naturally occurring) amino acid residue.

[0909] In some embodiments, X at position 73 is N, H, Q, or K, and / or X at position 77 is any amino acid (SEQ ID NO: 524).

[0910] In some embodiments, the X at position 73 is N or H, and / or the X at position 77 is M or T (Sequence ID 525).

[0911] In other exemplary embodiments, either X at positions 73 and 77 of SEQ ID NO: 523 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in SEQ ID NO: 325 or SEQ ID NO: 390.

[0912] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 526, SEQ ID NO: 527, or SEQ ID NO: 528 (for example, Table 15I (Table 28)).

[0913] [ka]

[0914] In an exemplary embodiment, X at positions 11 and 13 of Sequence ID No. 526 is independently any (e.g., naturally occurring) amino acid residue.

[0915] In some embodiments, X at position 11 is M, L, I, or V, and / or X at position 13 is any amino acid residue (SEQ ID NO: 527).

[0916] In some embodiments, the 11th position X is M or L, and / or the 13th position X is Q or L (Sequence ID 528).

[0917] In another exemplary embodiment, either position 11 or 13 of SEQ ID NO: 526 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in SEQ ID NO: 397 or SEQ ID NO: 398.

[0918] In one aspect of this disclosure, a single-domain antibody variant includes a variant having the amino acid sequence described in SEQ ID NO: 529, SEQ ID NO: 530, or SEQ ID NO: 531 (for example, Table 15J (Table 29)).

[0919] In the exemplary embodiment, X at positions 11-89 in Sequence ID No. 529 is each independently any (e.g., naturally occurring) amino acid residue.

[0920] In some embodiments, X at position 11 is M, L, I, or V; X at position 76 is N, K, E, Q, or D; X at position 78 is any amino acid residue, and / or X at position 89 is D, E, N, or Q (SEQ ID NO: 530).

[0921] In some embodiments, the X at position 11 is M or L; the X at position 76 is N or K; the X at position 78 is M or T, and / or the X at position 89 is D or E (Sequence ID 531).

[0922] In another exemplary embodiment, any one of the X at positions 11-89 of SEQ ID NO: 529 is a conserved or non-conserved amino acid substitution of an amino acid residue found at the corresponding position in SEQ ID NO: 311 or SEQ ID NO: 389.

[0923] In exemplary embodiments, the binder or its antigen-binding domain may include the amino acid sequence of CDR3 described in SEQ ID NO: 126, SEQ ID NO: 185, or SEQ ID NO: 186.

[0924] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 is as described in SEQ ID NOs: 126, SEQ ID NOs: 185, or SEQ ID NOs: 186.

[0925] In exemplary embodiments, the binder or its antigen-binding domain may include the FR2 amino acid sequence described in SEQ ID NO: 121 or SEQ ID NO: 180.

[0926] In another exemplary embodiment, the binder or its antigen-binding domain may include the amino acid sequence of CDR3 described in SEQ ID NO: 126 and the amino acid sequence of FR2 described in SEQ ID NO: 121.

[0927] In another exemplary embodiment, the binder or its antigen-binding domain may include the amino acid sequence of CDR3 described in SEQ ID NO: 185 and the amino acid sequence of FR2 described in SEQ ID NO: 180.

[0928] In another exemplary embodiment, the binder or its antigen-binding domain may include the amino acid sequence of CDR3 described in SEQ ID NO: 186 and the amino acid sequence of FR2 described in SEQ ID NO: 180.

[0929] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, wherein the amino acid sequence of CDR3 is as described in SEQ ID NOs: 126, 185, or 186, and the amino acid sequence of FR2 is as described in SEQ ID NOs: 121 or 180. In accordance with this disclosure, the amino acid sequences of CDR3 and FR2 are on the same polypeptide chain. Alternatively, in accordance with this disclosure, the amino acid sequences of CDR3 and FR2 are on separate polypeptide chains.

[0930] In accordance with this disclosure, the amino acid sequence of CDR3 is as described in SEQ ID NO: 48, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 220, SEQ ID NO: 230, SEQ ID NO: 238, SEQ ID NO: 246, SEQ ID NO: 303, SEQ ID NO: 310, SEQ ID NO: 317, SEQ ID NO: 324, SEQ ID NO: 331, SEQ ID NO: 338, SEQ ID NO: 345, SEQ ID NO: 352, SEQ ID NO: 396, SEQ ID NO: 404, SEQ ID NO: 411, SEQ ID NO: 418, SEQ ID NO: 425, SEQ ID NO: 432, SEQ ID NO: 439, SEQ ID NO: 446, SEQ ID NO: 453, SEQ ID NO: 460, SEQ ID NO: 467, SEQ ID NO: 490, or SEQ ID NO: 501.

[0931] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143 to 146, or SEQ ID NOs: 211 or 213, and the CDR3 amino acid sequence is as described in SEQ ID NOs: 48, 51, 54, 58, 61, 220, 230, 238, 246, 303, 310, 317, 324, 331, 338, 345, 352, 396, 404, 411, 418, 425, 432, 439, 446, 453, 460, 467, 490, or 501.

[0932] In accordance with this disclosure, the amino acid sequence of FR2 is as described in SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 505.

[0933] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146 or SEQ ID NOs: 211 or 213, wherein the FR2 amino acid sequence is as described in SEQ ID NOs: 116, 117, 118, 119, 120, 354, 355, 356, 360, 361, or 505.

[0934] The binder or antigen-binding domain may contain the amino acid sequence of CDR1 described in SEQ ID NO: 122, SEQ ID NO: 181, or SEQ ID NO: 182.

[0935] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR1 is as described in SEQ ID NOs: 122, SEQ ID NOs: 181, or SEQ ID NOs: 182.

[0936] In accordance with this disclosure, the amino acid sequence of CDR1 is as described in SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 236, SEQ ID NO: 244, SEQ ID NO: 301, SEQ ID NO: 308, SEQ ID NO: 315, SEQ ID NO: 322, SEQ ID NO: 329, SEQ ID NO: 336, SEQ ID NO: 343, SEQ ID NO: 350, SEQ ID NO: 394, SEQ ID NO: 402, SEQ ID NO: 409, SEQ ID NO: 416, SEQ ID NO: 423, SEQ ID NO: 430, SEQ ID NO: 437, SEQ ID NO: 444, SEQ ID NO: 451, SEQ ID NO: 458, SEQ ID NO: 465, SEQ ID NO: 488, or SEQ ID NO: 500.

[0937] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143 to 146, or SEQ ID NOs: 211 or 213, and the CDR1 amino acid sequence is as described in SEQ ID NOs: 52, 55, 56, 59, 62, 218, 228, 236, 244, 301, 308, 315, 322, 329, 336, 343, 350, 394, 402, 409, 416, 423, 430, 437, 444, 451, 458, 465, 488, or 500.

[0938] The binder or antigen-binding domain may contain the amino acid sequence of CDR2 described in SEQ ID NO: 125, SEQ ID NO: 183, or SEQ ID NO: 184.

[0939] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146 or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR2 is as described in SEQ ID NOs: 125, SEQ ID NOs: 183 or SEQ ID NOs: 184.

[0940] In accordance with this disclosure, the amino acid sequence of CDR2 is as described in SEQ ID NO: 47, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 219, SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 245, 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: 395, SEQ ID NO: 403, SEQ ID NO: 410, SEQ ID NO: 417, SEQ ID NO: 424, SEQ ID NO: 431, SEQ ID NO: 438, SEQ ID NO: 445, SEQ ID NO: 452, SEQ ID NO: 459, SEQ ID NO: 466, or SEQ ID NO: 489.

[0941] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143 to 146, or SEQ ID NOs: 211 or 213, and the CDR2 amino acid sequence is as described in SEQ ID NOs: 47, 50, 53, 57, 60, 123, 124, 219, 229, 237, 245, 302, 309, 316, 323, 330, 337, 344, 351, 395, 403, 410, 417, 424, 431, 438, 445, 452, 459, 466, or 489.

[0942] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 may be SEQ ID NOs: 48, 51, 54, 58, 61, 220, 230, 238, 246, 303, 310, 317, 324, 331, 338, 34 5. The amino acid sequence of FR2 is as described in SEQ ID NO: 352, SEQ ID NO: 396, SEQ ID NO: 404, SEQ ID NO: 411, SEQ ID NO: 418, SEQ ID NO: 425, SEQ ID NO: 432, SEQ ID NO: 439, SEQ ID NO: 446, SEQ ID NO: 453, SEQ ID NO: 460, or SEQ ID NO: 467, and the amino acid sequence of FR2 is as described in any one of SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, or SEQ ID NO: 360, or SEQ ID NO: 505.

[0943] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 may be SEQ ID NOs: 48, 51, 54, 58, 61, 220, 230, 238, 246, 303, 310, 317, 324, 331, 338, 345, or 352, 396, 4 The amino acid sequence of FR2 is as described in one of the following: 04, SEQ ID NO: 411, SEQ ID NO: 418, SEQ ID NO: 425, SEQ ID NO: 432, SEQ ID NO: 439, SEQ ID NO: 446, SEQ ID NO: 453, SEQ ID NO: 460, or SEQ ID NO: 467; the amino acid sequence of FR2 is as described in one of the following: SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO: 505; the amino acid sequence of CDR1 is as described in SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: The amino acid sequence of CDR2 is as described in any one of the following: number 59, SEQ ID NO: 62, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 236, SEQ ID NO: 244, SEQ ID NO: 301, SEQ ID NO: 308, SEQ ID NO: 315, SEQ ID NO: 322, SEQ ID NO: 329, SEQ ID NO: 336, SEQ ID NO: 343, or SEQ ID NO: 350, SEQ ID NO: 394, SEQ ID NO: 402, SEQ ID NO: 409, SEQ ID NO: 416, SEQ ID NO: 423, SEQ ID NO: 430, SEQ ID NO: 437, SEQ ID NO: 444, SEQ ID NO: 451, SEQ ID NO: 458, SEQ ID NO: 465, or SEQ ID NO: 488, and the amino acid sequence of CDR2 is as described in SEQ ID NO: 47, As described in any one of the following sequence numbers: 50, 53, 57, 60, 123, 124, 219, 229, 237, 245, 302, 309, 316, 323, 330, 337, 344, 351, 395, 403, 410, 417, 424, 431, 438, 445, 452, 459, or 466.

[0944] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 is as described in SEQ ID NOs: 142, SEQ ID NOs: 191, or SEQ ID NOs: 192.

[0945] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, where the amino acid sequence of FR2 is as described in SEQ ID NOs: 175, 193, or 194.

[0946] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, wherein the amino acid sequence of CDR3 is as described in SEQ ID NOs: 142, 191, or 192, and the amino acid sequence of FR2 is as described in SEQ ID NOs: 175, 193, or 194.

[0947] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143 to 146, or SEQ ID NOs: 211 or 213, the CDR3 amino acid sequence being as described in SEQ ID NOs: 3, 6, 9, 12, 15, 141, 217, 227, 235, 243, 300, 307, 314, 321, 328, 335, 342, 349, 393, 401, 408, 415, 422, 429, 436, 443, 450, 457, 464, 487, or 499.

[0948] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, where the FR2 amino acid sequence is as described in SEQ ID NOs: 170, 171, 172, 173, 174, 357, 358, or 359. In specific embodiments, the FR2 amino acid sequence is as described in SEQ ID NOs: 170 or 171.

[0949] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR1 is as described in SEQ ID NOs: 128, SEQ ID NOs: 187, or SEQ ID NOs: 188.

[0950] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143 to 146, or SEQ ID NOs: 211 or 213, the CDR1 amino acid sequence being as described in SEQ ID NOs: 1, 4, 7, 10, 13, 127, 215, 225, 233, 241, 298, 305, 312, 319, 326, 333, 340, 391, 399, 406, 413, 420, 427, 434, 441, 448, 455, 462, or 485.

[0951] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR2 is as described in SEQ ID NOs: 140, SEQ ID NOs: 189, or SEQ ID NOs: 190.

[0952] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, where the CDR2 amino acid sequence is defined in SEQ ID NOs: 2, 5, 8, 11, 14, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, sequence number As described in code 216, sequence number 226, sequence number 234, sequence number 242, sequence number 299, sequence number 306, sequence number 313, sequence number 320, sequence number 327, sequence number 334, sequence number 341, sequence number 348, sequence number 392, sequence number 400, sequence number 407, sequence number 414, sequence number 421, sequence number 428, sequence number 435, sequence number 442, sequence number 449, sequence number 456, sequence number 463, sequence number 486, sequence number 492, sequence number 495, or sequence number 498.

[0953] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 is defined in SEQ ID NOs: 3, 6, 12, 15, 141, 217, 227, 235, 243, 300, 307, 314, 321, 328, 335, 342, The amino acid sequence of FR2 is as described in any one of sequence numbers 349, 393, 401, 408, 415, 422, 429, 436, 443, 450, 457, 464, 487, or 499, and the amino acid sequence of FR2 is as described in any one of sequence numbers 170, 171, 172, 173, 174, 357, 358, or 359.

[0954] In exemplary embodiments, a single-domain antibody or its antigen-binding fragment may have an amino acid sequence defined in any one of SEQ ID NOs: 143-146, or SEQ ID NOs: 211 or 213, and the amino acid sequence of CDR3 is defined in SEQ ID NOs: 3, 6, 9, 12, 15, 141, 217, 227, 235, 243, 300, 307, 314, 321, 328, 335, 342, or 349, 393, sequence number The amino acid sequence of FR2 is as described in one of the following: SEQ ID NO. 401, SEQ ID NO. 408, SEQ ID NO. 415, SEQ ID NO. 422, SEQ ID NO. 429, SEQ ID NO. 436, SEQ ID NO. 443, SEQ ID NO. 450, SEQ ID NO. 457, SEQ ID NO. 464, SEQ ID NO. 487, or SEQ ID NO. 499; the amino acid sequence of FR2 is as described in one of the following: SEQ ID NO. 170, SEQ ID NO. 171, SEQ ID NO. 172, SEQ ID NO. 173, SEQ ID NO. 17...

Claims

1. A binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds to trophoblast cell surface antigen 2 (TROP2), a. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 225, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 226, SEQ ID NO: 234, SEQ ID NO: 242, SEQ ID NO: 320, SEQ ID NO: 392, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 227, or; b. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 228, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 245, SEQ ID NO: 323, or SEQ ID NO: 395, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO:

230. A binder containing a binder.

2. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO:

539.

3. The binder according to claim 1 or 2, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO:

540.

4. A binder according to any one of claims 1 to 3, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO:

541.

5. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 514 to 516.

6. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 517 to 519.

7. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 520 to 522.

8. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 523 to 525.

9. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence described in any one of SEQ ID NOs. 526 to 528.

10. A binder according to any one of claims 1 to 9, wherein at least one of one or more antigen-binding domains comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 373, SEQ ID NO: 232, SEQ ID NO: 231, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 372, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 247, SEQ ID NO: 388, SEQ ID NO: 325, SEQ ID NO: 390, SEQ ID NO: 397, or SEQ ID NO:

398.

11. A binder according to any one of claims 1 to 10, wherein at least one of one or more antigen-binding domains comprises an amino acid sequence that is at least 90% identical to the amino acid sequence described in SEQ ID NO: 373, SEQ ID NO: 232, SEQ ID NO: 231, SEQ ID NO: 295, SEQ ID NO: 296, SEQ ID NO: 297, SEQ ID NO: 372, SEQ ID NO: 374, SEQ ID NO: 375, SEQ ID NO: 376, SEQ ID NO: 377, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 247, SEQ ID NO: 388, SEQ ID NO: 325, SEQ ID NO: 390, SEQ ID NO: 397, or SEQ ID NO:

398.

12. A binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds to trophoblast cell surface antigen 2 (TROP2), a. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 488, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 490, b. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 485, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 486, SEQ ID NO: 492, or SEQ ID NO: 495, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 487, c. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 500, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 501, d. Heavy chain complementarity determination region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 485, heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 498 or SEQ ID NO: 486, and heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 499, e. CDRH1 having the amino acid sequence described in SEQ ID NO: 305, CDRH2 having the amino acid sequence described in SEQ ID NO: 306, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 307, f. CDRH1 having the amino acid sequence described in SEQ ID NO: 308, CDRH2 having the amino acid sequence described in SEQ ID NO: 309, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

310. g. CDRH1 having the amino acid sequence described in SEQ ID NO: 312, CDRH2 having the amino acid sequence described in SEQ ID NO: 313, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

314. h. CDRH1 having the amino acid sequence described in SEQ ID NO: 315, CDRH2 having the amino acid sequence described in SEQ ID NO: 316, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 317, i. CDRH1 having the amino acid sequence described in SEQ ID NO: 326, CDRH2 having the amino acid sequence described in SEQ ID NO: 327, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 328, j. CDRH1 having the amino acid sequence described in SEQ ID NO: 329, CDRH2 having the amino acid sequence described in SEQ ID NO: 330, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 331, k. CDRH1 having the amino acid sequence described in SEQ ID NO: 333, CDRH2 having the amino acid sequence described in SEQ ID NO: 334, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

335. l. CDRH1 having the amino acid sequence described in SEQ ID NO: 336, CDRH2 having the amino acid sequence described in SEQ ID NO: 337, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 338, m. CDRH1 having the amino acid sequence described in SEQ ID NO: 340, CDRH2 having the amino acid sequence described in SEQ ID NO: 341, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

342. n. CDRH1 having the amino acid sequence described in SEQ ID NO: 343, CDRH2 having the amino acid sequence described in SEQ ID NO: 344, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

345. o. CDRH1 having the amino acid sequence described in SEQ ID NO: 347, CDRH2 having the amino acid sequence described in SEQ ID NO: 348, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

349. p. CDRH1 having the amino acid sequence described in SEQ ID NO: 350, CDRH2 having the amino acid sequence described in SEQ ID NO: 351, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 352, q. CDRH1 having the amino acid sequence described in SEQ ID NO: 399, CDRH2 having the amino acid sequence described in SEQ ID NO: 400, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 401, r. CDRH1 having the amino acid sequence described in SEQ ID NO: 402, CDRH2 having the amino acid sequence described in SEQ ID NO: 403, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 404, s. CDRH1 having the amino acid sequence described in SEQ ID NO: 406, CDRH2 having the amino acid sequence described in SEQ ID NO: 407, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 408, t. CDRH1 having the amino acid sequence described in SEQ ID NO: 409, CDRH2 having the amino acid sequence described in SEQ ID NO: 410, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

411. u. CDRH1 having the amino acid sequence described in SEQ ID NO: 413, CDRH2 having the amino acid sequence described in SEQ ID NO: 414, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

415. v. CDRH1 having the amino acid sequence described in SEQ ID NO: 416, CDRH2 having the amino acid sequence described in SEQ ID NO: 417, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 418, w. CDRH1 having the amino acid sequence described in SEQ ID NO: 420, CDRH2 having the amino acid sequence described in SEQ ID NO: 421, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

422. x. CDRH1 having the amino acid sequence described in SEQ ID NO: 423, CDRH2 having the amino acid sequence described in SEQ ID NO: 424, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

425. y. CDRH1 having the amino acid sequence described in SEQ ID NO: 427, CDRH2 having the amino acid sequence described in SEQ ID NO: 428, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

429. z. CDRH1 having the amino acid sequence described in SEQ ID NO: 430, CDRH2 having the amino acid sequence described in SEQ ID NO: 431, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 432, aa. CDRH1 having the amino acid sequence described in SEQ ID NO: 434, CDRH2 having the amino acid sequence described in SEQ ID NO: 435, and / or CDRH3 having the amino acid sequence described in SEQ ID NO:

436. bb. CDRH1 having the amino acid sequence described in SEQ ID NO: 437, CDRH2 having the amino acid sequence described in SEQ ID NO: 438, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 439, cc. CDRH1 having the amino acid sequence described in SEQ ID NO: 441, CDRH2 having the amino acid sequence described in SEQ ID NO: 442, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 443, dd. CDRH1 having the amino acid sequence described in SEQ ID NO: 444, CDRH2 having the amino acid sequence described in SEQ ID NO: 445, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 446, ee. CDRH1 having the amino acid sequence described in SEQ ID NO: 448, CDRH2 having the amino acid sequence described in SEQ ID NO: 449, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 450, ff. CDRH1 having the amino acid sequence described in SEQ ID NO: 451, CDRH2 having the amino acid sequence described in SEQ ID NO: 452, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 453, gg. CDRH1 having the amino acid sequence described in SEQ ID NO: 455, CDRH2 having the amino acid sequence described in SEQ ID NO: 456, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 457, hh. CDRH1 having the amino acid sequence described in SEQ ID NO: 458, CDRH2 having the amino acid sequence described in SEQ ID NO: 459, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 460, ii. CDRH1 having the amino acid sequence described in SEQ ID NO: 462, CDRH2 having the amino acid sequence described in SEQ ID NO: 463, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 464, jj. CDRH1 having the amino acid sequence described in SEQ ID NO: 465, CDRH2 having the amino acid sequence described in SEQ ID NO: 466, and / or CDRH3 having the amino acid sequence described in SEQ ID NO: 467 A binder containing a binder.

13. The binder according to claim 12, wherein at least one of the one or more antigen-binding domains comprises a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 490, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in any one of SEQ ID NO: 493, SEQ ID NO: 496, or SEQ ID NO:

491.

14. The binder according to claim 12, wherein at least one of the one or more antigen-binding domains comprises a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 500, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 489, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 501, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in either SEQ ID NO: 503 or SEQ ID NO:

502.

15. The binder according to claim 12, wherein at least one of the one or more antigen-binding domains comprises a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence described in SEQ ID NO: 308, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence described in SEQ ID NO: 309, a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence described in SEQ ID NO: 310, and an amino acid sequence that is at least 80% identical to the amino acid sequence described in either SEQ ID NO: 311 or SEQ ID NO:

389.

16. The binder according to claim 1 or 12, wherein at least one of the one or more antigen-binding domains comprises a humanized framework region.

17. The binder according to claim 1 or 12, wherein at least one of the one or more antigen-binding domains comprises a human framework region.

18. The binder according to any one of claims 1 to 17, wherein at least one of the one or more antigen-binding domains comprises a humanized heavy chain variable region skeleton comprising the amino acid sequence described in SEQ ID NO: 211, SEQ ID NO: 477, or SEQ ID NO:

478.

19. The binder according to any one of claims 1 to 17, wherein at least one of the one or more antigen-binding domains comprises a humanized heavy chain variable region skeleton comprising the amino acid sequence described in SEQ ID NO: 213, SEQ ID NO: 479, or SEQ ID NO:

480.

20. A binder according to any one of claims 1 to 17, wherein at least one of one or more antigen-binding domains comprises an amino acid sequence described in SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, or SEQ ID NO: 271, or comprises a humanized heavy chain variable region skeleton comprising one to five amino acid substitutions, additions and / or deletions thereto.

21. A binder according to any one of claims 1 to 17, wherein at least one of one or more antigen-binding domains comprises the amino acid sequence described in SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 474, SEQ ID NO: 475 and SEQ ID NO: 476, or comprises a humanized heavy chain variable region skeleton comprising one to five amino acid substitutions, additions and / or deletions thereto.

22. The binder according to any one of claims 1 to 21, wherein the heavy chain variable region includes a framework region 2 (FR2) containing the sequence described in SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 354, SEQ ID NO: 355, SEQ ID NO: 356, SEQ ID NO: 360, SEQ ID NO: 361, or SEQ ID NO:

505.

23. A binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds to trophoblast cell surface antigen 2 (TROP2), and SEQ ID NOs: 373, 493, 503, 221, 222, 223, 224, 363, 364, 365, 366, 367, 368, 369, 370, 371, 469, 470, 471, 472, 473, 304, 231, 232, 295, 296, 297, 372, 374, 375, 376, 3 A binder comprising the amino acid sequence described in 77, SEQ ID NO: 378, SEQ ID NO: 379, SEQ ID NO: 380, SEQ ID NO: 381, SEQ ID NO: 382, ​​SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 383, SEQ ID NO: 384, SEQ ID NO: 385, SEQ ID NO: 386, SEQ ID NO: 387, SEQ ID NO: 247, SEQ ID NO: 388, SEQ ID NO: 325, SEQ ID NO: 390, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 311, SEQ ID NO: 389, SEQ ID NO: 318, SEQ ID NO: 332, SEQ ID NO: 339, SEQ ID NO: 346, SEQ ID NO: 353, SEQ ID NO: 405, SEQ ID NO: 412, SEQ ID NO: 419, SEQ ID NO: 426, SEQ ID NO: 433, SEQ ID NO: 440, SEQ ID NO: 447, SEQ ID NO: 454, SEQ ID NO: 461, SEQ ID NO: 468, SEQ ID NO: 491, SEQ ID NO: 496, or SEQ ID NO:

502.

24. The binder according to claim 24, comprising a dimerizing domain.

25. The binder according to any one of claims 1 to 24, wherein at least one of the one or more antigen-binding domains is a heavy chain variable region of a single-domain antibody.

26. The binder according to any one of claims 1 to 25, wherein all antigen-binding domains of the binder are heavy chain variable regions of one or more single-domain antibodies.

27. The binder according to claim 26 or 27, wherein one or more heavy chain variable regions are derived from camelid VH or VHH.

28. A binder according to any one of claims 1 to 27, comprising two or more antigen-binding domains.

29. A monospecific binder according to any one of claims 1 to 28.

30. A binder according to any one of claims 1 to 28, which is bispecific.

31. A binder according to any one of claims 1 to 28, which is polyspecific.

32. The binder according to claim 30 or 31, comprising one or more antigen-binding domains that bind to CD47, and / or one or more antigen-binding domains that bind to PD-1.

33. The binder according to any one of claims 1 to 32, wherein the binder comprises two polypeptide chains, each polypeptide chain comprising, in an N-terminal to C-terminal manner, a) one or more antigen-binding domains, b) optionally a linker, and c) a dimerizing domain.

34. The binder contains two polypeptide chains, each polypeptide chain having the amino acid sequence of formula I in the N-terminus to C-terminus manner: X-[(Ab a )-(L b )] m -(DD)-[(L c )-(Ab d )] n -Y (wherein m is 0, 1, 2 or an integer greater than 2; n is an integer greater than 2, or 0, 1, 2; m and n are not both 0 at the same time; Ab a Ab d Each of these represents an antigen-binding domain, and at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3); X or Y may or may not exist independently, and include an amino acid sequence; L b and L c each independently includes one or more linkers; and (DD stands for dimerization domain) A binder according to any one of claims 1 to 32, comprising:

35. The binder according to claim 24, 33, or 34, wherein the dimerization domain comprises the CH3 domain of an antibody or a portion thereof.

36. The binder according to claim 35, wherein the dimerization domain comprises the CH2 domain of the antibody or a portion thereof.

37. The binder according to claim 35 or 36, wherein the CH3 domain and / or CH2 domain are derived from human IgG1, human IgG2, human IgG3, or human IgG4 heavy chain.

38. The binder according to any one of claims 24 or 33 to 37, wherein the dimerizing domain comprises an amino acid sequence that is at least 80% identical to the amino acid sequence described in SEQ ID NO: 212 or SEQ ID NO:

506.

39. The binder according to any one of claims 1 to 38, wherein the binder comprises at least two antigen-binding domains, the at least two antigen-binding domains being identical or different.

40. A binder according to any one of claims 1 to 39, comprising Fc or an Fc moiety capable of binding to an Fc receptor of a monocyte or macrophage.

41. A binder according to any one of claims 1 to 40, wherein one or more antigen-binding domains are arranged in tandem.

42. A binder according to any one of claims 1 to 41, comprising a single-domain antibody.

43. A binder according to any one of claims 1 to 42, wherein the binder is a single-domain antibody.

44. A binder according to any one of claims 1 to 43, which is naked.

45. A binder according to any one of claims 1 to 44, which is conjugated to the treatment area.

46. A binder according to any one of claims 1 to 45, conjugated to a detectable portion.

47. A binder according to any one of claims 1 to 46, wherein one or more antigen-binding domains or binders specifically bind to human TROP2.

48. The binder according to any one of claims 1 to 47, which is a single-domain antibody comprising two identical heavy chain variable regions or two identical heavy chains.

49. A pharmaceutical composition comprising a binder or single-domain antibody according to any one of claims 1 to 48, and a pharmaceutically acceptable carrier.

50. A nucleic acid or set of nucleic acids encoding a binder or a single-domain antibody according to any one of claims 1 to 49.

51. A vector comprising the nucleic acid of claim 50.

52. Cells expressing the binder or single-domain antibody according to any one of claims 1 to 51.

53. A cell comprising the nucleic acid described in claim 50 or the vector described in claim 51.

54. A kit comprising a binder or a single-domain antibody according to any one of claims 1 to 53.

55. A kit comprising the nucleic acid described in claim 50, or the vector described in claim 51, or the cells described in claim 52 or claim 53.

56. A method for treating a disorder or disease, comprising administering a binder or single-domain antibody according to any one of claims 1 to 55, or a pharmaceutical composition comprising a binder or single-domain antibody according to any one of claims 1 to 55.

57. The method according to claim 56, wherein the disorder or disease is related to the expression or overexpression of TROP2.

58. The method according to claim 57, wherein the disorder or disease is cancer, solid tumor, epithelial cancer, lung cancer, metastatic lung cancer, small cell lung cancer, non-small cell lung cancer, myeloma, prostate cancer, breast cancer, triple-negative breast cancer, colon cancer, pancreatic cancer, glioblastoma, cervical cancer, colorectal cancer, stomach cancer, ovarian cancer, thyroid cancer, bladder cancer, uterine cancer, esophageal cancer, blood cancer, or head and neck cancer.

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