Binding agents targeting trop2-expressing tumor cells

Abstract

The present disclosure relates generally to binding agents, such as antibodies and antigen binding fragments thereof, capable of binding to trophoblast cell surface antigen-2 (TROP2). Also disclosed herein are binding agents capable of targeting tumor cells expressing TROP2 and their use for the treatment of cancer. A single domain antibody that specifically binds to an amino acid residue of the extracellular domain of TROP2 is provided.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480035021.9 (22) Application Date 2024.04.04 (30) Priority Data 63 / 457,092 2023.04.04 US 63 / 535,531 2023.08.30 US 63 / 598,055 2023.11.10 US (85) PCT International Application Entering National Phase Date 2025.11.25 (86) PCT International Application Application Data PCT / CA2024 / 050435 2024.04.04 (87) PCT International Application Publication Data WO2024 / 207112 EN 2024.10.10 (71) Applicant: Canadian company, Kiso Road Biotechnology Co., Ltd. Address: Canada (72) Inventors: Hou Wenyang, Yao Shugang, L. Dacruz, D.S. Yang (74) Patent Agency: China Council for the Promotion of International Trade Patent & Trademark Office Co., Ltd. 11038 Patent Attorney: Xie Lifei (51) Int.Cl. C07K 16 / 30 (2006.01) A61K 39 / 395 (2006.01) A61K 47 / 68 (2006.01) A61K 49 / 00 (2006.01) A61P 35 / 00 (2006.01) C07K 16 / 28 (2006.01) C07K 16 / 46 (2006.01) C12N 15 / 13 (2006.01) (54) Invention Title: Binders Targeting Tumor Cells Expressing TROP2 (57) Abstract: This disclosure generally relates to binders capable of binding trophoblast cell surface antigen-2 (TROP2), such as antibodies and antigen-binding fragments thereof. This document also discloses binders capable of targeting tumor cells expressing TROP2 and their use in the treatment of cancer. Single-domain antibodies that specifically bind to amino acid residues of the extracellular domain of TROP2 are provided. Claims 7 pages, Description 226 pages, Sequence Listing (Electronic Publication), Drawings 68 pages, CN 121419995 A 2026.01.27 CN 1 21 41 99 95 A 1. A binding agent comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds trophoblast cell surface antigen-2 (TROP2), and comprising: a. a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:225, having the amino acid sequences shown in SEQ ID NO:226, SEQ ID NO:234, SEQ ID NO:242, SEQ ID NO:320, and SEQ ID NO:392.The conjugate according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO: 227, and the conjugate according to claim 2, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO: 227, or the conjugate according to claim 3; or; b. a heavy chain complementarity-determining region 1 (CDRH1) comprising the amino acid sequence shown in SEQ ID NO: 228, a heavy chain complementarity-determining region 2 (CDRH2) comprising the amino acid sequence shown in SEQ ID NO: 229, SEQ ID NO: 237, SEQ ID NO: 245, or SEQ ID NO: 323, or SEQ ID NO: 395, and a heavy chain complementarity-determining region 3 (CDRH3) comprising the amino acid sequence shown in SEQ ID NO: 230. 2. The conjugate according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO: 539. 3. The conjugate according to claim 1 or 2, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO: 540. 4. The conjugate 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 shown in SEQ ID NO: 541. 5. The binder of claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any one of SEQ ID NO: 514-516. 6. The binder of claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any one of SEQ ID NO: 517-519. 7. The binder of claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any one of SEQ ID NO: 520-522. 8. The binder of claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any one of SEQ ID NO: 523-525. 9. The binder of claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any one of SEQ ID NO: 526-528. 10. The binder according to any one of claims 1 to 9, wherein at least one of the one or more antigen-binding domains comprises a domain corresponding to 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 ...The amino acid sequences shown in 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 are at least 80% identical. 11. The binder according to any one of claims 1 to 10, wherein at least one of the one or more antigen-binding domains comprises 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:385, SEQ ID NO:386, SEQ ID NO:387, SEQ ID NO:247, SEQ ID NO:388, SEQ ID NO:389, SEQ ID NO:380, SEQ ID NO:381, SEQ ID NO:382, SEQ ID NO:239, SEQ ID NO:247, SEQ ID NO:388, SEQ ID NO:389 ... 12. A binding agent 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 comprising: a. a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:490.b. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:485, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:486, SEQ ID NO:492 or SEQ ID NO:495, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:487; c. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:500, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:501; d. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:485, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:498 or SEQ ID NO:486, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:499. e. CDRH1 having the amino acid sequence shown in SEQ ID NO:305, CDRH2 having the amino acid sequence shown in SEQ ID NO:306, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:307; f. CDRH1 having the amino acid sequence shown in SEQ ID NO:308, CDRH2 having the amino acid sequence shown in SEQ ID NO:309, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:310; g. CDRH1 having the amino acid sequence shown in SEQ ID NO:312, CDRH2 having the amino acid sequence shown in SEQ ID NO:313, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:314; h. CDRH1 having the amino acid sequence shown in SEQ ID NO:315, CDRH2 having the amino acid sequence shown in SEQ ID NO:316, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:317; i. CDRH1 having the amino acid sequence shown in SEQ ID NO:326, CDRH2 having the amino acid sequence shown in SEQ ID NO:305, CDRH2 having the amino acid sequence shown in SEQ ID NO:306, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:307. CDRH2 having the amino acid sequence shown in NO:327 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:328; j. CDRH1 having the amino acid sequence shown in SEQ ID NO:329, CDRH2 having the amino acid sequence shown in SEQ ID NO:330 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:331;k. CDRH1 having the amino acid sequence shown in SEQ ID NO:333, CDRH2 having the amino acid sequence shown in SEQ ID NO:334, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:335; l. CDRH1 having the amino acid sequence shown in SEQ ID NO:336, CDRH2 having the amino acid sequence shown in SEQ ID NO:337, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:338; m. CDRH1 having the amino acid sequence shown in SEQ ID NO:340, CDRH2 having the amino acid sequence shown in SEQ ID NO:341, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:342; n. CDRH1 having the amino acid sequence shown in SEQ ID NO:343, CDRH2 having the amino acid sequence shown in SEQ ID NO:344, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:345; o. CDRH1 having the amino acid sequence shown in SEQ ID NO:347, CDRH2 having the amino acid sequence shown in SEQ ID NO:348, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:335. CDRH3 having the amino acid sequence shown in SEQ ID NO:349; Claims 2 / 7, page 3, CN 121419995 A p. CDRH1 having the amino acid sequence shown in SEQ ID NO:350, CDRH2 having the amino acid sequence shown in SEQ ID NO:351, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:352; q. CDRH1 having the amino acid sequence shown in SEQ ID NO:399, CDRH2 having the amino acid sequence shown in SEQ ID NO:400, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:401; r. CDRH1 having the amino acid sequence shown in SEQ ID NO:402, CDRH2 having the amino acid sequence shown in SEQ ID NO:403, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:404; s. CDRH1 having the amino acid sequence shown in SEQ ID NO:406, CDRH2 having the amino acid sequence shown in SEQ ID NO:407, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:408; t. CDRH1 having the amino acid sequence shown in SEQ ID NO:409, CDRH2 having the amino acid sequence shown in SEQ ID NO:410 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:411;u. CDRH1 having the amino acid sequence shown in SEQ ID NO:413, CDRH2 having the amino acid sequence shown in SEQ ID NO:414, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:415; v. CDRH1 having the amino acid sequence shown in SEQ ID NO:416, CDRH2 having the amino acid sequence shown in SEQ ID NO:417, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:418; w. CDRH1 having the amino acid sequence shown in SEQ ID NO:420, CDRH2 having the amino acid sequence shown in SEQ ID NO:421, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:422; x. CDRH1 having the amino acid sequence shown in SEQ ID NO:423, CDRH2 having the amino acid sequence shown in SEQ ID NO:424, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:425; y. CDRH1 having the amino acid sequence shown in SEQ ID NO:427, CDRH2 having the amino acid sequence shown in SEQ ID NO:415, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:416, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:417; CDRH2 having the amino acid sequence shown in SEQ ID NO:428 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:429; z. CDRH1 having the amino acid sequence shown in SEQ ID NO:430, CDRH2 having the amino acid sequence shown in SEQ ID NO:431 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:432; aa. CDRH1 having the amino acid sequence shown in SEQ ID NO:434, CDRH2 having the amino acid sequence shown in SEQ ID NO:435 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:436; bb. CDRH1 having the amino acid sequence shown in SEQ ID NO:437, CDRH2 having the amino acid sequence shown in SEQ ID NO:438 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:439; cc. CDRH1 having the amino acid sequence shown in SEQ ID NO:441, CDRH2 having the amino acid sequence shown in SEQ ID NO:442 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:443; dd. CDRH1 having the amino acid sequence shown in SEQ ID NO:444, CDRH2 having the amino acid sequence shown in SEQ ID NO:445, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:446; ee. having the amino acid sequence shown in SEQ ID NO:446.CDRH1 having the amino acid sequence shown in SEQ ID NO:448, CDRH2 having the amino acid sequence shown in SEQ ID NO:449, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:450; ff. CDRH1 having the amino acid sequence shown in SEQ ID NO:451, CDRH2 having the amino acid sequence shown in SEQ ID NO:452, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:453; gg. CDRH1 having the amino acid sequence shown in SEQ ID NO:455, CDRH2 having the amino acid sequence shown in SEQ ID NO:456, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:457; hh. CDRH1 having the amino acid sequence shown in SEQ ID NO:458, CDRH2 having the amino acid sequence shown in SEQ ID NO:459, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:460; ii. CDRH1 having the amino acid sequence shown in SEQ ID NO:462, and the amino acid sequence shown in SEQ ID NO:463. (Claims 3 / 7, Page 4, CN) 121419995 A sequence of CDRH2 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:464; jj. CDRH1 having the amino acid sequence shown in SEQ ID NO:465, CDRH2 having the amino acid sequence shown in SEQ ID NO:466 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:467. 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 shown in SEQ ID NO:488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:490, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:493, SEQ ID NO:496, or SEQ ID NO:491. 14. The binder of 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 shown in SEQ ID NO: 500, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 489, and an amino acid sequence shown in SEQ ID NO: 501.The heavy chain complementation-determining region 3 (CDRH3) of the sequence and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:503 or SEQ ID NO:502. 15. The binder of claim 12, wherein at least one of the one or more antigen-binding domains comprises a heavy chain complementation-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:308, a heavy chain complementation-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:309, and a heavy chain complementation-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:310, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:311 or SEQ ID NO:389. 16. The binder of claim 1 or 12, wherein at least one of the one or more antigen-binding domains comprises a humanized frame region. 17. The binder of claim 1 or 12, wherein at least one of the one or more antigen-binding domains comprises a human frame 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 backbone, the backbone comprising the amino acid sequence shown 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 backbone, the backbone comprising the amino acid sequence shown in SEQ ID NO:213, SEQ ID NO:479, or SEQ ID NO:480. 20. 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 backbone, the backbone comprising 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, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:26 ...The amino acid sequence shown in SEQ ID NO:269, SEQ ID NO:270 or SEQ ID NO:271, or an amino acid sequence comprising 1 to 5 amino acid substitutions, additions and / or deletions of the amino acid sequence. 21. 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 backbone, the backbone comprising 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:285, SEQ ID NO:286, 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:295, SEQ ID NO:296, 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:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:288, SEQ ID NO:289, SEQ ID NO:29 ... The amino acid sequences shown in SEQ ID NO:474, SEQ ID NO:475, and SEQ ID NO:476, or containing amino acid sequences with 1 to 5 amino acid substitutions, additions, and / or deletions. 22. The binding agent according to any one of the preceding claims, wherein the heavy chain variable region comprises a frame region 2 (FR2), the frame region 2 comprising the sequences shown 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 trophoblast cell surface antigen-2 (TROP2), and comprises SEQ ID NO:373, SEQ ID NO:493, SEQ ID NO:503, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:25.NO:363, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:304, 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: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, 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 The amino acid sequence shown in NO:491, SEQ ID NO:496, or SEQ ID NO:502. 24. The binder according to claim 24, wherein the binder comprises a dimerizing domain. 25. The binder according to any of the preceding claims, 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 the preceding claims, 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 the one or more heavy chain variable regions are derived from Camelidae VH or VHH. 28. The binder according to any one of the preceding claims, wherein the binder comprises two or more antigen-binding domains. 29. The binder according to any one of the preceding claims, wherein the binder is single-specific. 30. The binder according to any one of claims 1 to 28, wherein the binder is bispecific. 31. The binder according to any one of claims 1 to 28, wherein the binder is multispecific. 32. The binder according to claim 30 or 31, wherein the binder comprises one or more CD47-binding antigen-binding domains and / or one or more PD-1-binding antigen-binding domains. 33. The binder according to any one of claims 1 to 32, wherein the binder comprises two polypeptide chains, and each polypeptide chain comprises, from N-terminus to C-terminus: a) one or more antigen-binding domains, b) optional linkers, and c) dimerization domains. 34. The binder according to any one of claims 1 to 32, wherein the binder comprises two polypeptide chains, and wherein each polypeptide chain comprises an amino acid sequence of formula I in an N-terminal to C-terminal manner: X-[(Aba)-(Lb)]m-(DD)-[(Lc)-(Abd)]n-Y where m is an integer of 0, 1, 2 or greater than 2; where n is an integer of 0, 1, 2 or greater than 2; where m and n are not simultaneously 0; where Aba and Abd each represent an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3); where X or Y is present or absent independently and comprises an amino acid sequence; where Lb and Lc each independently comprise one or more linkers; and where DD represents a dimerizing domain. 35. The binder according to claim 24, 33 or 34, wherein the dimerizing domain comprises the CH3 domain of an antibody or a portion thereof. 36. The binder of claim 35, wherein the dimerizing domain comprises the CH2 domain or a portion thereof of the antibody. 37. The binder of claim 35 or 36, wherein the CH3 domain and / or CH2 domain are derived from the heavy chain of human IgG1, human IgG2, human IgG3, or human IgG4. 38. The binder of any one of claims 24 or 33 to 37, wherein the dimerizing domain comprises the same as SEQ ID NO.39. The binder according to any one of the preceding claims, wherein the binder comprises at least two antigen-binding domains, and wherein the at least two antigen-binding domains are identical or different. 40. The binder according to any one of the preceding claims, wherein the binder comprises an Fc or Fc moiety capable of binding to an Fc receptor on monocytes and / or macrophages. 41. The binder according to any one of the preceding claims, wherein the one or more antigen-binding domains are arranged in tandem. 42. The binder according to any one of the preceding claims, wherein the binder comprises a single-domain antibody. 43. The binder according to any one of the preceding claims, wherein the binder is a single-domain antibody. 44. The binder according to any one of the preceding claims, wherein the binder is naked. 45. The binder according to any one of the preceding claims, wherein the binder is conjugated to a therapeutic portion. 46. The binder according to any one of the preceding claims, wherein the binder is conjugated to a detectable portion. 47. The binder according to any one of the preceding claims, wherein the one or more antigen-binding domains or the binder specifically bind to human TROP2. 48. A conjugate according to any one of the preceding claims, wherein the conjugate is a single-domain antibody comprising two identical heavy chain variable regions or two identical heavy chains. 49. A pharmaceutical composition comprising the conjugate or single-domain antibody according to any one of the preceding claims, and a pharmaceutically acceptable carrier. 50. A nucleic acid or nucleic acid set encoding the conjugate or single-domain antibody according to any one of the preceding claims. Claims 6 / 7 pages 7 CN 121419995 A 51. A vector comprising the nucleic acid of claim 50. 52. A cell expressing the conjugate or single-domain antibody according to any one of the preceding claims. 53. A cell comprising the nucleic acid of claim 50 or the vector of claim 51. 54. A kit comprising the conjugate or single-domain antibody according to any one of the preceding claims. 55. A kit comprising the nucleic acid of claim 50, the vector of claim 51, or the cell of claim 52 or 53. 56. A method of treating a condition or disease comprising administering the conjugate or single-domain antibody according to any one of the preceding claims, or a pharmaceutical composition comprising the conjugate or single-domain antibody according to any one of the preceding claims. 57. The method of claim 56, wherein the condition or disease is associated with the expression or overexpression of TROP2. 58. The method of claim 57, wherein the condition or disease is cancer, a solid tumor, epithelial cancer, or lung cancer.Cancer, metastatic lung cancer, small cell lung cancer, non-small cell lung cancer, myeloma, prostate cancer, breast cancer, triple-negative breast cancer, rectal cancer, pancreatic cancer, glioblastoma, cervical cancer, colorectal cancer, gastric cancer, ovarian cancer, thyroid cancer, gastric cancer, bladder cancer, uterine cancer, esophageal cancer, hematologic malignancy, or head and neck cancer. Claims 7 / 7 pages 8 CN 121419995 A Binders for Targeting Tumor Cells Expressing TROP2 Technical Field

[0001] This disclosure generally relates to binders capable of binding to trophoblast cell surface antigen-2 (TROP2), such as antibodies and antigen-binding fragments thereof. Binders capable of targeting tumor cells expressing TROP2 and their use in the treatment of cancer are also disclosed herein. Single-domain antibodies that specifically bind to amino acid residues of the extracellular domain of TROP2 are provided. Background Art

[0002] Trophoblast cell surface antigen-2 (TROP2) is a membrane-bound protein expressed at low levels in normal adult tissues. TROP2 possesses pleiotropic functions, including inducing cell signaling and cell-cell incompatibility by regulating interactions with extracellular and intracellular proteins. TROP2 has an epidermal growth factor (EGF)-like domain followed by a 23-amino acid transmembrane domain and a cytoplasmic tail. The EGF-like domain contains a cysteine-rich domain, a thyroglobulin type 1 domain, and a cysteine-deficient domain. In TROP2 deficiency, its role in normal tissues appears to be compensated by the expression of EpCAM, which is highly correlated with TROP2. TROP2 is highly expressed in a wide range of advanced epithelial cancers, including breast and pancreatic cancer (Stepan et al., J. Histochem and Cytochem, 2011, the entire contents of which are incorporated herein by reference). In several in vitro and in vivo models, TROP2 overexpression has been shown to confer oncogenic behavior. TROP2 upregulation in solid tumors is associated with increased tumor invasiveness, metastasis, and a decline in overall survival in many refractory cancers, making it an attractive target for cancer therapy.

[0003] TROP2-positive cancers have been successfully targeted clinically using antibody-drug conjugates (ADCs) rather than naked functional antibodies. ADCs have associated drug toxicity, off-target effects, and payload delivery problems in healthy tissues. Given the dose-limiting side effects, narrow therapeutic window, and efficacy limitations of ADCs, there is an urgent need to improve anti-TROP2 therapeutic agents (if any) for patients who do not respond well to current therapies.

[0004] Camelidae and cartilaginous fish naturally produce antibodies composed of functional homodimeric heavy chain antibodies (HCAbs) (Hamers-Casterman et al., 1993; Muyldermans and Smider, 2016). The heavy chain of HCAbs lacks a first constant.The HCAb contains a CH1 domain and differs from classic antibodies only in that the light chain pairing typically involves some amino acid substitutions (Muyldermans et al., 1994; Vu et al., 1997). These substitutions (Val37Phe / Tyr, Gly44Glu, Leu45Arg, and Trp47Gly) are present in frame region 2 (FR2). The antigen-binding fragment of the HCAb is called VHH or nanobody®. VHH has a molecular weight of approximately 15 kDa, which makes it suitable for applications requiring enhanced tissue penetration or rapid clearance, such as radioisotope-based imaging. However, for therapeutic applications, it is generally necessary to increase the half-life of VHH in order to minimize renal clearance and optimize therapeutic efficacy (De Vlieger et al., Antibodies 8(1), 1–22, 2019). Although methods to increase the VHH half-life have been employed, such as PEGylation, N-glycosylation, HSA or other carrier protein fusion, these constructs have introduced immunogenicity or have limited success rates.

[0005] Variable regions of single-domain antibodies have been used as building blocks to prepare bispecific and multispecific antibodies. In some studies, bivalent constructs have shown increased affinity or cohesion compared to monovalent forms (Conrath et al., 2001; Coppieters et al., 2006; Hmila et al., 2008; Simmons et al., 2006 and Hultberg et al., 2011, Jähnichen et al. (2010), Fridy et al., 2014).

[0006] Novel TROP2 binders comprising antibodies and their antigen-binding fragments, such as single-domain antibodies and their antigen-binding fragments. Specification 1 / 226 Page 9 CN 121419995 A Summary of the Invention

[0007] Therefore, this disclosure generally relates to binders comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains specifically binds trophoblast cell surface antigen-2 (TROP2).

[0008] The binder, or at least one of the one or more binding domains thereof, binds to human TROP2. Therefore, the binders of this disclosure can be used to treat human diseases or conditions associated with the expression or overexpression of TROP2.

[0009] In some embodiments, the binders of this disclosure may be able to bind to the extracellular domain (ECD) of TROP2, said extracellular domain being as shown by amino acids 27 to 274 of TROP2 (e.g., SEQ ID NO:39 or SEQ ID NO:40), see Goldenbenberg et al., 2018, Ikeda, M. et al., 2015, the entire contents of which are incorporated herein by reference.

[0010] In some embodiments, one or more antigen-binding domains of the binder include the complementarity-determining region (CDR), heavy chain variable region, or heavy chain of a single-domain antibody (sdAb).

[0011] The single-domain antibodies of this disclosure have unique binding characteristics and activities. For example, the complementarity-determining region 3 (CDR3) of the sdAb forms a loop that adapts to a groove within the cysteine-rich domain (CRD) of the extracellular domain (ECD) of TROP2. Amino acid residues of CDR3 and frame region 2 (FR2) interact with amino acid residues of the cysteine-rich domain of TROP2 and together form a clamp around the CRD as a two-site antibody. These unique properties endow the sdAbs disclosed herein with high affinity and a slow dissociation rate. Surprisingly, amino acid residues of complementarity-determining region 1 (CDR1) and complementarity-determining region 2 (CDR2) do not appear to interact with amino acid residues of the CRD. Therefore, it is assumed that changes in the amino acid sequences of CDR1 and CDR2 will not affect binding to TROP2. Therefore, the one or more antigen-binding domains contain at least a CDR3 amino acid residue.

[0012] In some cases, the one or more antigen-binding domains may contain a CDR3 amino acid residue and a frame region 2 (FR2) amino acid residue.

[0013] In other cases, the one or more antigen-binding domains may contain a CDR3 amino acid residue and a frame region 2 (FR2) amino acid residue, and optionally contain a CDR1 and / or CDR2 amino acid residue.

[0014] In a further embodiment, the one or more antigen-binding domains may contain CDR1, CDR2, and CDR3 amino acid residues.

[0015] In yet another further embodiment, the one or more antigen-binding domains may contain CDR1, FR2, CDR2, and CDR3 amino acid residues.

[0016] In some embodiments, the CDR1, CDR2, CDR3, and FR2 amino acid residues correspond to the corresponding residues of the single-domain antibodies disclosed herein.

[0017] According to this disclosure, CDRs can 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)).

[0018] Alternatively, CDRs can be identified using the IMGT numbering scheme (e.g., Lefranc, M.-P., The Immunologist, 7, 132-136 (1999)).

[0019] In some cases, CDR1, CDR2, CDR3 and / or FR2 correspond to Kabat CDR1, CDR2, CDR3 and / or FR2.

[0020] In other cases, CDR1, CDR2, CDR3 and / or FR2 correspond to IMGT CDR1, CDR2, CDR3 and / or FR2.

[0021] In some embodiments, one or more antigen-binding domains may contain amino acid residues of the variable region.

[0022] For example, in some embodiments, one or more antigen-binding domains may contain the amino acid sequence of Camelidae VH or VHH. In other embodiments, one or more antigen-binding domains may contain the amino acid sequence of mouse VH. In yet another embodiment, one or more antigen-binding domains may contain the amino acid sequence of human VH. In a further embodiment, one or more antigen-binding domains may comprise an amino acid sequence of a humanized VH or VHH (e.g., a humanized camelid VH or VHH, or a humanized mouse VH).

[0023] In some cases, this disclosure relates to anti-TROP2 single-domain antibodies capable of inducing antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP). Furthermore, in other cases, this disclosure relates to anti-TROP2 single-domain antibodies capable of internalization, making them suitable for delivering drug payloads to the tumor microenvironment or to tumor cells. In still other cases, this disclosure relates to anti-TROP2 single-domain antibodies that interfere with / block TROP2 function.

[0024] Although the conjugates of this disclosure may have activity against tumor cells even when unconjugated, this disclosure also covers the conjugation of conjugates to therapeutic portions. Conjugation may be particularly beneficial when the conjugate is inactive or has low activity against tumor cells.

[0025] Therefore, this disclosure relates to unconjugated (naked) single-domain antibodies capable of binding TROP2. This disclosure also relates to antibody-drug conjugates (ADCs) comprising a single-domain antibody capable of binding TROP2 and a drug payload.

[0026] The binding function of an sdAb is conferred by a single polypeptide chain, i.e., the heavy chain variable region. This unique property provides format flexibility. For example, multiple sdAbs or sdAb antigen-binding domains can be fused into a single polypeptide chain conferring multivalent and / or multispecificity. Furthermore, multiple polypeptide chains can be assembled to increase the diversity (multi-site binding) or affinity for TROP2 interactions. Additionally, the sdAb antigen-binding domain can be fused to any type of polypeptide chain, including, for example, but not limited to, fusion with the heavy and / or light chains of a natural antibody or its antigen-binding fragment, fusion with a protein scaffold, fusion with an immune cell modulator, etc.

[0027] Therefore, this disclosure relates not only to the single-domain antibodies disclosed herein, but also more generally to binders comprising one or more antigen-binding domains of the single-domain antibodies disclosed herein.

[0028] Furthermore, this disclosure relates to binders that compete with the single-domain antibodies of this disclosure. For example, a single-domain antibody is provided that competes with one of the various single-domain antibodies disclosed herein for binding to at least one epitope comprising an amino acid residue of an extracellular domain (ECD) of TROP2.

[0029] The binder or the antigen-binding domain of the binder can be derived from a variety of sources, including from animals capable of producing single-domain antibodies, such as camels, sharks, transgenic animals, screening of antibody libraries, etc. For example, antibodies can be obtained from hybridomas after immunization of rodents (mice, rats, etc.), or from large mammals such as camels. In some embodiments, the animal can be immunized with the TROP2 antigen.

[0030] According to 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 chains. In some instances, the two heavy chain variable regions are linked to a dimerizing domain to allow dimer formation.

[0031] In some embodiments, the binder comprises a polypeptide chain comprising, from N-terminus to C-terminus: a) an antigen-binding domain of a single-domain antibody, b) an optional linker, and c) a dimerizing domain.

[0032] In some embodiments, the binder comprises a polypeptide chain comprising, from N-terminus to C-terminus: a) a dimerizing domain, b) an optional linker, and c) an antigen-binding domain of a single-domain antibody.

[0033] In other embodiments, the binder comprises a polypeptide chain comprising, from N-terminus to C-terminus: a) an antigen-binding domain of a single-domain antibody, b) a linker, c) a dimerizing domain, and one or more d) linkers and e) an antigen-binding domain of a single-domain antibody.

[0034] In some embodiments, the antigen-binding domain a) and the antigen-binding domain 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.

[0035] In yet other embodiments, the binder may have the format shown in Formula I, II, III, IIIa and IIIb, IV, V, VI, VII or VIII disclosed herein.

[0036] According to this disclosure, the binder may contain additional amino acid sequences or portions (small molecules, labels, etc.) at the N-terminus and / or C-terminus. For example, according to this disclosure, the binder may contain additional antigenic domains of single-domain antibodies at the N-terminus or C-terminus.A binding domain. Additional antigen-binding domains may be separated from the core by one or more linkers.

[0037] According to this disclosure, a binding or antigen-binding domain may contain the sequence shown herein. According to this disclosure, a single domain may contain the sequence shown herein.

[0038] In some embodiments, at least one of the binding agents or one or more antigen-binding domains comprises CDR1, CDR2, CDR3, FR2, or a combination thereof of the heavy chain variable region shown in any of the following SEQ ID NO: SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:247, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:311, SEQ ID NO:318, SEQ ID NO:325, SEQ ID NO:332, SEQ ID NO:339, SEQ ID NO:346, SEQ ID NO:353, 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 ...9, SEQ ID NO:379, 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:379, SEQ ID NO:379, SEQ ID NO:379, SEQ ID NO:373, SEQ ID NO:374, SEQ ID NO:375, SEQ ID NO:376, SEQ ID NO: 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:491, SEQ ID NO:493, SEQ ID NO:496, SEQ ID NO:502 or SEQ ID NO:503.

[0039] 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 comprises an amino acid represented by any of the following SEQ ID NOs.Amino acid sequences that are 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, or at least 99% identical or the same as the following sequences: SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:247, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:311, SEQ ID NO:318, SEQ ID NO:325, SEQ ID NO:332, SEQ ID NO:339, SEQ ID NO:346, SEQ ID NO:353, 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:379, SEQ ID NO:379, SEQ ID NO:379, SEQ ID NO:379, SEQ ID NO:379, SEQ ID NO:370 ...0, SEQ ID NO:379, SEQ ID NO:370, SEQ ID NO:370, SEQ ID NO:379, SEQ ID NO:370, SEQ ID NO:370, SEQ ID NO:370, SEQ ID NO:379, 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:491, SEQ ID NO:493, SEQ ID NO:496, SEQ ID NO:502 or SEQ ID NO:503.

[0040] In some embodiments, at least one of the binders or one or more antigen-binding domains comprises a variable region having the sequence shown herein (including the original sequence or humanized sequence), and comprises 1 to 5 amino acid substitutions, 1 to 5 amino acid deletions, or 1 to 5 amino acid additions thereto.

[0041] Specific embodiments of this disclosure include binders, such as single-domain antibodies, comprising one or more antigen-binding domains, wherein at least one of said one or more antigen-binding domains binds trophoblast cell surface antigen-2 (TROP2), and comprises:Specification 4 / 226 pages 12 CN 121419995 A

[0042] a) Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:225, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequences shown 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-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:227, or;

[0043] b) Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:228, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequences shown 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-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:230.

[0044] In a more specific embodiment of this disclosure, at least one of one or more antigen-binding domains of the binder or single-domain antibody comprises:

[0045] a) a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:225, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequences shown in SEQ ID NO:226, SEQ ID NO:234, SEQ ID NO:242, SEQ ID NO:320, SEQ ID NO:392, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:227, or;

[0046] b) a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:228, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:229, SEQ ID NO:237, SEQ ID NO:245 or SEQ ID NO:323 or SEQ ID NO:395, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:227, or SEQ ID NO:228, or SEQ ID NO:229, SEQ ID NO:237, SEQ ID NO:245 or SEQ ID NO:323 or SEQ ID NO:395, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:226, SEQ ID NO:234, SEQ ID NO:242, SEQ ID NO:320, SEQ ID NO:395, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:227, or SEQ ID NO: The heavy chain complementarity-determining region 3 (CDRH3) of the amino acid sequence shown in NO:230, and;

[0047] with 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 IDThe amino acid sequences shown in 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 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 shown in 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.

[0048] In other specific embodiments, at least one of one or more antigen-binding domains of the binder or single-domain antibody comprises: a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:490, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:493, SEQ ID NO:496, or SEQ ID NO:491.

[0049] In yet another specific embodiment, at least one of one or more antigen-binding domains of the binder or single-domain antibody comprises: a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 500, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 501; and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in either SEQ ID NO: 503 or SEQ ID NO: 502.

[0050] In a further specific embodiment, at least one of one or more antigen-binding domains of the binder or single-domain antibody comprises: a heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO: 308, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 309, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 310; and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 501.The amino acid sequence shown in any of the embodiments of NO:311 or SEQ ID NO:389, page 5 / 226, 13 CN 121419995 A, is at least 80% identical.

[0051] In some cases, at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO:539.

[0052] In an exemplary embodiment, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO:540.

[0053] In other exemplary embodiments, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in SEQ ID NO:541.

[0054] In an exemplary embodiment, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown in any of SEQ ID NO:514-516.

[0055] In other exemplary embodiments, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence as shown in any one of SEQ ID NO: 517-519.

[0056] In other exemplary embodiments, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence as shown in any one of SEQ ID NO: 520-522.

[0057] In exemplary embodiments, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence as shown in any one of SEQ ID NO: 523-525.

[0058] In other exemplary embodiments, the binder of any of the foregoing embodiments, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence as shown in any one of SEQ ID NO: 526-528.

[0059] In exemplary embodiments, the binder or antigen-binding domain of this disclosure may have ADCP activity against cells expressing human TROP2 and differentiation cluster 47 (CD47) (Uniprot accession number Q08722-1). SEQ ID NO:176 provides an example and non-limiting embodiment of the CD47 antigen.

[0060] Therefore, this disclosure covers conjugates comprising one or more antigen-binding domains capable of binding TROP2 and one or more antigen-binding domains capable of binding CD47 and / or PD-1.

[0061] In some embodiments, the conjugate is an antibody or an antigen-binding fragment thereof (e.g., a single-domain antibody or an antigen-binding fragment thereof).

[0062] This disclosure also relates to conjugators capable of competing with one or more conjugators disclosed herein.

[0063] Other aspects and embodiments of this disclosure relate to compositions or pharmaceutical compositions comprising conjugators disclosed herein and pharmaceutically acceptable carriers.

[0064] Still other aspects and embodiments of this disclosure relate to pharmaceutical compositions comprising conjugated conjugators disclosed herein and pharmaceutically acceptable carriers.

[0065] Still other aspects and embodiments of this disclosure relate to nucleic acids or groups of nucleic acids encoding conjugators disclosed herein.

[0066] Further aspects and embodiments of this disclosure relate to vectors comprising nucleic acids disclosed herein, or groups of vectors each comprising nucleic acids disclosed herein.

[0067] Further aspects and embodiments of this disclosure relate to cells expressing conjugators disclosed herein.

[0068] In some embodiments, cells are engineered to express conjugators, such as antibodies or antigen-binding fragments or antibody-like molecules thereof. Therefore, this disclosure covers isolated cells expressing antibodies or antigen-binding fragments or antibody-like molecules thereof.

[0069] In other embodiments, cells are engineered to express a binding agent, such as a chimeric antigen receptor (CAR) construct. Therefore, this disclosure covers T cells, NK cells, monocytes, or macrophages expressing a chimeric antigen receptor (CAR) construct.

[0070] Further aspects and embodiments of this disclosure relate to cells comprising the nucleic acids or vectors disclosed herein.

[0071] Further aspects and embodiments of this disclosure relate to kits comprising the binding agents disclosed herein.

[0072] In some embodiments, the kit may comprise a binding agent that binds TROP2 and a binding agent that binds macrophage markers. For example, the kit may comprise an antibody that specifically binds TROP2 or an antigen-binding fragment thereof. In some cases, the kit may comprise a binding agent as described herein. The kit may also comprise an antibody that binds macrophage markers, such as, but not limited to, an antibody that binds CD68 (human CD68) or an antigen-binding fragment thereof.

[0073] Further aspects and embodiments of this disclosure relate to kits comprising the nucleic acids or vectors disclosed herein.

[0074] Further aspects and embodiments of this disclosure relate to kits comprising the cells disclosed herein.

[0075] In another aspect and embodiment, this disclosure relates to methods of treating a condition or disease, comprising administering the conjugates, compositions, or pharmaceutical compositions disclosed herein.

[0076] In some embodiments, the TROP2 conjugate disclosed herein is administered to a subject suffering from cancers selected from the group consisting of...Agents, compositions, or pharmaceutical compositions: epithelial cancers, 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, gastric cancer, bladder cancer, uterine cancer, esophageal cancer, head and neck cancer, hematologic cancers.

[0077] In other embodiments, the TROP2 binder, composition, or pharmaceutical composition disclosed herein is administered to a subject with metastatic cancer.

[0078] Other aspects of this disclosure relate to methods of treating cancer, including administering the binder disclosed herein to a subject with a tumor expressing TROP2 and expressing one or more macrophage markers (e.g., but not limited to CD68).

[0079] In other aspects and embodiments, this disclosure relates to methods of preparing the binder disclosed herein by transforming cells with one or more vectors containing nucleic acids disclosed herein, and optionally conjugating the binder to a therapeutic portion, a detectable portion, or a protein or nanoparticle that allows for an extended half-life.

[0080] Other scope, availability, and advantages of this disclosure will become apparent from the non-limiting detailed description given below. However, it should be understood that while these detailed descriptions indicate exemplary embodiments of this disclosure, they are provided by way of example only with reference to the accompanying drawings. Brief Description of the Drawings

[0081] Figures 1A-D: Illustrations showing the change in tumor volume over time 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 PBS.

[0082] Figure 1E: Histograms show the binding of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs to MDA-MB-453, MDA-MB-231, BxPC3, and A375 cells compared to the AR47A6.4.2 anti-TROP2 monoclonal antibody, analyzed by flow cytometry. The negative control is an sdAb binding to HEWL (NC).

[0083] Figure 2A: Illustration of in vivo dose / response studies of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs treated weekly in an MDA-MB-453-implanted NCG mouse tumor model at doses of 0.5 mg / kg, 2 mg / kg, 8 mg / kg, and 30 mg / kg ip. The negative control is PBS.

[0084] Figure 2B: Once-weekly (QW) administration of KD004 at a dose of 8 mg / kg ip in MDA-MB-453-implanted NCG mice (instruction manual page 7 / 226)Figure 2C: Illustration of the weekly in vivo treatment of large tumors in a tumor model. The negative control is PBS. Treatment was interrupted at approximately day 70 and replaced with PBS, and the mice in the negative control treatment were treated with KD004 at 8 mg / kg QW.

[0085] Figure 2D: Illustration of the antitumor effects of various treatment regimens of KD004 at a dose of 8 mg / kg at the indicated frequency in an MDA-MB-453-implanted NCG mouse tumor model. The negative control is PBS.

[0086] Figure 2D: Illustration of the antitumor effects of various treatment regimens of KD002 or KD005 at a dose of 0.1 mg / kg ip in an MDA-MB-453-implanted NCG mouse tumor model. The negative control is PBS.

[0087] Figure 2E: In vivo pharmacokinetic parameters of anti-TROP2.

[0088] Figure 3: A graph showing the change in tumor volume over time in MDA-MB-453-implanted NCG mice treated with KD004 at 8 mg / kg ip at indicated intervals in the presence or absence of human PBMCs. The negative control was PBS.

[0089] Figure 4A: A histogram showing the cell viability of MDA-MB-453 cells after treatment with 1 µM KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb for 12 days compared to AR47A6.4.2 anti-TROP2 monoclonal antibody. The negative control included sdAb bound to HEWL (NC) or untreated cells.

[0090] Figure 4B: Images of MDA-MB-453 cells at 4X resolution after 12 days of treatment with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb. Negative controls included sdAbs that bound HEWL (NC) or untreated cells.

[0091] Figure 5A: Histograms showing cell viability of MDA-MB-453 cells reflecting ADCC activity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs. Negative controls included untreated cells (without Ab), effectorless cells, target-free cells, or sdAbs that bound HEWL (NC). Comparisons included cells treated with human IgG1 anti-TROP2 antibodies generated from sequences derived from clones hTINA-1 and h7E6.

[0092] Figure 5B: In vivo testing of sdAb tumor growth inhibition (TGI) in NCG and SCID mice with different effector functional backgrounds and sc cells implanted with MDA-MB-231 or MDA-MB-453 cells. KD002 or KD004 was administered when the average tumor volume reached 100 mm3.

[0093] Figures 6A-B: Histograms of internalization of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb (Figure 6A) or AR47A6.4.2 (Figure 6B) in MDA-MB-453 cells after 4 or 24 hours. Negative controls included sdAbs bound to HEWL (NC) or culture medium alone.

[0094] Figure 6C: Illustration showing the internalization of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs in MDA-MB-453 cells. Internalization was assessed by conjugation of HCAbs with pH-sensitive probes and monitored by flow cytometry (Em. 560 nm).

[0095] Figure 6D: Confocal microscopic images of MDA-MB-453 cells showing KD002 and KD005 antibody binding and internalization, taken at 0, 6, and 24 hours. A 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 confocal fluorescence microscopy. Scale bar, 10 μm.

[0096] Figures 7A-D: The binding of KD001, KD002, KD003, KD004, KD005, KD006 or KD007 anti-TROP2 sdAbs to CHO (Figure 7A) or HEK parental cells (Figure 7C) or CHO expressing human TROP2 (Figure 7B) or HEK cells expressing human TROP2 (Figure 7D) was assessed by flow cytometry compared with negative controls (sdAbs that bind to HEWL (NC)) or monoclonal antibodies AR47A6.4.2, hTINA-1, h7E6 and hRS7 controls.

[0097] Figure 7E: The bar graph shows 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) proteins.

[0098] Figure 8: The graph shows the competitive assay of binding to recombinant human TROP2 performed on each of the His-tagged KD001 (Figure 8A), KD002 (Figure 8B), KD003 (Figure 8C), KD004 (Figure 8D), or KD005 (Figure 8E) and KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs. Instructions for Use, page 8 / 226, CN 121419995 A

[0099] Figure 9A: The graph shows the competitive assay of binding to recombinant human TROP2 between His-tagged KD004 and hRS7 or AR47A6.4.2 antibodies.

[0100] Figure 9B: A graph showing a competitive assay of binding to recombinant human TROP2 between His-tagged KD006 and KD007 anti-TROP2 sdAbs.

[0101] Figure 10A: A schematic diagram showing various constructs for locating the binding domains of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs.

[0102] Figure 10B: Images of immunoblotting performed with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs or with AR47A6.4.2 and hRS7 on the extracellular domain (ECD) and cysteine-deficient domain (CPD) of human TROP2. Negative controls included sdAbs that bound to HEWL (NC).

[0103] Figures 11A-E: Images of immunoblotting performed with KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb on different forms of recombinant human TROP2 (non-denatured, linear, reduced, and deglycosylated using peptidyl N-glycosidase F).

[0104] Figures 12A-12D: Graphs showing the binding of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAb on recombinant cynomolgus / 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 included sdAbs binding to HEWL. Positive controls included anti-TROP2 polyclonal antibodies.

[0105] Figure 12E: The table shows the affinity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs for human TROP2 and cynomolgus monkey TROP2 as shown by SPR.

[0106] Figure 12F: The table shows the affinity of KD001, KD002, KD003, KD004, or KD005 anti-TROP2 sdAbs for human TROP2, where ka, kd, and KD are indicated.

[0107] Figures 13A-13B: The histograms show the binding of 100 nM of the specified TROP2 antibody to HEK cells transfected with TROP2 constructs truncated with the various cysteine-rich domains as indicated.

[0108] Figure 13C: The graph shows tumor growth in a T.Tn xenograft model in SCID mice after weekly treatment with anti-TROP2 antibody at 8 mg / kg ip. Treatment with KD006 induced a 48.6% inhibition of tumor growth in the T.Tn xenograft model.

[0109] Figure 14A: Illustration of antibody-mediated phagocytic activity against MDA-MB-453 tumors by IgG1 or IgG4 versions of KD002 and KD004.

[0110] Figure 14B: Graph showing CD47 and TROP2 expression levels on MDA-MB-453, MDA-MB-231, and NCI-H292 cells as measured by staining with anti-CD47 (Biolegend, catalog number 323124) and anti-TROP2 (Biolegend, catalog number 363804) antibodies diluted to 1 / 100. The data are shown as fold changes in CD47 and TROP2 levels relative to isotype control staining.

[0111] Figures 14C-E: The figures illustrate the dose-response ADCP activities of the KD002 and KD004 TROP2 antibodies against MDA-MB-453 (Figure 14C), MDA-MB-231 (Figure 14D), and NCI-H292 (Figure 14E) cells. ADCP was measured using the Promega ADCP Assay Kit (Promega, catalog number CS314906). The test antibodies were serially diluted from 1.88 nM at a dilution of 1 / 2.2. The TROP2 antibodies KD002 and KD004 showed high levels of ADCP activity against MDA-MB-453 cells, while their ADCP activity against MDA-MB-231 and NCI-H292 cells was relatively low.

[0112] Figure 14F: The illustration shows 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. Measurements were performed at a 1 nM antibody concentration.

[0113] Figure 15A: The illustration shows the CD47 expression levels on MDA-MB-231 and NCI-H292 cells after CD47 knockdown using siRNA. CD47 siRNA and control siRNA were used for the knockdown assay (Thermofisher CD47 silencer, selected from pre-designed siRNA (catalog number 4392421) and a negative control siRNA (catalog number AM4635)).

[0114] Figures 15B-C: These illustrations show the dose-response ADCP activity of KD002 and KD005 TROP2 antibodies against MDA-MB-231 (Figure 15B) and NCI-H292 (Figure 15C) cells after CD47 knockdown. The antibodies were serially diluted from 2.1 nM at a 1 / 2.5 dilution.

[0115] Figure 15D: This illustration shows the effect of KD002 and KD005 TROP2 antibodies on CD47 expression after inhibition or non-inhibition (siNC) with siRNA (siCD47).KD005 and its corresponding IgG4 versions (KD017 and KD019) showed ADCP activity against MDA-MB-453, MDA-MB-231, and NCI-H292 cells. The assays were performed at an antibody concentration of 1 nM.

[0116] Figure 15E: This figure illustrates the in vivo efficacy of the TROP2-CD47 bispecific antibody in an MDA-MB-231 xenograft model in SCID mice. Mice were treated with KD004 at 8 mg / kg once weekly, and KD065 and KD066 were administered ip at the same molar concentration (11 mg / kg).

[0117] Figure 16A: This figure shows M1 and M2 macrophages differentiated from THP-1 cells after treatment with PMA and cytokines. Following PMA treatment, THP-1 cells showed upregulation of CD68. Following treatment with IFN-γ and LPS, M1 cells showed upregulation of CD38 and CD86. Following treatment with IL-4 and IL-13, M2 macrophages showed upregulation of CD206 and downregulation of CD38.

[0118] Figure 16B: Bar graphs show the percentage of M1 or M2 macrophages phagocytosed upon treatment with KD002, KD004, or KD005 at 10 nM, as indicated by gating on pHrodo-positive and Celltrace Violet-positive cells.

[0119] Figure 17A: Bar graphs show the binding of 25 nM TROP2 antibody to recombinant TROP2 protein as shown by ELISA using anti-IgG4 Fc secondary antibody.

[0120] Figure 17B: Graphs show the antitumor effects of KD002 and KD017 at a dose of 8 mg / kg iv in an MDA-MB-453-implanted NCG mouse tumor model. The negative control was PBS.

[0121] Figure 18A: This figure illustrates the dose-response ADCP activity of KD002 and KD005 TROP2 antibodies against MDA-MB-453 (top), MDA-MB-468 (middle), and T.Tn cells (bottom). ADCP was measured using the Promega ADCP Assay Kit (Promega, catalog number CS314906).

[0122] Figure 18B: This figure illustrates the antitumor activity of KD002 and KD005 in xenograft tumor models of MDA-MB-453 (top), MDA-MB-468 (middle), and TTn (bottom). Antibodies were tested weekly at 8 mg / kg per ip.

[0123] Figure 19A: F4 / 80 antibody against cells collected from COLO205, CAPAN1, MDA-MB-231, TTn, and MDA-MB-453.Micrographs of xenograft tumors from a xenograft model stained with mouse macrophages using IHC. The xenograft model was treated with TROP2 antibodies KD002 and KD005 at a dose level of 8 mg / kg. Tumors were collected at the end of the study.

[0124] Figure 19B: Microarray of human colon adenocarcinoma tumor tissue with KD067, KD068, or isotype control KD069 antibodies. Microarrays of various human tumor tissues were purchased from Novus Biologics (catalog number NBP2-42056) and stained with antibodies at 2 µg / ml.

[0125] Figure 19C: Microarray of human invasive ductal carcinoma of the breast tumor tissue with KD067, KD068, or isotype control KD069 antibodies. Microarrays of various human tumor tissues were purchased from Novus Biologics (catalog number NBP2-42052) and stained with antibodies at 2 µg / ml.

[0126] Figure 19D: Human head and neck tumor tissue microarray (H&N squamous cell carcinoma) with KD067, KD068 or isotype control KD069 antibodies. Multiple human tumor tissue microarrays were purchased from Novus Biologics (catalog number NBP2-42056) and stained with antibodies at 2 µg / ml.

[0127] Figure 19E: Human renal transitional cell carcinoma microarray with KD067, KD068 or isotype control KD069 antibodies. Multiple human tumor tissue microarrays were purchased from Novus Biologics (catalog number NBP2-42052) and stained with antibodies at 2 µg / ml. Specification 10 / 226 pages 18 CN 121419995 A

[0128] Figure 20A: X-ray crystal structure of the extracellular domain of the TROP2 protein with a translucent surface. The relative positions of the cysteine-rich domain (CRD), thyroglobulin domain (TY-1), and cysteine-deficient domain (CPD) are indicated.

[0129] Figure 20B: X-ray cocrystal of KD012 with human TROP2 (surface presentation shown). Contact regions on TROP2 are shown in dark gray on the surface of TROP2. Contacts between KD012 and TROP2 are mainly found in the CRD of TROP2 and in CDR3 and frame 2 of KD012. CDR and frame 2 are indicated by arrows.

[0130] Figure 20C: X-ray cocrystal structure of human TROP2 and KD012. KD102 is depicted as a band diagram. Selected residues on KD012 are shown, with interacting residues on TROP2 shown in dark gray.

[0131] Figure 20D: Amino acid residues involved in the interaction between KD012 and human TROP2 (within 4.5 Å; CDRs annotated according to IMGT numbers).

[0132] Figures 21A-21B: Dose-response ELISA binding of KD002 to a humanized variant of the recombinant human TROP2 protein. The TROP2 antibody was serially diluted from 200 nM to 0.00256 nM at a 1 / 5 dilution.

[0133] Figures 22A-22B: Dose-response ELISA binding of KD005 to a humanized variant of the recombinant human TROP2 protein. The TROP2 antibody was serially diluted from 200 nM to 0.00256 nM at a 1 / 5 dilution.

[0134] Figures 23A-23B: Bar graphs show the relative optical units of the humanized variant of KD002 compared to KD002 at 0.5 nM (Figure 23A) or 0.1 nM (Figure 23B), as measured using the Promega ADCP reporter assay.

[0135] Figures 24A-24B: Bar graphs showing the relative light units of KD005 and the humanized variant of KD005 at 0.5 nM (Figure 24A) or 0.1 nM (Figure 24B), as measured using the Promega ADCP reporter assay.

[0136] Figures 24C-24I: Dose-response ADCP assays 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).

[0137] Figures 25-25B: Bar graphs show the relative optical units of the humanized variant of KD002 compared to KD002 at 0.5 nM (Figure 25A) or 0.1 nM (Figure 25B), as measured using the Promega ADCC reporter assay.

[0138] Figures 26A-26B: Bar graphs show the relative optical units of KD005 and the humanized variant of KD005 at 0.5 nM (Figure 26A) or 0.1 nM (Figure 26B), as measured using the Promega ADCC reporter assay.

[0139] Figure 27: Alignment between the variable regions of KD001, KD002, KD003, KD004, and KD005.

[0140] Figure 28: Alignment and common sequence of the KD002 variant.

[0141] Figure 29: Alignment of KD002 variants selected for their ADCC and ADCP activities.

[0142] Figure 30: Alignment and common sequences of KD005 variants.

[0143] Figure 31: Alignment of KD005 variants selected for their binding activity.

[0144] Figure 32: Alignment of KD005 variants selected for their ADCP activity.

[0145] Figure 33: Alignment of KD005 variants selected for their ADCC activity.

[0146] Figure 34: Alignment of KD005 variants selected based on all characteristics.

[0147] Figure 35: KD005 resulted in complete tumor regression and a long-lasting response in a large Colo201 colorectal cancer xenograft model in SCID mice.

[0148] Figure 36: KD005 treatment resulted in tumor growth inhibition in an NCI-H2228 non-small cell lung cancer xenograft model in SCID mice.

[0149] Figure 37: KD005 treatment resulted in tumor growth inhibition in a Cal27 head and neck cancer xenograft model in SCID mice.

[0150] Figure 38: KD005 treatment resulted in tumor regression in a Colo201 colorectal cancer xenograft model in SCID mice. Treatment with the loss-of-effect LALAPG Fc mutant KD005 did not affect tumor growth. Instructions for Use, Page 11 / 226, 19 CN 121419995 A

[0151] Figure 39: KD005 treatment induced tumor regression in an MDA-MB-453 breast cancer xenograft model in NCG mice. KD005 treatment did not alter tumor growth in an MDA-MB-453 xenograft model implanted in mice lacking all Fcg receptors (FcgRko).

[0152] Figure 40A: Table of EC50 values ​​(nM) of KD005, KD001, KD132, KD134 binding to recombinant human TROP2 protein.

[0153] Figure 40B: Table of EC50 values ​​(nM) of KD001, KD132, KD134 binding to MDA-MB-453 and COLO 201 cell lines.

[0154] Figure 40C: Table of EC50 values ​​(nM) of ADCP activity of KD001, KD132, and KD134 in MDA-MB-453 and COLO 201 cells.

[0155] Figures 41A-D: Tumor regression induced in MDA-MB-453 (A-B) and COLO201 (C-D) xenograft models by treatment with KD133, KD135, and KD136 compared to the positive control KD005 and the negative isotype control with specificity for egg white lysozyme (HEWL).

[0156] Figure 42A: Table of EC50 values ​​(nM) of KD005, KD137, KD138, and KD139 binding to recombinant human TROP2 protein.

[0157] Figure 42B: Table of EC50 values ​​(nM) of KD005, KD138, KD139 binding to MDA-MB-453 and COLO 201 cell lines.

[0158] Figure 42C: Table of EC50 values ​​(nM) of ADCP activity of KD005, KD138, KD139 in MDA-MB-453 and COLO 201 cells.

[0159] Figures 43A-D: Comparison with positive control KD005 and negative isotypes with specificity for egg white lysozyme (HEWL).In contrast, treatment with KD137, KD138, and KD139 induced tumor regression in MDA-MB-453 (A-B) and COLO201 (C-D) xenograft models.

[0160] Figures 44A-B: Binding of KD142 and KD143 to recombinant human TROP2 protein (A), and binding to MDA-MB-453 cells and NCI-H292 cells (B).

[0161] Figure 45: Comparison of heavy chain variable regions in Tables 15E and 15F. Detailed Description

[0162] Definitions

[0163] Unless otherwise indicated herein or obviously contradicted by the context, the terms “an”, “a”, and “the”, and similar references used in the context of describing embodiments (especially in the context of the claims) shall be understood to cover both singular and plural.

[0164] Unless specifically stated or obvious from the context, the term “or” as used herein shall be understood as inclusive and covers both “or” and “and”.

[0165] The term “and / or” as used herein shall be considered as specifically disclosing each of the specified features or components (in the presence or absence of another feature or component).

[0166] Unless otherwise indicated, the terms “comprising,” “having,” “including,” and “containing” shall be understood as open-ended terms (i.e., meaning “including but not limited to”). The term “composed of” is considered closed-ended.

[0167] As used herein, the term “TROP2” refers to the TROP2 protein or a polypeptide containing the TROP2 amino acid sequence. The term “TROP2” covers the human TROP2 protein and any protein having at least 80% identity with the human TROP2 protein.

[0168] The term “anti-TROP2 antibody” refers to any form of antibody that binds to the TROP2 protein (e.g., the TROP2 antigen) or a portion thereof, including but not limited to “anti-TROP2 single-domain antibody”.

[0169] The term "binding agent" refers to a molecule that comprises a polypeptide moiety (e.g., a polypeptide chain) and is capable of specifically binding an antigen, or that comprises a domain (e.g., an antigen-binding domain) capable of specifically binding an antigen.

[0170] The term "antibody" is used in the broadest sense and covers a wide variety of antibody forms and structures, including any immunoglobulin, monoclonal antibody, polyclonal antibody, bivalent antibody, monovalent antibody, bispecific antibody, multispecific (multispecific) antibody, conventional antibody, single-domain antibody, single-chain antibody, heavy-chain antibody, nanobody, full-length antibody, humanized antibody, chimeric antibody that binds a specific antigen, and any antigen-binding fragment exhibiting the desired antigen-binding activity. Antibodies can be naturally occurring (natural) or products of recombinant technologies.

[0171] As used herein, the terms "single-domain antibody" and "heavy-chain antibody" are used interchangeably. The term "single-domain antibody" includes naturally occurring antibodies.Naturally occurring single-domain antibodies or heavy-chain antibodies, and single-domain antibodies in which a naturally occurring constant region is replaced by a dimerizing domain disclosed herein; and variants, such as humanized or chimeric single-domain antibodies. When a single-domain antibody has a dimerizing domain, it typically consists of two polypeptide chains.

[0172] "Naturally occurring single-domain antibodies" include antibodies produced by camels (cameloid antibodies) or sharks. "Naturally occurring single-domain antibodies" also encompass antibodies produced by transgenic animals modified to express heavy-chain antibodies. Exemplary embodiments of transgenic animals are provided in International Application No. PCT / CA2021 / 050951, filed July 21, 2021, and published on January 20, 2022, under WO2022 / 011457, the entire contents of which are incorporated herein by reference.

[0173] It should be understood herein that 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”, “KD030”, “KD031”, “KD032”, and “KD033” are used interchangeably with the terminology used in this document. "KD034", "KD035", "KD036", "KD037", "KD038", "KD039", "KD040", "KD041", "KD042", "KD043", "KD044", "KD045", "KD046", "KD047", "KD048", "KD049", "KD050", "KD051", "KD052", "KD053", "KD054", "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", "KD096", "KD097", "KD098", "KD099", "KD100", "KD101", "KD102", "KD103", "KD104", "KD105", "KD106", "KD107", "KD108", "KD109", "KD110", "KD111", "KD112", "KD113", "KD114", "KD115", "KD116", "KD117", "KD118", "KD119", "DK120", "KD121", "KD122", "KD123", "KD124", "KD125", "KD126", Any one of “KD127”, “KD128”, “KD129”, “KD130”, “KD131”, “KD132”, “KD133”, “KD134”, “KD135”, “KD136”, “KD137”, “KD138”, “KD139”, “KD140”, or “KD141” refers to a single-domain antibody containing two heavy chains, each having the corresponding heavy chain amino acid sequence shown in any one of Tables 15A to 15K, Table 16, Table 18 or Table 19, or each having the corresponding variable region amino acid sequence shown in any one of Tables 15A to 15K, Table 16, Table 18 or Table 19.

[0174] The terms “KD065” or “KD066” refer to a binder comprising two chains, each having the corresponding amino acid sequence of the polypeptide chain shown in SEQ ID NO: 150 or SEQ ID NO: 151, or each having the corresponding variable region amino acid sequence of the polypeptide chain. In some embodiments, either KD065 or KD066 may have the corresponding variable region amino acid sequence of KD065 or KD66 and hinge and / or dimerizing domains (e.g., CH2 and / or CH3) different from those of KD065 or KD66.

[0175] As used herein, the term “subject in need” means a subject suffering from or suspected of having a condition or disease associated with TROP2, TROP2 expression (e.g., expression in tissues, cells, or serum), or TROP2 overexpression. The term "subject in need" also refers to a subject suffering from a condition or disease that could benefit from treatment with a TROP2-targeting binder, or a subject suspected of having such a condition or disease. "Subject in need" includes subjects with cancer or suspected of having cancer.

[0176] For the purposes of this invention, the term "treatment" refers to both therapeutic treatment and preventative or tumor prevention measures. Subject in needSubjects to be treated include subjects who already have the condition and subjects who are susceptible to the condition; and subjects who will prevent the condition. Therefore, subjects in need of treatment include subjects who already have cancer and subjects who are susceptible to cancer or will manage cancer.

[0177] The term “natural” as used in the context of a sequence refers to a sequence that exists in nature.

[0178] The term “about” or “approximately” with respect to a given value means taking into account variations in that value. In some embodiments, the term “about” or “approximately” will generally mean a range within + / - 10%, + / - 5%, + / - 4%, + / - 3%, + / - 2%, or + / - 1% of a given value or range.

[0179] In this document, it should be understood that expressions referring to a range of values ​​in the form of, for example, “from A to B” include each single value and any subranges that constitute and include such ranges. For example, the expression "from 1 to 10" includes subranges, such as, but not limited to, "from 2 to 10", "from 2 to 9", "from 3 to 6", "from 5 to 7" and any single value contained therein, and includes 1 and 10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0180] In this document, it should be understood that the term "at least" with respect to a given value means including both the value and the superordinate 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%".

[0181] As used herein, the term “humanized” means that a binder (e.g., 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.

[0182] As used herein, the expression “fully humanized frame region” means that the amino acid sequence of all frame regions of a given binder (e.g., an antibody or antigen-binding fragment) is identical to the amino acid sequence of a variable region of a human antibody (e.g., a variable region sequence of a human germline antibody).

[0183] As used herein, the expression “partially humanized” means that one or more of the binder (e.g., 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.The amino acid sequences of multiple or all of the frame regions are not identical to the amino acid sequences of the variable regions of human antibodies (e.g., the variable regions of human germline antibodies (e.g., the sequence of the variable region of human germline antibodies)).

[0184] In some cases, humanized, fully humanized, or partially humanized binders (e.g., antibodies or antigen-binding fragments) may contain CDR or CDR amino acid residues of the variable regions of human antibodies (e.g., the sequence of the variable region of human germline antibodies).

[0185] Thus, “partially humanized frame regions” encompass, for example, but not limited to, human frame regions containing reversion mutations that reintroduce the original amino acid at its original position. “Partially humanized frame regions” also encompass, for example, but not limited to, human frame regions in which one or more amino acid residues have been substituted, added, or deleted.

[0186] As used herein, the term “affinity” refers to the strength of the non-covalent interaction between the binder or its antigen-binding domain and the antigen. “Affinity” is expressed, for example, by the KD value, which is the ratio (koff / kon) of the dissociation rate to the binding rate when the binding between the antigen and the binder (e.g., an antibody, such as a single-domain antibody) reaches equilibrium. Affinity can be determined using any conventional techniques known in the art, including but not limited to surface plasmon resonance methods, micro-thermophoresis methods, HPLC-MS methods, and flow cytometry (e.g., FACS) methods. Antibodies typically have a KD value ≤10⁻⁶ M (e.g., ≤5×10⁻⁷ M, ≤2×10⁻⁷ M, ≤10⁻⁷ M, ≤5×10⁻⁸ M, ≤2×10⁻⁸ M, ≤10⁻⁸ M, ≤5×10⁻⁹ M, ≤4×10⁻⁹ M, ≤3×10⁻⁹ M, ≤2×10⁻⁹ M, or ≤10⁻⁹ M and any value ≤10⁻⁶). Preferably, the antibody has a KD value in the nanomolar range or lower (e.g., ≤ 9×10⁻⁹ M, ≤ 8×10⁻⁹ M, ≤ 7×10⁻⁹ M, ≤ 6×10⁻⁹ M, ≤ 5×10⁻⁹ M, ≤ 4×10⁻⁹ M, ≤ 3×10⁻⁹ M, ≤ 2×10⁻⁹ M, ≤ 1×10⁻⁹ M or lower, e.g., ≤ 1×10⁻¹⁰ M, ≤ 1×10⁻¹¹ M, ≤ 1×10⁻¹² M). Even more preferably, the antibody has a KD value in the picomolar range or lower (e.g., ≤ 9 × 10⁻¹² M, ≤ 8 × 10⁻¹² M, ≤ 7 × 10⁻¹² M, ≤ 6 × 10⁻¹² M, ≤ 5 × 10⁻¹² M, ≤ 4 × 10⁻¹² M, ≤ 3 × 10⁻¹² M, ≤ 2 × 10⁻¹² M, ≤ 1 × 10⁻¹² M or lower).

[0187] The terms “specific binding” or “specific binding” as used herein refer to the binding between two molecules (e.g., antibodies).A non-random binding reaction between the antibody and the antigen. Specific binding can be characterized by binding affinity, with a KD value ≤10⁻⁶ M (e.g., ≤5×10⁻⁷ M, ≤2×10⁻⁷ M, ≤10⁻⁷ M, ≤5×10⁻⁸ M, ≤2×10⁻⁸ M, ≤10⁻⁸ M, ≤5×10⁻⁹ M, ≤4×10⁻⁹ M, ≤3×10⁻⁹ M, ≤2×10⁻⁹ M, or ≤10⁻⁹ M and any value ≤10⁻⁶) indicating specific binding between a binder (e.g., an antibody, such as a single-domain antibody) and TROP2 (e.g., human TROP2).

[0188] As used herein, the terms “competitive binding” and “competing 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.

[0189] As used herein, the term “epitope” refers to a specific group of atoms or amino acid residues on an antigen to which an antibody (e.g., a single-domain antibody) binds. If two antibodies exhibit competitive binding to an antigen, they may bind the same or closely related epitopes within the antigen. Epitopes may be linear or conformational (i.e., including spaced amino acid residues). For example, if an antibody or antigen-binding fragment blocks the binding of a reference antibody (e.g., a single-domain antibody) to an antigen by at least 85%, or at least 90%, or at least 95%, the antibody or antigen-binding fragment may be considered to bind 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 antibody of the present invention for binding to human TROP2.

[0190] As used herein, the term “sequence identity” refers to the degree of identity between two nucleic acid or two amino acid sequences when compared optimally and when mutations such as substitution, insertion, or deletion are appropriate. Sequence identity may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0191] The term "antigen-binding fragment" as used in the expressions "antibody or antigen-binding fragment thereof" or "single-domain antibody or antigen-binding fragment thereof" refers to a fragment of an antibody or single-domain antibody that covers an antigen-binding domain and may or may not include other portions of the antibody or single-domain antibody, such as amino acid residues in the hinge region, amino acid residues in the constant region, or portions of the Fc region. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact antibody (e.g., a single-domain antibody).

[0192] The antigen-binding fragment of a single-domain antibody includes, for example, a CDR and a sequence covering the CDR, such as the entire variable region or a portion thereof, or the entire heavy chain or a portion thereof, and may include or exclude other portions of the antibody.

[0193] The term "antigen-binding domain" refers to a portion of an antibody involved in antigen binding and includes, for example, one or more CDRs, one or more frame regions (FRs), or the entire variable region. In the case of its single-domain antibody, the term "antigen-binding domain" refers to a portion of a single-domain antibody involved in antigen binding and includes, for example, one or more of CDR1 (CDRH1), CDR2 (CDRH2), or CDR3 (CDRH3), one or more frame regions FR1, FR2, FR3, FR4, or the entire variable region (VH or VHH). In the case of its natural antibody, the term "antigen-binding domain" refers to a portion of the natural antibody involved in antigen binding and includes, for example, one or more of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, one or more light chain or heavy chain framework regions FR1, FR2, FR3, FR4, or one or both entire variable regions (heavy chain variable region (VH) and / or light chain variable region specification 15 / 226 page 23 CN 121419995 A (VL)).

[0194] As used herein, the terms "CDRH1", "CDRH2", and "CDRH3" refer to CDR1, CDR2, or CDR3 of the antibody heavy chain, respectively.

[0195] TROP2 Antigen

[0196] The antibody or antigen-binding fragment of this disclosure can be obtained, for example, by immunizing an animal with the TROP2 antigen.

[0197] Alternatively, the antibody or antigen-binding fragment of this disclosure can be obtained from an antibody library.

[0198] Binders that bind to TROP2 can be identified by computer simulation, in vitro and / or in vivo methods. Several in vitro methods rely on the binding of a candidate antibody or its antigen-binding fragment to an antigen. Thus, anti-TROP2 antibodies or their antigen-binding fragments can be identified by methods involving binding to the TROP2 antigen disclosed herein and / or by using the assays disclosed herein.

[0199] In one embodiment, the TROP2 antigen comprises a human TROP2 protein (e.g., SEQ ID NO:39) or a TROP2 homolog or variant thereof, the sequence of which is at least 80% identical to that of the human TROP2 protein or a portion thereof (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.

[0200] In one embodiment, the TROP2 antigen is human TROP2, a TROP2 homolog, a TROP2 variant having a sequence at least 80% identical to that of human TROP2, or a fragment thereof.

[0201] In one exemplary embodiment, the TROP2 homolog is cynomolgus monkey TROP2.

[0202] In another exemplary embodiment, the TROP2 homolog is rhesus monkey TROP2.

[0203] In some embodiments, the TROP2 antigen contains the extracellular domain of TROP2 (e.g., human TROP2, TROP2 homologs, or variants thereof).

[0204] In some embodiments, the TROP2 antigen contains the extracellular domain (ECD) of TROP2 (as shown by amino acids 27 to 274 of TROP2 (e.g., SEQ ID NO:39 or SEQ ID NO:40), see Goldenbenberg et al., 2018, Ikeda, M. et al., 2015, the entire contents of which are incorporated herein by reference).

[0205] In some embodiments, the TROP2 antigen contains epitopes common to human TROP2 and cynomolgus monkey TROP2.

[0206] In some embodiments, the TROP2 antigen contains epitopes similar to those of human TROP2 and cynomolgus monkey TROP2.

[0207] In some embodiments, the TROP2 antigen comprises amino acid residues 27-274 of human TROP2 or a TROP2 homolog, or fragments thereof. Therefore, the TROP2 antigen may comprise a polypeptide comprising a human TROP2 amino acid sequence composed of amino acid residues 27-274, or fragments thereof.

[0208] In other embodiments, the TROP2 antigen comprises amino acid residues 27-146 of human TROP2 or a TROP2 homolog, or fragments thereof. Therefore, the TROP2 antigen may comprise a polypeptide comprising a human TROP2 amino acid sequence composed of amino acid residues 27-146, or fragments thereof.

[0209] In yet another embodiment, the TROP2 antigen comprises amino acid residues 27-73 of human TROP2 or a TROP2 homolog, or fragments thereof. Therefore, the TROP2 antigen may comprise a polypeptide comprising a human TROP2 amino acid sequence composed of amino acid residues 27-73, or fragments thereof.

[0210] According to this disclosure, the TROP2 antigen may comprise at least 10 amino acid residues of human TROP2.

[0211] More specifically, the TROP2 antigen is a fragment of at least 10 amino acid residues of the human TROP2 amino acid sequence consisting of amino acid residues 27-146.

[0212] According to this disclosure, the TROP2 antigen comprises 10 to 110 amino acid residues of the human TROP2 amino acid sequence consisting of amino acid residues 27-146, including, for example, 10 to 100 amino acid residues, 10 to 90 amino acid residues, 10 to 80 amino acid residues, etc. (Specification 16 / 226 pages 24 CN 121419995 A)Amino acid residues, 10 to 70 amino acid residues, 10 to 60 amino acid residues, 10 to 50 amino acid residues, 10 to 40 amino acid residues, 10 to 30 amino acid residues, 10 to 29 amino acid residues, 10 to 28 amino acid residues, 10 to 27 amino acid residues, 10 to 26 amino acid residues, 10 to 25 amino acid residues, 10 to 24 amino acid residues, 10 to 23 amino acid residues, 10 to 22 amino acid residues, 10 to 21 amino acid residues, 10 to 20 amino acid residues, 10 to 19 amino acid residues, 10 to 18 amino acid residues, 10 to 17 amino acid residues, 10 to 16 amino acid residues, 10 to 15 amino acid residues, 10 to 14 amino acid residues, 10 to 13 amino acid residues, 10 to 12 amino acid residues, 10 to 11 amino acid residues.

[0213] For example, the TROP2 antigen contains at least 10 amino acid residues of the human TROP2 amino acid sequence consisting of amino acid residues 27-73.

[0214] According to this disclosure, the TROP2 antigen comprises 10 to 45 amino acid residues of the human TROP2 amino acid sequence consisting of amino acid residues 27-73, including, for example, 10 to 40 amino acid residues, 10 to 35 amino acid residues, 10 to 30 amino acid residues, 10 to 29 amino acid residues, 10 to 28 amino acid residues, 10 to 27 amino acid residues, 10 to 26 amino acid residues, 10 to 25 amino acid residues, 10 to 24 amino acid residues, 10 to 23 amino acid residues, 10 to 22 amino acid residues, 10 to 21 amino acid residues, 10 to 20 amino acid residues, 10 to 19 amino acid residues, 10 to 18 amino acid residues, 10 to 17 amino acid residues, 10 to 16 amino acid residues, 10 to 15 amino acid residues, 10 to 14 amino acid residues, 10 to 13 amino acid residues, 10 to 12 amino acid residues, and 10 to 11 amino acid residues.

[0215] In some cases, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 27-56 of human TROP2 or fragments thereof.

[0216] In other cases, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 27-66 of human TROP2 or fragments thereof.

[0217] In still other cases, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 47-56 of human TROP2 or fragments thereof.

[0218] In still other cases, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 60-66 of human TROP2 or fragments thereof.

[0219] In still other cases, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 47-66 of human TROP2 or fragments thereof.Human TROP2 amino acid sequence.

[0220] For example, the TROP2 antigen comprises a human TROP2 amino acid sequence consisting of amino acid residues 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.

[0221] In some cases, the TROP2 antigen may not include the cysteine-deficient domain (CPD) of TROP2.

[0222] Exemplary and non-limiting embodiments of the TROP2 antigen are provided in SEQ ID NO:164.

[0223] Therefore, in some embodiments, the binder of this disclosure or the antigen-binding domain of the binder is capable of binding human TROP2.

[0224] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder is capable of binding both human TROP2 and cynomolgus monkey TROP2.

[0225] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder preferentially binds human TROP2 rather than mouse TROP2, or has a higher affinity for human TROP2 than for mouse TROP2.

[0226] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder preferentially binds human TROP2 rather than rat TROP2, or has a higher affinity for human TROP2 than for rat TROP2. Specification 17 / 226 pages 25 CN 121419995 A

[0227] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder binds amino acid residues common to the extracellular domains of human TROP2 and cynomolgus monkey TROP2.

[0228] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder binds amino acid residues not present in the extracellular domain of mouse TROP2.

[0229] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder binds amino acid residues not present in the extracellular domain of rat TROP2.

[0230] In some embodiments, the binder of this disclosure or the antigen-binding domain of the binder binds amino acid residues present in the extracellular domains of human TROP2, cynomolgus monkey TROP2, mouse TROP2, and rat TROP2.

[0231] Therefore, the binder or the antigen-binding domain of the binder is capable of binding human TROP2 and cynomolgus monkey TROP2.

[0232] In an exemplary embodiment, the binder or the antigen-binding domain of the binder is capable of binding the denatured form of human TROP2.

[0233] In another exemplary embodiment, the binder or the antigen-binding domain of the binder is capable of binding to the reduced form of human TROP2.

[0234] In other exemplary embodiments, the binder or the antigen-binding domain of the binder is capable of binding to the deglycosylated form of human TROP2.

[0235] Binder

[0236] Exemplary embodiments of the binders of this disclosure include the polypeptide chains disclosed herein.

[0237] Exemplary embodiments of the binders of this disclosure include antibodies or antigen-binding fragments thereof.

[0238] Thus, exemplary embodiments of the binders include, but are not limited to, natural antibodies or antigen-binding fragments thereof, or single-domain antibodies or antigen-binding fragments thereof, or variants thereof, such as humanized, chimeric, or human antibodies or antigen-binding fragments thereof, or humanized or chimeric single-domain antibodies or antigen-binding fragments thereof.

[0239] In particular, single-domain antibodies or antigen-binding fragments thereof disclosed herein and binders comprising one or more antigen-binding domains of one or more single-domain antibodies disclosed herein are considered.

[0240] This disclosure also covers single-domain antibodies that compete with one or more single-domain antibodies having the amino acid sequences disclosed herein for binding to the TROP2 antigen.

[0241] 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 immune cell modulators.

[0242] Thus, 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.

[0243] Further 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.

[0244] Still further exemplary embodiments of the binders of this disclosure include immune cell modulators comprising one or more antigen-binding domains of one or more monoantibodies disclosed herein.

[0245] This disclosure also covers binders, including, for example, but not limited to, antibodies or antigen-binding fragments thereof, and single-domain antibodies or antigen-binding fragments thereof that compete with single-domain antibodies having the amino acid sequences disclosed herein.

[0246] This disclosure therefore relates to a binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains comprises an antigen-binding fragment thereof capable of binding trophoblast cell surface antigen-2 (TROP2).

[0247] In some embodiments, the binder of this disclosure is capable of binding the extracellular domain (ECD) of TROP2. Specification 18 / 226 pages 26 CN 121419995 A

[0248] In some embodiments, the binder of this disclosure is capable of binding the cysteine-rich structure of TROP2.

[0249] In some embodiments, the binder of this disclosure is capable of binding an epitope containing an amino acid of the extracellular domain of TROP2.

[0250] In some embodiments, the binder of this disclosure is capable of binding an epitope containing an amino acid of the cysteine-rich domain of TROP2 and / or the thyroglobulin type 1 domain.

[0251] In some embodiments, the binder of this disclosure is capable of binding an epitope comprising an amino acid residue contained in amino acid sequence 27-274 of TROP2 or a fragment thereof.

[0252] In some embodiments, the binder of this disclosure is not capable of binding the cysteine-deficient domain (CPD) of TROP2.

[0253] In some embodiments, the binder of this disclosure is not capable of binding amino acids 146 to 274 of TROP2.

[0254] In some embodiments, the binder comprises an antigen-binding domain of a single-domain antibody capable of binding TROP2.

[0255] In some embodiments, the binder comprises a single-domain antibody.

[0256] In other embodiments, the binder is a single-domain antibody.

[0257] In some embodiments, the antigen-binding domain of the binder comprises a CDR of the single-domain antibody.

[0258] In some embodiments, the antigen-binding domain of the binder is a variable region of the single-domain antibody. In some cases, the variable region may be a camelid VH or VHH. In some cases, the variable region includes, for example, a camelid CDR and a frame region, such as a camelid frame region, a humanized frame region, a human frame region, or a mouse frame region.

[0259] In some embodiments, the binder comprises a variable region comprising a camelid-derived CDR and a humanized frame region.

[0260] In other embodiments, the binder comprises a variable region comprising a camelid-derived CDR and a human frame region.

[0261] In some embodiments, the binder comprises two heavy chain variable regions of the single-domain antibody, each heavy chain variable region comprising a dimerizing domain, thereby forming a dimer.

[0262] In some embodiments, the humanization level of a single-domain antibody can be calculated based on sequence identity with the closest human VH germline gene. Therefore, the percentage of humanization score is based on frame regions 1-3 (FR1, FR2, FR3) and CDR1 and CDR2. According to one embodiment, the sequences of CDR3 and FR4 are not used to assess the humanization level of the single-domain antibody disclosed herein.

[0263] In an exemplary embodiment, the humanization level of the antigen-binding domain is at least 80%.

[0264] In other exemplary embodiments, the humanization level of the variable region of the single-domain antibody of this disclosure is at least 80%.

[0265] In some embodiments, the binder of this disclosure may comprise one or more polypeptide chains, each polypeptide...The chain independently comprises the amino acid sequence of Formula I from N- to C-terminus:

[0266] X-[(Aba)-(Lb)]m-(DD)-[(Lc)-(Abd)]n-Y

[0267] where m is an integer of 0, 1, 2 or greater than 2;

[0268] where n is an integer of 0, 1, 2 or greater than 2;

[0269] where m and n are not both 0;

[0270] where Aba and Abd each represent an antigen-binding domain, wherein at least one antigen-binding domain is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2) or antigen-binding domain 3 (ABD3); Specification 19 / 226 Page 27 CN 121419995 A

[0271] where X or Y may be present or absent independently, and comprises an amino acid sequence;

[0272] where Lb and Lc each independently comprise one or more linkers; and

[0273] Wherein DD represents a dimerization domain.

[0274] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises Formula II: X-(Aba1)-(Lb1)-(DD)-(Lc1)-(Abd1)-Y (Formula II).

[0275] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises Formula III: X-(Aba1)-(Lb1)-(DD)-(Lc1)-(Abd1)-(Lc2)-(Abd2)-Y (Formula III).

[0276] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises Formula IV: X-(Aba1)-(Lb2)-(Aba2)-(Lb1)-(DD)-(Lc1)-(Abd1)-Y (Formula IV).

[0277] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises formula V: X-(Aba1)-(Lb2)-(Aba2)-(Lb1)-(DD)-(Lc1)-(Abd1)-(Lc2)-(Abd2)-Y (Formula V).

[0278] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises formula VI: X-(Aba1)-(Lb2)-(Aba2)-(Lb1)-(DD)-(Lc1)-(Abd1)-(Lc2)-(Abd2)-(Lc3)-(Abd3)-Y (Formula VI).

[0279] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises formula VII: X-(Aba1)-(Lb3)-(Aba2)-(Lb2)-(Aba3)-(Lb1)-(DD)-(Lc1)-(Abd1)-(Lc2)-(Abd2)-Y (Formula VII)VII).

[0280] In some embodiments, the binder comprises one or more polypeptide chains, wherein at least one polypeptide bond comprises Formula VIII: X-(Aba1)-(Lb3)-(Aba2)-(Lb2)-(Aba3)-(Lb1)-(DD)-(Lc1)-(Abd1)-(Lc2)-(Abd2)-(Lc3)-(Abd3)-Y (Formula VIII).

[0281] In some embodiments, Aba1, Aba2, Aba3, Abd1, Abd2, or Abd3 each independently comprises an antigen-binding domain.

[0282] In some embodiments, Lb1 comprises one or more linkers and / or a hinge region of an antibody or its antigen-binding fragment. In some embodiments, the hinge region is a hinge region of a naturally occurring antibody. In exemplary embodiments, the hinge may comprise an amino acid sequence or a portion thereof shown in SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:481. In other embodiments, the hinge region is a mutated or synthetic hinge region as 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%, or at least 99% identical to the hinge of a naturally occurring antibody. In one exemplary embodiment, the mutated or synthetic hinge may have the sequence shown in SEQ ID NO:481 or a portion thereof.

[0283] In some embodiments, the hinge region may be replaced by a linker or spacer. In some embodiments, the hinge region, linker, or spacer is optional.

[0284] In some embodiments, Lb2, Lb3, Lc1, Lc2, and Lc3 each independently contain one or more linkers disclosed herein.

[0285] Dimerization Domain and Linker

[0286] According to this disclosure, the binder may consist of two polypeptide chains, each polypeptide chain containing one or more antigen-binding domains and a dimerization domain. The one or more antigen-binding domains may be separated by a linker.

[0287] Exemplary embodiments of the dimerizing domain and the linker are provided in PCT / CA2020 / 051753, filed December 18, 2020, and disclosed on June 24, 2021, under WO2021 / 119832A1, the entire contents of which are incorporated herein by reference.

[0288] In some embodiments, the dimerizing domain allows the formation of homodimers. Specification 20 / 226 pages 28 CN 121419995 A

[0289] In some embodiments, the dimerizing domain allows the formation of heterodimers.

[0290] In some embodiments, the dimerizing domain comprises an amino acid sequence adjacent to the amino acid sequence of the antigen-binding domain.

[0291] In some embodiments, the dimerizing domain includes an immunoglobulin dimerizing domain. Other dimerizing domains known to those skilled in the art are considered herein, including leucine zippers, etc.

[0292] In some embodiments, the dimerizing domain includes an IgG, IgM, IgA, IgD, or IgE dimerizing domain (derived from human or animal IgG, IgM, IgA, IgD, or IgE).

[0293] In some embodiments, the dimerizing domain comprises a constant region of an antibody or a portion thereof.

[0294] In some embodiments, the constant region or a portion thereof is derived from the IgG1, IgG2, IgG3, or IgG4 heavy chain or a combination thereof. For example, the constant region or a portion thereof comprises the amino acid sequence of the IgG1, IgG2, IgG3, or IgG4 heavy chain or a combination thereof. In some cases, 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.

[0295] In some embodiments, the constant region or a portion thereof is derived from the human IgG1 heavy chain.

[0296] In some embodiments, the constant region or a portion thereof is derived from the human IgG2 heavy chain.

[0297] In some embodiments, the constant region or a portion thereof is derived from the human IgG3 heavy chain.

[0298] In some embodiments, the constant region or a portion thereof is derived from the human IgG4 heavy chain.

[0299] In some embodiments, the binder comprises the Fc region of an antibody or a portion thereof.

[0300] In some embodiments, the binder comprises an Fc region or a portion thereof containing Fc modification or not containing Fc modification.

[0301] In an exemplary embodiment, the binder may comprise an Fc or a portion thereof capable of binding to Fc receptors on monocytes and / or macrophages.

[0302] In another exemplary embodiment, the binder may comprise an Fc or a portion thereof capable of binding to Fc receptors on CD68+ macrophages.

[0303] In other embodiments, the binder comprises a modified or mutated Fc region or a portion thereof. For example, the Fc region or a portion thereof may be modified or mutated to alter one or more characteristics of the binder. In an exemplary embodiment, the Fc region or a portion thereof is not glycosylated. In other exemplary embodiments, the Fc region or a portion thereof includes one or more of the Fc modifications listed in Table A.

[0304] In other exemplary embodiments, the Fc region or a portion thereof is altered or mutated to increase ADCC activity. For example, fucosylation (e.g., N297 according to the EU numbering system) can lead to increased FcRgII binding on NK cells and can significantly 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 a fucosylated Fc region or a portion thereof. In yet another embodiment, the Fc region of the binder...Or a portion thereof lacks fucose residues. In another embodiment, the Fc region of the binder or a portion thereof lacks core fucose residues. In a further embodiment, the Fc region of the binder or a portion thereof completely lacks core fucose residues.

[0305] In some embodiments, the dimerizing domain comprises a CH3 domain.

[0306] In some embodiments, the CH3 domain is a native CH3 domain or a mutated CH3 domain.

[0307] In some embodiments, the dimerizing domain also comprises a CH2 domain.

[0308] In some embodiments, the dimerizing domain comprises the CH3 domain of the antibody. The dimerizing domain may also comprise the CH2 domain of the antibody.

[0309] In some embodiments, the CH2 domain is a native CH2 domain or a mutated CH2 domain.

[0310] In some embodiments, the dimerizing domain comprises a native CH2 domain and a native CH3 domain.

[0311] In some embodiments, the dimerizing domain comprises a native CH2 domain and a mutated CH3 domain. Specification 21 / 226 pages 29 CN 121419995 A

[0312] In some embodiments, the dimerizing domain comprises a mutated CH2 domain and a natural CH3 domain.

[0313] In some embodiments, the dimerizing domain comprises a mutated CH2 domain and a mutated CH3 domain.

[0314] In some embodiments, the CH3 domain may have the sequence shown in SEQ 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.

[0315] In other embodiments, the CH3 domain may have the sequence shown in SEQ 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.

[0316] In some embodiments, the CH2 domain may have the sequence shown in SEQ 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.

[0317] In some embodiments, the CH2 domain may have the sequence shown in SEQ 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.

[0318] In some embodiments, the dimerizing domain may comprise a CH3 domain and a CH2 domain, wherein the CH3 domain has the sequence shown in SEQ 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, and the CH2 domain has the sequence shown in SEQ ID NO:37 or the sequence shown in SEQ ID NO:42.The sequence or the sequence thereof is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical.

[0319] In some embodiments, the dimerizing domain may comprise a CH3 domain and a CH2 domain, wherein the CH3 domain has the sequence shown in SEQ ID NO:43 or the sequence thereof is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical, and the CH2 domain has the sequence shown in SEQ ID NO:37 or the sequence shown in SEQ ID NO:42 or the sequence thereof is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical.

[0320] In some embodiments, the dimerizing domain may comprise the sequence shown in SEQ ID NO:212 or the sequence thereof is at least 80% identical, at least 85% identical, at least 90% identical, or at least 99% identical.

[0321] In some embodiments, the dimerizing domain may comprise the sequence shown in SEQ ID NO:506 or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical.

[0322] In some embodiments, the dimerizing domain may comprise the sequence shown in SEQ ID NO:214 or a sequence that is at least 80%, at least 85%, at least 90%, or at least 99% identical.

[0323] In some embodiments, the dimerizing domain comprises a hinge region, such as the hinge region of a naturally occurring antibody or its antigen-binding fragment, or a mutated or synthetic hinge region.

[0324] In some embodiments, the dimerizing domain comprises a peptide linker.

[0325] In some embodiments, the linker is an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid residues in length.

[0326] In some embodiments, the linker is non-cleavable.

[0327] In some embodiments, the adapter comprises one or more GGGGS units (SEQ ID NO: 206).

[0328] In some embodiments, the adapter may comprise the formula (X(PAPAP))nKA (SEQ ID NO: 207), where n is an integer selected from 1 to 10, where X may or may not be present, and is A. In some embodiments, the adapter may comprise the sequence shown in SEQ ID NO: 163.

[0329] In some embodiments, the adapter is an antibody hinge or a portion thereof. For example, in some embodiments, the adapter may comprise a human IgG1 hinge sequence or a portion thereof. In some embodiments, the adapter may comprise a human IgG2 hinge sequence or a portion thereof. In some embodiments, the adapter may comprise a human IgG3 hinge sequence or a portion thereof. In another embodiment, the adapter may comprise a human IgG4 hinge sequence or a portion thereof.

[0330] Antibody and Antigen Binding Fragment Specification 22 / 226 pages 30 CN 121419995 A

[0331] In some embodiments, the binder is an anti-TROP2 antibody or an antigen-binding fragment thereof. It should be understood herein that the following represent exemplary and non-limiting embodiments of the antibody or its antigen-binding fragment.

[0332] Antibodies or their antigen-binding fragments (or sequences thereof) can be obtained by immunizing animals (including camels, llamas, dromedary camels, alpacas, and transgenic animals) with the TROP2 antigen as disclosed herein. For example, single-domain antibodies or their antigen-binding fragments (or sequences thereof) can be obtained by immunizing animals (e.g., dromedary camels, camels, llamas, alpacas, and rabbits) with a TROP2 antigen (e.g., an ECD or a fragment thereof). Alternatively, a single-domain antibody or a sequence of its antigen-binding domain can be obtained by immunizing a transgenic animal capable of expressing a single-domain antibody (see International Application No. PCT / CA2021 / 050951, filed July 12, 2021, and published on January 20, 2022, under WO2022 / 011457 A1, the entire contents of which are incorporated herein by reference).

[0333] In some cases, the antibody or its antigen-binding fragment (or sequence thereof) is obtained from the serum or tissues (e.g., bone marrow, spleen, and / or lymph nodes) of an immunized animal.

[0334] Hybridomas can be generated by fusing cells of an immunized animal with malignant and immortalized plasma cells (e.g., myeloma cells lacking hypoxanthine-guanine-phosphoribotransferase (HGPRT), which allows cells to grow in HAT medium (hypoxanthine-aminopterin-thymidine)). Splenic cells are particularly suitable for generating hybridomas. The antibody or its antigen-binding fragment can be isolated from the hybridoma.

[0335] 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 encoding the same amino acid sequence but having different nucleic acid sequences compared to the original nucleic acid sequence may be generated.

[0336] When the nucleic acid sequence encoding the amino acid sequence of an antibody is obtained, recombinant expression of the antibody can be achieved by transfecting one or more expression vectors into immortalized mammalian cell lines.

[0337] Alternatively, the sequence of an antibody or its antigen-binding fragment can also be obtained by screening an established antibody library for sequences having desired characteristics, such as homology or identity with a reference antibody.

[0338] The antibodies of this disclosure include natural or intact antibodies that specifically bind to TROP2. Natural or intact antibodies include recombinantly expressed antibodies isolated from immunized animals or hybridomas.

[0339] Exemplary embodiments of the binders of this disclosure include specific binding to at least one cell containing TROP2.Antibodies or antigen-binding fragments thereof that bind to an epitope of an amino acid containing an outer structural domain (ECD). Other exemplary embodiments of the binding agents of this disclosure include antibodies or antigen-binding fragments thereof that specifically bind to at least one epitope of an amino acid containing a cysteine-rich structural domain of TROP2. In some embodiments, the antibody or antigen-binding fragment thereof binds to the TROP2 antigen disclosed herein.

[0340] In one embodiment, the antibody of this disclosure specifically binds to human TROP2. In another embodiment, the antibody of this disclosure specifically binds to cynomolgus monkey TROP2. In another embodiment, the antibody of this disclosure specifically binds to rhesus monkey TROP2.

[0341] More specifically, the antibody of this disclosure specifically binds to the ECD or CRD of human TROP2.

[0342] Natural antibodies or intact antibodies comprise two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as α, δ, ε, γ, and μ, each consisting of a variable region (VH) and first, second, third, and optionally fourth constant regions (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 backbone of the Y is composed of the second and third constant regions of the two heavy chains linked together by disulfide bonds. Each arm of the Y includes the variable region and first constant region of a single heavy chain, which binds the 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 in both chains generally contain three highly variable loops called complementarity-determining regions (CDRs) (light chain CDRs, including CDRL1, CDRL2, and CDRL3; heavy chain CDRs, including CDRH1, CDRH2, and CDRH3). The CDR boundaries of antibody-antigen binding fragments disclosed in this paper can be defined or identified according to the rules of Kabat, IMGT, Chothia, or Al-Lazikani. Three CDRs are inserted between flanking elongations called frame regions (FRs) (light chain FRs include LFR1, LFR2, LFR3, and LFR4; heavy chain FRs include HFR1, HFR2, HFR3, and HFR4), which are more conserved than the CDRs and form a scaffold supporting highly variable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), and IgA1 (α1 heavy chain).

[0343] The antibodies of this disclosure include chimeric antibodies that specifically bind TROP2. Chimeric antibodies are recombinant proteins containing variable regions, said variable regions including complementarity-determining regions (CDRs) derived from antibodies of one or more species (e.g., rodent antibodies), while the constant domains of said antibody molecule are derived from constant domains of another species (e.g., human antibodies).

[0344] The antibodies of this disclosure include humanized antibodies that specifically bind TROP2. Humanized antibodies are recombinant proteins in which CDRs or CDR residues from an antibody of one species (e.g., rodent antibodies) are transferred from the heavy and light variable chains of the rodent antibody to the human heavy chain variable region and light chain variable region (e.g., frame region sequence). The constant domains of the antibody molecule are derived from the constant domains of the human antibody. In some embodiments, a limited number of frame region amino acid residues from the parental (rodent) antibody may be substituted into the human antibody frame region sequence. In other instances, since simply transferring a mouse CDR into a human FR typically results in a decrease or even loss of antibody affinity, additional modifications may be required to restore the original affinity of the mouse antibody. This can be achieved by replacing one or more human residues in the FR region with mouse residues 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, this disclosure covers humanized antibodies that compete with the single-domain antibodies disclosed herein.

[0345] The antibodies of this disclosure include human antibodies that specifically bind to TROP2. Complete human antibodies can be obtained from transgenic nonhuman animals. Methods for generating complete human antibodies using combinatorial methods or transgenic animals transformed with human immunoglobulin loci are known in the art. In one alternative, phage display technology can be used to generate human antibodies. Complete human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and function in vivo as substantially endogenous human antibodies. In some embodiments, this disclosure covers human antibodies that compete with the single-domain antibodies disclosed herein.

[0346] The antibodies of this disclosure include single-domain antibodies (sdAbs) that specifically bind to TROP2. As used herein, the terms "single-domain antibody" and "heavy chain antibody" are used interchangeably. The term "single-domain antibody" is used in its broad sense and includes naturally occurring single-domain antibodies or variants thereof, such as, but not limited to, humanized single-domain antibodies or chimeric single-domain antibodies. A single-domain antibody is called chimeric when it has a constant region of another species. In one embodiment, the single-domain antibody is a chimeric single-domain antibody. In some embodiments, the chimeric single-domain antibody comprises a humanized variable region and a human constant region. In another embodiment, the chimeric single-domain antibody comprises a variable region of a natural single-domain antibody. The term "single-domain antibody" also includes mutant versions of naturally occurring single-domain antibodies.Mutated versions of humanized single-domain antibodies and chimeric single-domain antibodies, wherein the amino acid sequence includes amino acid substitutions, deletions and / or additions.

[0347] Single-domain antibodies (sdAbs) are typically composed of two heavy chains. Single-domain antibodies are about 12-15 kDa in size (about 110 amino acids in length). sdAbs can selectively bind to target antigens, just like full-length antibodies, and have similar affinity for the antigen. However, due to their smaller size, they may be able to penetrate solid tumors better. The smaller size also contributes to the stability of sdAbs, which are more resistant to pH and temperature extremes than full-size antibodies (Van Der Linden, Specification 24 / 226, 32 CN 121419995 A et al., 1999, Biochim Biophys Act 1431:37-46). Single-domain antibodies were initially developed following the discovery of fully functional antibodies without light chains in camelids (camels, alpacas, llamas) (e.g., Hamsen et al., 2007, Appl Microbiol Biotechnol 77:13-22). 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 to 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 by the Fc domain of a human IgG antibody or a portion thereof. In some embodiments, the heavy chain constant region of a naturally occurring single-domain antibody is replaced by a human IgG heavy chain constant region comprising CH1, CH2, and CH3 domains and optionally a CH4 domain, and may optionally include a hinge region. In some embodiments, the heavy chain constant region of a naturally occurring single-domain antibody is replaced by the CH3 or CH2-CH3 domain of a human IgG heavy chain, and may optionally include a hinge region.

[0348] The antigen-binding properties of a single-domain antibody are conferred by the variable region of the heavy chain. Therefore, a single heavy chain or even a single heavy chain variable region (VH or VHH) may be sufficient to specifically bind an antigen. However, single-domain antibodies are typically composed of two heavy chains. The two heavy chains can be assembled via dimerizing domains, such as the constant region of class G human immunoglobulins. The two heavy chains can also be expressed as a single polypeptide chain.

[0349] Therefore, in some embodiments, the binder includes an antibody and its antigen-binding fragment, such as, but not limited to, single-domain antibodies from camelids or sharks; human antibodies, including IgG (including human IgG1, human IgG2, human IgG3, human IgG4)), human IgM, human IgA (including human IgA1 and human IgA2), human IgE, human IgD; animal antibodies, including, for example, IgG (IgG1, IgG2a, IgG2b, IgG2c ...IgG2b, IgG2c, IgG3, IgG4), IgM, IgA, IgE, and IgD, and variants of similar forms (e.g., chimeric, humanized, or human).

[0350] In other embodiments, the binders of this disclosure encompass antigen-binding fragments, such as, but not limited to, Fab, Fab', F(ab')2, complementarity-determining regions, variable regions including VH, VHH, VL, and analogues thereof.

[0351] The antigen-binding fragment antigen is prepared by known techniques, such as by proteolysis of a full-length antibody or by expression in Escherichia coli or another host encoding the DNA of said fragment. The antigen-binding fragment can be obtained by digesting a full-length antibody with pepsin or papain using conventional methods. For example, the antigen-binding fragment can be produced by enzymatic cleavage of an antibody with pepsin to obtain a fragment of approximately 100 kD, denoted as F(ab')2. The F(ab')2 fragment can be further cleaved using a thiol reducing agent and optionally a blocking group for the thiol group generated by disulfide bond cleavage to produce a Fab' monovalent fragment of approximately 50 Kd. Alternatively, enzymatic cleavage using papain can directly produce two monovalent Fab fragments and an Fc fragment.

[0352] In some embodiments, the antibody binding TROP2 can be bispecific or multispecific. A “multispecific antibody” is an antibody that can simultaneously bind to at least two targets with different structures (e.g., two or more different antigens, two or more different epitopes on the same antigen, or haptens and / or antigens or epitopes). A “multivalent antibody” is an antibody that can simultaneously bind to at least two targets having the same or different structures. Titer indicates how many antigen-binding domains an antibody has; i.e., monovalent, bivalent, trivalent, or multivalent. Multivalentity provides the advantage of multiple interactions when the antibody binds to an antigen, thereby increasing the affinity for binding to the antigen. Specificity indicates how many antigens or epitopes the antibody can bind; i.e., monospecific, bispecific, trispecific, or multispecific. Using these definitions, natural antibodies (e.g., IgG) are bivalent because they have two antigen-binding domains (each consisting of amino acid residues from the variable regions of the light and heavy chains), but are monospecific because each antigen-binding domain binds to the same epitope. Multispecific multivalent antibodies include antibodies having more than one antigen-binding domain with different specificities. A “bispecific antibody” is an antibody that can simultaneously bind to two targets with different structures. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) may have at least one antigen-binding domain that specifically binds to TROP2. Specification 25 / 226 pages 33 CN 121419995 A

[0353] In addition to recombinant technology, various methods for generating bispecific or multispecific antibodies are known, such as, for example, US patentsThe embodiments disclosed in patent number 7,405,320 are incorporated herein by reference. Bispecific antibodies can be produced by a tetravalent hybridoma method involving the fusion of two different hybridomas, each producing a monoclonal antibody that recognizes a different antigenic site. Another method for preparing bispecific antibodies uses a heterobifunctional crosslinking agent to chemically conjugate two different monoclonal antibodies.

[0354] Single-domain antibodies

[0355] An exemplary embodiment of the binder of this disclosure is a single-domain antibody. In this document, it should be understood that the following represents exemplary and non-limiting embodiments of a single-domain antibody or an antigen-binding fragment thereof.

[0356] Single-domain antibodies have unique binding characteristics. For example, the complementarity-determining region 3 (CDR3) of the sdAb disclosed herein forms a loop that adapts to a groove within the cysteine-rich domain (CRD) of the extracellular domain (ECD) of TROP2. The amino acid residues of CDR3 and the amino acid residues of the frame region 2 (FR2) interact with the amino acid residues of the cysteine-rich domain of TROP2 and together form a clamp around the CRD. These unique properties provide sdAb with high affinity for TROP2 and a slow dissociation rate. 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 CRD.

[0357] The single-domain antibodies of this disclosure also possess anticancer activities, such as antibody-dependent cytotoxicity (ADCC) and / or antibody-dependent phagocytosis (ADCP) activities. The single-domain antibodies of this disclosure can also be conjugated for therapeutic or diagnostic purposes.

[0358] Exemplary embodiments of the conjugates of this disclosure include a single-domain antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing an amino acid of the extracellular domain (ECD) of TROP2. Other exemplary embodiments of the conjugates of this disclosure include a single-domain antibody or an antigen-binding fragment thereof that specifically binds to at least one epitope containing a cysteine-rich domain of TROP2. In some embodiments, the single-domain antibody or its antigen-binding fragment binds to the TROP2 antigen disclosed herein.

[0359] In one embodiment, the single-domain antibody of this disclosure specifically binds to human TROP2. In another embodiment, the single-domain antibody of this disclosure specifically binds to cynomolgus monkey TROP2. In yet another embodiment, the single-domain antibody of this disclosure specifically binds to rhesus monkey TROP2.

[0360] More specifically, the single-domain antibody of this disclosure specifically binds to the ECD or CRD of human TROP2.

[0361] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:54 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of SEQ ID NO:54.

[0362] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:52 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:53.

[0363] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:48 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:47.

[0364] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:220 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:218 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:219.

[0364] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:220, the amino acid sequence of FR2 shown in SEQ ID NO:117, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:218 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:219.

[0365] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:490 (page 26 / 226 of the specification, CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:505, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:488 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:489.

[0366] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:501 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:500 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:489.

[0367] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:230 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:228 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:229.

[0368] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:230 and the amino acid sequence of FR2 shown in SEQ ID NO:361, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:228 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:229.

[0369] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:238 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:236 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:237.

[0370] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:246 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:244 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:245.

[0371] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:303 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:301 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:302.

[0372] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:310 and the amino acid sequence of FR2 shown in SEQ ID NO:360, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:308 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:309.

[0373] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:317 and the amino acid sequence of FR2 shown in SEQ ID NO:354, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:315 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:316.

[0374] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:324 and SEQ ID NO:315.The amino acid sequence of FR2 shown in NO:116 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:322 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:323.

[0375] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:331 and the amino acid sequence of FR2 shown in SEQ ID NO:355 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:329 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:330.

[0376] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:338 and the amino acid sequence of FR2 shown in SEQ ID NO:116 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:336 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:337.

[0377] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:345 and the amino acid sequence of FR2 shown in SEQ ID NO:356, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:343 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:344.

[0378] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:352 (page 27 / 226, CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:350 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:351.

[0379] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:396 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:394 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:395.

[0380] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:404 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:394 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:395.The amino acid sequence of CDR1 shown in SEQ ID NO: 402 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 403.

[0381] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 411 and the amino acid sequence of FR2 shown in SEQ ID NO: 116 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 409 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 410.

[0382] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 418 and the amino acid sequence of FR2 shown in SEQ ID NO: 116 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 416 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 416.

[0383] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:425 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:423 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:424.

[0384] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:432 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:430 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:431.

[0385] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:439, the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:437 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:438.

[0386] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:446, the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:444 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:445.

[0387] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises SEQ ID NO:439, the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:444 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:445.The amino acid sequence of CDR3 shown in NO:453 and the amino acid sequence of FR2 shown in SEQ ID NO:360, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:451 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:452.

[0388] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:460 and the amino acid sequence of FR2 shown in SEQ ID NO:360, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:458 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:459.

[0389] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:467 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:465 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:466.

[0390] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:58, the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:56 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:57.

[0391] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:61 (page 28 / 226 of specification 36 CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:59 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:60.

[0392] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:51 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:50.

[0393] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:48 and the amino acid sequence of FR2 shown in SEQ ID NO:116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:62.The amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 47.

[0394] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 48 and the amino acid sequence of FR2 shown in SEQ ID NO: 117 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 47.

[0395] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 48 and the amino acid sequence of FR2 shown in SEQ ID NO: 118 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 47.

[0396] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 48, the amino acid sequence of FR2 shown in SEQ ID NO: 119, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 47.

[0397] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 48, the amino acid sequence of FR2 shown in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 124.

[0398] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 48, the amino acid sequence of FR2 shown in SEQ ID NO: 117, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 55 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 123.

[0399] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 51, the amino acid sequence of FR2 shown in SEQ ID NO: 116, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 50.

[0400] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 51, and optionally the amino acid sequence of FR2 shown in SEQ ID NO: 116 and / or the amino acid sequence of CDR1 shown in SEQ ID NO: 62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 50.The amino acid sequence of CDR3 and the amino acid sequence of FR2 shown in SEQ ID NO:117 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:50.

[0401] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:51 and the amino acid sequence of FR2 shown in SEQ ID NO:118 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:50.

[0402] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:51 and the amino acid sequence of FR2 shown in SEQ ID NO:120 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:62 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:50.

[0403] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:5.

[0404] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 (page 29 / 226, CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:130.

[0405] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:131.

[0406] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of SEQ ID NO:131.The amino acid sequence of CDR2 shown in NO:133.

[0407] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:131.

[0408] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:132.

[0409] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6, the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:133.

[0410] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6, the amino acid sequence of FR2 shown in SEQ ID NO:172, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:133.

[0411] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6, the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:134.

[0412] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:4 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:135.

[0413] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:6 and the amino acid sequence of FR2 shown in SEQ ID NO:171.The amino acid sequence of FR2 shown in NO:174 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:127 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:129.

[0414] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:170 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:14.

[0415] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:170 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:137.

[0416] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:138.

[0417] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:217, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:215 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:216.

[0418] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:227, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:225 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:226.

[0419] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:235, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:233 and / or SEQ ID NO:226.

[0420] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:243, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:241 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:242.

[0421] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:300, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:298 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:299.

[0422] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:307 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:305 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:306.

[0423] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:314 and the amino acid sequence of FR2 shown in SEQ ID NO:357, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:312 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:313.

[0424] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:321 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:319 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:320.

[0425] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:328 and the amino acid sequence of FR2 shown in SEQ ID NO:358, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:326 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:327.

[0426] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:335 and SEQ ID NO:326.

[0427] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR2 shown in SEQ ID NO: 170 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 333 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 334.

[0428] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 349 and the amino acid sequence of FR2 shown in SEQ ID NO: 170 and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 347 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 348.

[0429] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:393, the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:391 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:392.

[0430] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:401 (page 31 / 226, CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:399 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:400.

[0431] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:408 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:406 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:407.

[0432] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:415 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:406 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:407.The amino acid sequence of CDR1 shown in SEQ ID NO: 413 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 414.

[0433] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 422 and the amino acid sequence of FR2 shown in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 420 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 421.

[0434] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO: 429 and the amino acid sequence of FR2 shown in SEQ ID NO: 170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO: 427 and / or the amino acid sequence of CDR2 shown in SEQ ID NO: 428.

[0435] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:436 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:434 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:435.

[0436] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:443 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:441 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:442.

[0437] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:450 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:448 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:449.

[0438] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:457 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:455 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:456.

[0439] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises SEQ ID NO:450 and / or the amino acid sequence of FR2 shown in SEQ ID NO:170.The amino acid sequence of CDR3 shown in NO:464 and the amino acid sequence of FR2 shown in SEQ ID NO:170, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:462 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:463.

[0440] In an exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:136.

[0441] In an exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:137.

[0442] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15, the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:138.

[0443] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 (page 32 / 226 of specification 40 CN 121419995 A), the amino acid sequence of FR2 shown in SEQ ID NO:171, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:139.

[0444] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR3 shown in SEQ ID NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:173, and optionally the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:137.

[0445] In some embodiments, the single-domain antibody or its antigen-binding fragment of this disclosure comprises the three CDRs of the single-domain antibody disclosed herein.

[0446] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:15 and / or the amino acid sequence of FR2 shown in SEQ ID NO:173.The amino acid sequence shown is CDR1, the heavy chain complementarity determination region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:2, and the heavy chain complementarity determination region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:3.

[0447] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:4, CDRH2 having the amino acid sequence shown in SEQ ID NO:5, and CDRH3 having the amino acid sequence shown in SEQ ID NO:6.

[0448] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:7, CDRH2 having the amino acid sequence shown in SEQ ID NO:8, and CDRH3 having the amino acid sequence shown in SEQ ID NO:9.

[0449] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:10, CDRH2 having the amino acid sequence shown in SEQ ID NO:11, and CDRH3 having the amino acid sequence shown in SEQ ID NO:12.

[0450] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:13, CDRH2 having the amino acid sequence shown in SEQ ID NO:14, and CDRH3 having the amino acid sequence shown in SEQ ID NO:15.

[0451] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:16, CDRH2 having the amino acid sequence shown in SEQ ID NO:17, and CDRH3 having the amino acid sequence shown in SEQ ID NO:18.

[0452] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:19, CDRH2 having the amino acid sequence shown in SEQ ID NO:20, and CDRH3 having the amino acid sequence shown in SEQ ID NO:21.

[0453] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:46, CDRH2 having the amino acid sequence shown in SEQ ID NO:47, and CDRH3 having the amino acid sequence shown in SEQ ID NO:48.

[0454] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:55, CDRH2 having the amino acid sequence shown in SEQ ID NO:47, and CDRH3 having the amino acid sequence shown in SEQ ID NO:48.CDRH2 having the amino acid sequence shown in SEQ ID NO:47 and CDRH3 having the amino acid sequence shown in SEQ ID NO:48.

[0455] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:49, CDRH2 having the amino acid sequence shown in SEQ ID NO:50, and CDRH3 having the amino acid sequence shown in SEQ ID NO:51.

[0456] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:62, CDRH2 having the amino acid sequence shown in SEQ ID NO:50, and CDRH3 having the amino acid sequence shown in SEQ ID NO:51.

[0457] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:52, CDRH2 having the amino acid sequence shown in SEQ ID NO:53, and CDRH3 having the amino acid sequence shown in SEQ ID NO:54.

[0458] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:56, CDRH2 having the amino acid sequence shown in SEQ ID NO:57, and CDRH3 having the amino acid sequence shown in SEQ ID NO:58.

[0459] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:59, CDRH2 having the amino acid sequence shown in SEQ ID NO:60, and CDRH3 having the amino acid sequence shown in SEQ ID NO:61.

[0460] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:63, CDRH2 having the amino acid sequence shown in SEQ ID NO:64, and CDRH3 having the amino acid sequence shown in SEQ ID NO:65.

[0461] In other exemplary embodiments, the single-domain antibody comprises CDRH1 having the amino acid sequence shown in SEQ ID NO:66, CDRH2 having the amino acid sequence shown in SEQ ID NO:67, and CDRH3 having the amino acid sequence shown in SEQ ID NO:68.

[0462] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:123, and SEQ ID NO:55.The amino acid sequence of CDR3 shown in NO:48.

[0463] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:124, and the amino acid sequence of CDR3 shown in SEQ ID NO:48.

[0464] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:130, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0465] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:131, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0466] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:132, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0467] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:133, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0468] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:134, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0469] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:135, and the amino acid sequence of CDR3 shown in the specification (page 34 / 226, CN 121419995 A) shown in SEQ ID NO:6.

[0470] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:127, the amino acid sequence of CDR2 shown in SEQ ID NO:129, and the amino acid sequence of CDR3 shown in SEQ ID NO:6.

[0471] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:14, and the amino acid sequence of CDR3 shown in SEQ ID NO:15.

[0472] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:136, and the amino acid sequence of CDR3 shown in SEQ ID NO:15.

[0473] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:137, and the amino acid sequence of CDR3 shown in SEQ ID NO:15.

[0474] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:138, and the amino acid sequence of CDR3 shown in SEQ ID NO:15.

[0475] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:139, and the amino acid sequence of CDR3 shown in SEQ ID NO:15.

[0476] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:215, the amino acid sequence of CDR2 shown in SEQ ID NO:216, and the amino acid sequence of CDR3 shown in SEQ ID NO:217.

[0477] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:218, the amino acid sequence of CDR2 shown in SEQ ID NO:219, and the amino acid sequence of CDR3 shown in SEQ ID NO:220.

[0478] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:225, the amino acid sequence of CDR2 shown in SEQ ID NO:226, and the amino acid sequence of CDR3 shown in SEQ ID NO:227.

[0479] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:228, the amino acid sequence of CDR2 shown in SEQ ID NO:226, and the amino acid sequence of CDR3 shown in SEQ ID NO:227.The amino acid sequences of CDR1 shown in SEQ ID NO:229, CDR2 shown in SEQ ID NO:230, and CDR3 shown in SEQ ID NO:230 are described.

[0480] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:233, the amino acid sequence of CDR2 shown in SEQ ID NO:234, and the amino acid sequence of CDR3 shown in SEQ ID NO:235.

[0481] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:236, the amino acid sequence of CDR2 shown in SEQ ID NO:237, and the amino acid sequence of CDR3 shown in SEQ ID NO:238.

[0482] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:241, the amino acid sequence of CDR2 shown in SEQ ID NO:242, and the amino acid sequence of CDR3 shown in SEQ ID NO:243 (page 35 / 226, CN 121419995 A).

[0483] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:244, the amino acid sequence of CDR2 shown in SEQ ID NO:245, and the amino acid sequence of CDR3 shown in SEQ ID NO:246.

[0484] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:298, the amino acid sequence of CDR2 shown in SEQ ID NO:299, and the amino acid sequence of CDR3 shown in SEQ ID NO:300.

[0485] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:301, the amino acid sequence of CDR2 shown in SEQ ID NO:302, and the amino acid sequence of CDR3 shown in SEQ ID NO:303.

[0486] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:305, the amino acid sequence of CDR2 shown in SEQ ID NO:306, and the amino acid sequence of CDR3 shown in SEQ ID NO:307.

[0487] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:308.The amino acid sequences of CDR1 shown in SEQ ID NO:309, CDR2 shown in SEQ ID NO:310, and CDR3 shown in SEQ ID NO:310 are described.

[0488] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:312, the amino acid sequence of CDR2 shown in SEQ ID NO:313, and the amino acid sequence of CDR3 shown in SEQ ID NO:314.

[0489] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:315, the amino acid sequence of CDR2 shown in SEQ ID NO:316, and the amino acid sequence of CDR3 shown in SEQ ID NO:317.

[0490] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:319, the amino acid sequence of CDR2 shown in SEQ ID NO:320, and the amino acid sequence of CDR3 shown in SEQ ID NO:321.

[0491] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:322, the amino acid sequence of CDR2 shown in SEQ ID NO:323, and the amino acid sequence of CDR3 shown in SEQ ID NO:324.

[0492] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:326, the amino acid sequence of CDR2 shown in SEQ ID NO:327, and the amino acid sequence of CDR3 shown in SEQ ID NO:328.

[0493] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:329, the amino acid sequence of CDR2 shown in SEQ ID NO:330, and the amino acid sequence of CDR3 shown in SEQ ID NO:331.

[0494] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:333, the amino acid sequence of CDR2 shown in SEQ ID NO:334, and the amino acid sequence of CDR3 shown in SEQ ID NO:335.

[0495] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:336, the amino acid sequence of CDR2 shown in SEQ ID NO:334, and the amino acid sequence of CDR3 shown in SEQ ID NO:335.The amino acid sequence of CDR2 shown in NO:337 and the amino acid sequence of CDR3 shown in SEQ ID NO:338, page 36 / 226, CN 121419995 A.

[0496] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:340, the amino acid sequence of CDR2 shown in SEQ ID NO:341, and the amino acid sequence of CDR3 shown in SEQ ID NO:342.

[0497] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:343, the amino acid sequence of CDR2 shown in SEQ ID NO:344, and the amino acid sequence of CDR3 shown in SEQ ID NO:345.

[0498] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:347, the amino acid sequence of CDR2 shown in SEQ ID NO:348, and the amino acid sequence of CDR3 shown in SEQ ID NO:349.

[0499] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:350, the amino acid sequence of CDR2 shown in SEQ ID NO:351, and the amino acid sequence of CDR3 shown in SEQ ID NO:352.

[0500] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:391, the amino acid sequence of CDR2 shown in SEQ ID NO:392, and the amino acid sequence of CDR3 shown in SEQ ID NO:393.

[0501] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:394, the amino acid sequence of CDR2 shown in SEQ ID NO:395, and the amino acid sequence of CDR3 shown in SEQ ID NO:396.

[0502] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:399, the amino acid sequence of CDR2 shown in SEQ ID NO:400, and the amino acid sequence of CDR3 shown in SEQ ID NO:401.

[0503] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:402, the amino acid sequence of CDR2 shown in SEQ ID NO:394, and the amino acid sequence of CDR3 shown in SEQ ID NO:401.The amino acid sequence of CDR2 shown in NO:403 and the amino acid sequence of CDR3 shown in SEQ ID NO:404.

[0504] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:406, the amino acid sequence of CDR2 shown in SEQ ID NO:407, and the amino acid sequence of CDR3 shown in SEQ ID NO:408.

[0505] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:409, the amino acid sequence of CDR2 shown in SEQ ID NO:410, and the amino acid sequence of CDR3 shown in SEQ ID NO:411.

[0506] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:413, the amino acid sequence of CDR2 shown in SEQ ID NO:414, and the amino acid sequence of CDR3 shown in SEQ ID NO:415.

[0507] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:416, the amino acid sequence of CDR2 shown in SEQ ID NO:417, and the amino acid sequence of CDR3 shown in SEQ ID NO:418.

[0508] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:420, the amino acid sequence of CDR2 shown in SEQ ID NO:421, and the amino acid sequence of CDR3 shown in SEQ ID NO:422 (page 37 / 226, CN 121419995 A).

[0509] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:423, the amino acid sequence of CDR2 shown in SEQ ID NO:424, and the amino acid sequence of CDR3 shown in SEQ ID NO:425.

[0510] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:427, the amino acid sequence of CDR2 shown in SEQ ID NO:428, and the amino acid sequence of CDR3 shown in SEQ ID NO:429.

[0511] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:430, the amino acid sequence of CDR2 shown in SEQ ID NO:428, and the amino acid sequence of CDR3 shown in SEQ ID NO:429.The amino acid sequence of CDR2 shown in NO:431 and the amino acid sequence of CDR3 shown in SEQ ID NO:432.

[0512] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:434, the amino acid sequence of CDR2 shown in SEQ ID NO:435, and the amino acid sequence of CDR3 shown in SEQ ID NO:436.

[0513] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:437, the amino acid sequence of CDR2 shown in SEQ ID NO:438, and the amino acid sequence of CDR3 shown in SEQ ID NO:439.

[0514] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:441, the amino acid sequence of CDR2 shown in SEQ ID NO:442, and the amino acid sequence of CDR3 shown in SEQ ID NO:443.

[0515] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:444, the amino acid sequence of CDR2 shown in SEQ ID NO:445, and the amino acid sequence of CDR3 shown in SEQ ID NO:446.

[0516] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:448, the amino acid sequence of CDR2 shown in SEQ ID NO:449, and the amino acid sequence of CDR3 shown in SEQ ID NO:450.

[0517] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:451, the amino acid sequence of CDR2 shown in SEQ ID NO:452, and the amino acid sequence of CDR3 shown in SEQ ID NO:453.

[0518] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:455, the amino acid sequence of CDR2 shown in SEQ ID NO:456, and the amino acid sequence of CDR3 shown in SEQ ID NO:457.

[0519] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:458, the amino acid sequence of CDR2 shown in SEQ ID NO:459, and the amino acid sequence of CDR3 shown in SEQ ID NO:460.The amino acid sequence of CDR3 is shown.

[0520] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:462, the amino acid sequence of CDR2 shown in SEQ ID NO:463, and the amino acid sequence of CDR3 shown in SEQ ID NO:464.

[0521] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:465, the amino acid sequence of CDR2 shown in SEQ ID NO:466, and the amino acid sequence of CDR3 shown in SEQ ID NO:467.

[0522] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:485, the amino acid sequence of CDR2 shown in SEQ ID NO:486, and the amino acid sequence of CDR3 shown in SEQ ID NO:487.

[0523] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:488, the amino acid sequence of CDR2 shown in SEQ ID NO:489, and the amino acid sequence of CDR3 shown in SEQ ID NO:490.

[0524] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:485, the amino acid sequence of CDR2 shown in SEQ ID NO:492, and the amino acid sequence of CDR3 shown in SEQ ID NO:487.

[0525] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:485, the amino acid sequence of CDR2 shown in SEQ ID NO:495, and the amino acid sequence of CDR3 shown in SEQ ID NO:487.

[0526] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:485, the amino acid sequence of CDR2 shown in SEQ ID NO:498, and the amino acid sequence of CDR3 shown in SEQ ID NO:499.

[0527] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:500, the amino acid sequence of CDR2 shown in SEQ ID NO:489, and the amino acid sequence of CDR3 shown in SEQ ID NO:501.The amino acid sequence of CDR3 is shown.

[0528] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:485, the amino acid sequence of CDR2 shown in SEQ ID NO:486, and the amino acid sequence of CDR3 shown in SEQ ID NO:499.

[0529] In some embodiments, the single-domain antibody or its antigen-binding fragment of the present disclosure comprises the amino acid sequences of CDR1, CDR2, CDR3, and FR2 of the single-domain antibody disclosed herein.

[0530] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:52, the amino acid sequence of CDR2 shown in SEQ ID NO:53, the amino acid sequence of CDR3 shown in SEQ ID NO:54, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0531] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:47, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0532] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:56, the amino acid sequence of CDR2 shown in SEQ ID NO:57, the amino acid sequence of CDR3 shown in SEQ ID NO:58, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0533] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:59, the amino acid sequence of CDR2 shown in SEQ ID NO:60, the amino acid sequence of CDR3 shown in SEQ ID NO:61, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0534] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:62, the amino acid sequence of CDR2 shown in SEQ ID NO:50, the amino acid sequence of CDR3 shown in SEQ ID NO:51, and the amino acid sequence of FR2 shown in SEQ ID NO:116. Specification 39 / 226 pages 47 CN 121419995 A

[0535] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:47, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0536] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:218, the amino acid sequence of CDR2 shown in SEQ ID NO:219, the amino acid sequence of CDR3 shown in SEQ ID NO:220, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0537] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:218, the amino acid sequence of CDR2 shown in SEQ ID NO:219, the amino acid sequence of CDR3 shown in SEQ ID NO:220, and the amino acid sequence of FR2 shown in SEQ ID NO:117.

[0538] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:228, the amino acid sequence of CDR2 shown in SEQ ID NO:229, the amino acid sequence of CDR3 shown in SEQ ID NO:230, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0539] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:236, the amino acid sequence of CDR2 shown in SEQ ID NO:237, the amino acid sequence of CDR3 shown in SEQ ID NO:238, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0540] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:244, the amino acid sequence of CDR2 shown in SEQ ID NO:245, the amino acid sequence of CDR3 shown in SEQ ID NO:246, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0541] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:301, the amino acid sequence of CDR2 shown in SEQ ID NO:302, and the amino acid sequence of CDR2 shown in SEQ ID NO:303.The amino acid sequence of CDR3 shown is shown, and the amino acid sequence of FR2 shown in SEQ ID NO:116 is shown.

[0542] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:308, the amino acid sequence of CDR2 shown in SEQ ID NO:309, the amino acid sequence of CDR3 shown in SEQ ID NO:310, and the amino acid sequence of FR2 shown in SEQ ID NO:360.

[0543] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:315, the amino acid sequence of CDR2 shown in SEQ ID NO:316, the amino acid sequence of CDR3 shown in SEQ ID NO:317, and the amino acid sequence of FR2 shown in SEQ ID NO:354.

[0544] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:322, the amino acid sequence of CDR2 shown in SEQ ID NO:323, the amino acid sequence of CDR3 shown in SEQ ID NO:324, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0545] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:329, the amino acid sequence of CDR2 shown in SEQ ID NO:330, the amino acid sequence of CDR3 shown in SEQ ID NO:331, and the amino acid sequence of FR2 shown in SEQ ID NO:355.

[0546] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:336, the amino acid sequence of CDR2 shown in SEQ ID NO:337, the amino acid sequence of CDR3 shown in SEQ ID NO:338, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0547] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:343, the amino acid sequence of CDR2 shown in SEQ ID NO:344, the amino acid sequence of CDR3 shown in SEQ ID NO:345, and the amino acid sequence of FR2 shown in SEQ ID NO:356. Specification 40 / 226 pages 48 CN 121419995 A

[0548] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises SEQ ID NO:343, the amino acid sequence of CDR1 shown in SEQ ID NO:344, the amino acid sequence of CDR2 shown in SEQ ID NO:345, and the amino acid sequence of FR2 shown in SEQ ID NO:356.The amino acid sequence of CDR1 shown in SEQ ID NO:350, the amino acid sequence of CDR2 shown in SEQ ID NO:351, the amino acid sequence of CDR3 shown in SEQ ID NO:352, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0549] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:394, the amino acid sequence of CDR2 shown in SEQ ID NO:395, the amino acid sequence of CDR3 shown in SEQ ID NO:396, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0550] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:402, the amino acid sequence of CDR2 shown in SEQ ID NO:403, the amino acid sequence of CDR3 shown in SEQ ID NO:404, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0551] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:409, the amino acid sequence of CDR2 shown in SEQ ID NO:410, the amino acid sequence of CDR3 shown in SEQ ID NO:411, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0552] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:416, the amino acid sequence of CDR2 shown in SEQ ID NO:417, the amino acid sequence of CDR3 shown in SEQ ID NO:418, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0553] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:423, the amino acid sequence of CDR2 shown in SEQ ID NO:424, the amino acid sequence of CDR3 shown in SEQ ID NO:425, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0554] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:430, the amino acid sequence of CDR2 shown in SEQ ID NO:431, the amino acid sequence of CDR3 shown in SEQ ID NO:432, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0555] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:437, the amino acid sequence of CDR2 shown in SEQ ID NO:438, the amino acid sequence of CDR3 shown in SEQ ID NO:439, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0556] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:444, the amino acid sequence of CDR2 shown in SEQ ID NO:445, the amino acid sequence of CDR3 shown in SEQ ID NO:446, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0557] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:451, the amino acid sequence of CDR2 shown in SEQ ID NO:452, the amino acid sequence of CDR3 shown in SEQ ID NO:453, and the amino acid sequence of FR2 shown in SEQ ID NO:360.

[0558] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:458, the amino acid sequence of CDR2 shown in SEQ ID NO:459, the amino acid sequence of CDR3 shown in SEQ ID NO:460, and the amino acid sequence of FR2 shown in SEQ ID NO:360.

[0559] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:465, the amino acid sequence of CDR2 shown in SEQ ID NO:466, the amino acid sequence of CDR3 shown in SEQ ID NO:467, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0560] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:488, the amino acid sequence of CDR2 shown in SEQ ID NO:489, the amino acid sequence of CDR3 shown in SEQ ID NO:490, and the amino acid sequence of FR2 shown in SEQ ID NO:505. Specification 41 / 226 pages 49 CN 121419995 A

[0561] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:500, SEQ IDThe amino acid sequence of CDR2 shown in NO:489, the amino acid sequence of CDR3 shown in SEQ ID NO:501, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0562] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:47, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:117.

[0563] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:47, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:118.

[0564] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:47, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:119.

[0565] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:124, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:117.

[0566] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:55, the amino acid sequence of CDR2 shown in SEQ ID NO:123, the amino acid sequence of CDR3 shown in SEQ ID NO:48, and the amino acid sequence of FR2 shown in SEQ ID NO:117.

[0567] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:62, the amino acid sequence of CDR2 shown in SEQ ID NO:50, the amino acid sequence of CDR3 shown in SEQ ID NO:51, and the amino acid sequence of FR2 shown in SEQ ID NO:116.

[0568] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:62.The amino acid sequences of CDR1, CDR2, CDR3, and FR2 shown in SEQ ID NO:50, SEQ ID NO:51, and FR2 shown in SEQ ID NO:117 are shown.

[0569] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequences of CDR1, CDR2, CDR3, and FR2 shown in SEQ ID NO:118, as shown in SEQ ID NO:62.

[0570] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequences of CDR1, CDR2, CDR3, and FR2 shown in SEQ ID NO:120, as shown in SEQ ID NO:62, SEQ ID NO:50, SEQ ID NO:51, and FR2 shown in SEQ ID NO:120.

[0571] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:5, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0572] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:130, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0573] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:131, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:170. Specification page 42 / 226 50 CN 121419995 A

[0574] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:133, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0575] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:131, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0576] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:132, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0577] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:133, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0578] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:133, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:172.

[0579] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:134, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0580] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:4, the amino acid sequence of CDR2 shown in SEQ ID NO:135, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0581] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:127, the amino acid sequence of CDR2 shown in SEQ ID NO:129, the amino acid sequence of CDR3 shown in SEQ ID NO:6, and the amino acid sequence of FR2 shown in SEQ ID NO:174.

[0582] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:14, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0583] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:137, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0584] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:138, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0585] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:215, the amino acid sequence of CDR2 shown in SEQ ID NO:216, the amino acid sequence of CDR3 shown in SEQ ID NO:217, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0586] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:225, the amino acid sequence of CDR2 shown in SEQ ID NO:226, the amino acid sequence of CDR3 shown in SEQ ID NO:227, and the amino acid sequence of FR2 shown in SEQ ID NO:170. Specification 43 / 226 pages 51 CN 121419995 A

[0587] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:233, the amino acid sequence of CDR2 shown in SEQ ID NO:234, the amino acid sequence of CDR3 shown in SEQ ID NO:235, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0588] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:241, SEQ ID NO:234, and the amino acid sequence of FR2 shown in SEQ ID NO:235.The amino acid sequence of CDR2 shown in NO:242, the amino acid sequence of CDR3 shown in SEQ ID NO:243, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0589] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:298, the amino acid sequence of CDR2 shown in SEQ ID NO:299, the amino acid sequence of CDR3 shown in SEQ ID NO:300, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0590] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:305, the amino acid sequence of CDR2 shown in SEQ ID NO:306, the amino acid sequence of CDR3 shown in SEQ ID NO:307, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0591] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:312, the amino acid sequence of CDR2 shown in SEQ ID NO:313, the amino acid sequence of CDR3 shown in SEQ ID NO:314, and the amino acid sequence of FR2 shown in SEQ ID NO:357.

[0592] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:319, the amino acid sequence of CDR2 shown in SEQ ID NO:320, the amino acid sequence of CDR3 shown in SEQ ID NO:321, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0593] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:326, the amino acid sequence of CDR2 shown in SEQ ID NO:327, the amino acid sequence of CDR3 shown in SEQ ID NO:328, and the amino acid sequence of FR2 shown in SEQ ID NO:358.

[0594] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:333, the amino acid sequence of CDR2 shown in SEQ ID NO:334, the amino acid sequence of CDR3 shown in SEQ ID NO:335, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0595] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises SEQ ID NO:333.The amino acid sequence of CDR1 shown in NO:340, the amino acid sequence of CDR2 shown in SEQ ID NO:341, the amino acid sequence of CDR3 shown in SEQ ID NO:342, and the amino acid sequence of FR2 shown in SEQ ID NO:359.

[0596] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:347, the amino acid sequence of CDR2 shown in SEQ ID NO:348, the amino acid sequence of CDR3 shown in SEQ ID NO:349, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0597] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:391, the amino acid sequence of CDR2 shown in SEQ ID NO:392, the amino acid sequence of CDR3 shown in SEQ ID NO:393, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0598] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:399, the amino acid sequence of CDR2 shown in SEQ ID NO:400, the amino acid sequence of CDR3 shown in SEQ ID NO:401, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0599] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:406, the amino acid sequence of CDR2 shown in SEQ ID NO:407, the amino acid sequence of CDR3 shown in SEQ ID NO:408, and the amino acid sequence of FR2 shown in SEQ ID NO:170. Specification 44 / 226 pages 52 CN 121419995 A

[0600] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:413, the amino acid sequence of CDR2 shown in SEQ ID NO:414, the amino acid sequence of CDR3 shown in SEQ ID NO:415, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0601] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:420, the amino acid sequence of CDR2 shown in SEQ ID NO:421, the amino acid sequence of CDR3 shown in SEQ ID NO:422, and the amino acid sequence of SEQ ID NO:420.The amino acid sequence of FR2 shown in NO:170.

[0602] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:427, the amino acid sequence of CDR2 shown in SEQ ID NO:428, the amino acid sequence of CDR3 shown in SEQ ID NO:429, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0603] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:434, the amino acid sequence of CDR2 shown in SEQ ID NO:435, the amino acid sequence of CDR3 shown in SEQ ID NO:436, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0604] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:441, the amino acid sequence of CDR2 shown in SEQ ID NO:442, the amino acid sequence of CDR3 shown in SEQ ID NO:443, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0605] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:448, the amino acid sequence of CDR2 shown in SEQ ID NO:449, the amino acid sequence of CDR3 shown in SEQ ID NO:450, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0606] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:455, the amino acid sequence of CDR2 shown in SEQ ID NO:456, the amino acid sequence of CDR3 shown in SEQ ID NO:457, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0607] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:462, the amino acid sequence of CDR2 shown in SEQ ID NO:463, the amino acid sequence of CDR3 shown in SEQ ID NO:464, and the amino acid sequence of FR2 shown in SEQ ID NO:170.

[0608] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:136, and the amino acid sequence of FR2 shown in SEQ ID NO:170.The amino acid sequence of CDR3 shown in NO:15 and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0609] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13 and / or the amino acid sequence of CDR2 shown in SEQ ID NO:137, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0610] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:138, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0611] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:139, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:171.

[0612] In one exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the amino acid sequence of CDR1 shown in SEQ ID NO:13, the amino acid sequence of CDR2 shown in SEQ ID NO:137, the amino acid sequence of CDR3 shown in SEQ ID NO:15, and the amino acid sequence of FR2 shown in SEQ ID NO:173. Specification 45 / 226 pages 53 CN 121419995 A

[0613] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises a variable region of the single-domain antibody disclosed herein.

[0614] In other exemplary embodiments, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:22.

[0615] In other exemplary embodiments, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:23.

[0616] In other exemplary embodiments, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:24.

[0617] In yet another exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:25.

[0618] In yet another exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:26.

[0619] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:27.

[0620] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:28.

[0621] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:221.

[0622] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:222.

[0623] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:223.

[0624] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:224.

[0625] In yet another exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:231.

[0626] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:232.

[0627] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:239.

[0628] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:240.

[0629] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:247.

[0630] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:304.

[0631] In a further exemplary embodiment, the single-domain antibody includes a variable region having an amino acid sequence as shown in SEQ ID NO:311.

[0632] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:318.

[0633] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:325.

[0634] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:332.

[0635] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:339.

[0636] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:346.

[0637] In a further exemplary embodiment, the single-domain antibody comprises a variable region having an amino acid sequence as shown in SEQ ID NO:353.

[0638] In some embodiments, the single-domain antibody may comprise a sequence made of a combination of CDRH1, CDRH2, and CDRH3 shown in Table 23 or Table 24 and a heavy chain variable region backbone.

[0639] Therefore, in some embodiments, the single-domain antibody may comprise the sequences shown in Table 1A. Specification 47 / 226 pages 55 CN 121419995 A

[0640] Specification 48 / 226 pages 56 CN 121419995 A Specification 49 / 226 pages 57 CN 121419995 A Specification 50 / 226 pages 58 CN 121419995 A Specification 51 / 226 pages 59 CN 121419995 A

[0641] It should be understood from this document that the positions of the residues mentioned in Tables 23 and 24 take into account the insertion of the complete amino acid sequence of each of IMGT CDR1, CDR2 and CDR3 into the humanized heavy chain variable region backbone, and refer to the position of the amino acid residues of SEQ ID NO: 145 in Table 1C. Therefore, after inserting the complete amino acid sequences of IMGT CDR1, CDR2, and CDR3, IMGT frame 1 typically covers positions 1 to 25, IMGT frame 2 typically covers positions 34 to 50, IMGT frame 3 typically covers positions 58 to 95, and IMGT frame 4 typically covers positions 110-120. Since the length of the IMGT CDRs may vary slightly, the positioning of the frames may also change accordingly. However, those skilled in the art will be able to determine the position number of a given variable region by comparing it with SEQ ID NO:145 in Table 1C.

[0642] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:29.

[0643] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:71.

[0644] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:72.

[0645] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:73.

[0646] In another exemplary embodiment, the single-domain antibody comprises, as shown in SEQ ID NO: 10 ...

[0647] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:75.

[0648] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:76.

[0649] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:77.

[0650] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:78.

[0651] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:79.

[0652] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:80.

[0653] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:81.

[0654] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:82.

[0655] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:83. Specification 52 / 226 pages 60 CN 121419995 A

[0656] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:84.

[0657] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:85.

[0658] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:86.

[0659] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:87.

[0660] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:88.

[0661] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:89.

[0662] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:90.

[0663] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:91.

[0664] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:92.

[0665] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:93.

[0666] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:94.

[0667] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:95.

[0668] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:96.

[0669] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:97.

[0670] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:98.

[0671] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:99.

[0672] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:100.

[0673] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:101.

[0674] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:012.

[0675] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:103.

[0676] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:104.

[0677] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:105.

[0678] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:106.

[0679] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:107.

[0680] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:108.

[0681] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:109.

[0682] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:110.

[0683] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:111.

[0684] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:112.

[0685] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:113.

[0686] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:114.

[0687] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:115.

[0688] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:221.

[0689] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:222.

[0690] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:223.

[0691] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:224.

[0692] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:231.

[0693] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:232.

[0694] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:239. Specification 53 / 226 pages 61 CN 121419995 A

[0695] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:240.

[0696] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:247.

[0697] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:295.

[0698] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:296.

[0699] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:297.

[0700] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:304.

[0701] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:311.

[0702] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:318.

[0703] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:325.

[0704] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:332.

[0705] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:339.

[0706] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:346.

[0707] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:353.

[0708] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:362.

[0709] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:363.

[0710] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:364.

[0711] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:365.

[0712] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:366.

[0713] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:367.

[0714] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:368.

[0715] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:369.

[0716] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:370.

[0717] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:371.

[0718] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:372.

[0719] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:373.

[0720] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:374.

[0721] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:375.

[0722] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:376.

[0723] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:377.

[0724] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:378.

[0725] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:379.

[0726] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:380.

[0727] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:381.

[0728] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:382.

[0729] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:383.

[0730] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:384.

[0731] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:385.

[0732] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:386.

[0733] ​​In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:387. Specification page 54 / 226 62 CN 121419995 A

[0734] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:388.

[0735] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:389.

[0736] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:390.

[0737] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:397.

[0738] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:398.

[0739] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:405.

[0740] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:412.

[0741] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:419.

[0742] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:426.

[0743] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:433.

[0744] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:440.

[0745] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:447.

[0746] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:454.

[0747] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:461.

[0748] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:468.

[0749] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:469.

[0750] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:470.

[0751] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:471.

[0752] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:472.

[0753] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:473.

[0754] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:491.

[0755] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:493.

[0756] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:496.

[0757] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:502.

[0758] In another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:503.

[0759] In exemplary embodiments, the following sequences are specifically selected: SEQ ID NO: 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, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 397.Single-domain antibodies comprising the CDR or heavy chain variable region or a humanized version thereof shown in 398, 405, 412, 419, 426, 433, 440, 447, 454, 461, 468, and 318.

[0760] In another exemplary embodiment, the single-domain antibody or its antigen-binding fragment comprises the heavy chain of the single-domain antibody disclosed herein.

[0761] In an exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown as SEQ ID NO:29.

[0762] In an exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown as SEQ ID NO:30.

[0763] In yet another exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown as SEQ ID NO:31.

[0764] In a further exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown as SEQ ID NO:32.

[0765] In a further exemplary embodiment, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:33, as specified in document 55 / 226, page 63, CN 121419995 A.

[0766] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:34.

[0767] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:35.

[0768] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:44.

[0769] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:45.

[0770] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:482.

[0771] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:483.

[0772] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:484.

[0773] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:494.

[0774] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:497.

[0775] In other exemplary embodiments, the single-domain antibody comprises the amino acid sequence shown in SEQ ID NO:504.

[0776] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in any one of SEQ ID NO:22-28, 71-115, 143 to 146.

[0777] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in SEQ ID NO:22-28, 71-115, 143 to 146.The amino acid sequence shown in SEQ ID NO:295.

[0778] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in SEQ ID NO:304.

[0779] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in SEQ ID NO:311.

[0780] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in SEQ ID NO:353.

[0781] In some cases, the binder of this disclosure does not contain the amino acid sequence shown in SEQ ID NO:362.

[0782] In some cases, the binder of this disclosure may comprise the amino acid sequence shown in any one of SEQ ID NO:22-28, 71-115, 143 to 146, provided that the binder also comprises SEQ ID NO:221-224, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:247, SEQ ID NO:304, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:311, SEQ ID NO:318, SEQ ID NO:325, SEQ ID NO:332, SEQ ID NO:339, SEQ ID NO:346, SEQ ID NO:353, SEQ ID NO:363, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:369, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:369 ... NO:368, SEQ ID NO:369, SEQ ID NO:370, SEQ ID NO:371, 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, 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 IDThe amino acid sequence shown in any one of 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.

[0783] According to this disclosure, a single-domain antibody may comprise the amino acid sequence combination of Table 1A and a dimerization domain.

[0784] In some embodiments, the dimerization domain may comprise SEQ ID NO:38 or a variant thereof. In other embodiments, the dimerization domain may comprise SEQ ID NO:43 or a variant thereof. The dimerizing domain may also contain SEQ ID NO:37 or SEQ ID NO:179 or a variant thereof, and SEQ ID NO:36 or SEQ ID NO:42 or a variant thereof.

[0785] In another embodiment, the dimerizing domain may contain SEQ ID NO:212 or a variant thereof. In another embodiment, the dimerizing domain may contain SEQ ID NO:506 or a variant thereof. In a further embodiment, the dimerizing domain may contain SEQ ID NO:214 or a variant thereof.

[0786] Therefore, in an exemplary embodiment, the single-domain antibody may contain the amino acid sequence shown in Table 1B.

[0787] Table 1B

[0788] Specification 57 / 226 pages 65 CN 121419995 A Specification 58 / 226 pages 66 CN 121419995 A Specification 59 / 226 pages 67 CN 121419995 A Specification 60 / 226 pages 68 CN 121419995 A Specification 61 / 226 pages 69 CN 121419995 A Specification 62 / 226 pages 70 CN 121419995 A Specification 63 / 226 pages 71 CN 121419995 A Specification 64 / 226 pages 72 CN 121419995 A

[0789] Binders containing the antigen-binding domain of sdAb

[0790] The binders of this disclosure comprise one or more antigen-binding domains of an antibody or an antigen-binding fragment thereof that binds 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 instance, the binder may comprise a variable region or a portion thereof of a single domain disclosed herein.

[0791] Hereinafter, it should be understood that the following represent exemplary and non-limiting embodiments of binders comprising antigen-binding domains of single-domain antibodies.

[0792] In some embodiments, the binders of this disclosure comprise one or more antigen-binding domains that specifically bind trophoblast cell surface antigen-2 (TROP2). In some embodiments, the binders of this disclosure comprise one or more antigen-binding domains that bind to a target different from TROP2.

[0793] Exemplary embodiments of the binders of this disclosure include binders comprising one or more antigen-binding domains that specifically bind to an epitope of at least one amino acid comprising an extracellular domain (ECD) of TROP2. Other exemplary embodiments of the binders of this disclosure include binders comprising one or more antigen-binding domains that specifically bind to at least one epitope of an amino acid containing a cysteine-rich domain of TROP2. In some embodiments, the binder comprises one or more antigen-binding domains disclosed herein that specifically bind to the TROP2 antigen.

[0794] In one embodiment, the binder of this disclosure comprises one or more antigen-binding domains that specifically bind to human TROP2. In another embodiment, the binder of this disclosure comprises one or more antigen-binding domains that specifically bind to cynomolgus monkey TROP2. In yet another embodiment, the binder of this disclosure comprises one or more antigen-binding domains that specifically bind to rhesus monkey TROP2.

[0795] 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.

[0796] In other embodiments, the binder of this disclosure comprises one or more antigen-binding domains of a natural antibody (CDR and / or VL and VH) that binds to TROP2.

[0797] 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 indicated below.

[0798] Similarly, the CDR or variable regions of multiple single-domain antibodies can be expressed as a single polypeptide chain, thereby providing multivalentity and / or multispecificity for the binding agent. Furthermore, multiple polypeptide chains can be assembled to increase the diversity or affinity of interactions with TROP2. (See page 65 / 226 for specification)73 CN 121419995 A. As indicated below, numerous forms are considered.

[0799] Therefore, the antigen-binding fragments or antigen-binding domains of the single-domain antibodies disclosed herein can be used to generate monospecific, multispecific, monovalent, or multivalent binders.

[0800] For example, the variable region or CDR of a single-domain antibody can be fused with the Fc, CH3, or CH2-CH3 domain of the antibody to allow homodimerization, thereby generating a multivalent binder.

[0801] Alternatively, multiple copies of the variable region or CDR of a single-domain antibody can be fused in tandem with a desired spacer or adapter to generate multivalent and monospecific binders.

[0802] For example, the variable regions or CDRs of two single-domain antibodies can be fused with the Fc, CH3, or CH2-CH3 domain to allow heterodimerization, thereby generating multivalent and multispecific binders.

[0803] Alternatively, the variable regions or CDRs of two or more single-domain antibodies can be fused in tandem with a desired spacer or adapter to generate multivalent and multispecific binders.

[0804] The conjugates of this disclosure may be in the following forms: antibodies or antigen-binding fragments thereof, antibody-like molecules (natural antibodies fused with a single-domain antibody or with the antigen-binding domain of an sdAb, Fc-fusions, CH3-fusions and analogues thereof), fusions with a protein scaffold, immune cell modulators and analogues thereof.

[0805] The conjugates also include immune cell modulators, such as dual-affinity retargeting molecules (DART), chimeric antigen receptor (CAR) constructs, bispecific T cell conjugate constructs (BiTE), bispecific killer cell conjugates (BiKE), and trispecific killer cell conjugates containing scFv or VHH (TriKE).

[0806] The conjugates also cover fusions with a protein scaffold, including ankyrin repeats, the Z domain of staphylococcal protein A, type III fibronectin, knottin, and analogues. Exemplary embodiments of the binder include monospecific, bispecific (symmetric or asymmetric), trispecific, or multispecific antibodies, as well as monovalent, bivalent, trivalent, or multivalent antibodies, single-chain FVs (scFVs), and derivatives such as biantibodies, triantibodies, tetraantibodies, tandem bi-scFvs, tandem tri-scFvs, scFV-Fc, microantibodies (scFV-CH3), tandem biantibodies, bi-biantibodies, dimers, and analogs thereof, VHs or VHHs and derivatives such as tandem bispecific or multispecific VHHs, bivalent VHH-Fc fusions, VHH-hinge-CH2-CH3 fusions, bivalent CH3 fusions, VHH pentaantibodies, decaantibodies, and analogs thereof.

[0807] The single-chain Fv molecule (scFv) comprises 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). If the VL domain is the N-terminal portion of the scFv molecule,The scFv molecule is then represented as VL-L-VH, or VH-L-VL if the VH domain is the N-terminal portion of the scFv molecule. Methods for preparing scFv molecules and designing suitable peptide linkers are described in the following: U.S. Patent No. 4,704,692, U.S. Patent No. 4,946,778, R. Raag and M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995), and R. E. Bird and BW Walker, Single Chain Antibody Variable Regions, TIBTECH, Vol 9:132-137 (1991).

[0808] In some embodiments, the binder of this disclosure may have the form disclosed in PCT / CA2020 / 051753 (filed on 18 December 2020 and published on 24 June 2021 under WO2021 / 119832A1, the entire contents of which are incorporated herein by reference), such as formula Ia, formula Ib, formula Ic, formula II, formula III, formula IV, formula V, formula VI, formula VII or formula VIII and similar formulas, or the forms of formula I, formula II, formula III, formula IIIa and formula IIIb, formula IV, formula V, formula VI, formula VII or formula VIII disclosed herein.

[0809] The binders of this disclosure can be formed by assembling two polypeptide chains having the same conformation (having the same or different amino acid sequences) or different conformations, wherein the same or different conformations include those shown in Formula I, Formula II, Formula III, Formula IIIa and Formula IIIb, Formula IV, Formula V, Formula VI, Formula VII or Formula VIII disclosed herein. Specification 66 / 226 pages 74 CN 121419995 A

[0810] In some embodiments, the binders of this disclosure are monospecific. In some embodiments, the binders of this disclosure are multispecific.

[0811] The binders of this disclosure (including antibodies or antigen-binding fragments thereof) can be monospecific, multispecific, monovalent or multivalent.

[0812] Binders containing two or more antigen-binding domains of a monodomain antibody are considered multivalent. Multivalent single-domain antibodies can be monospecific (if all antigen-binding domains bind to the exact same epitope) or multispecific (if the antigen-binding domains bind to different epitopes on the same target or to different epitopes or targets). Binders containing only one antigen-binding domain are considered monovalent.

[0813] The production of binders (e.g., antibodies or antigen-binding fragments) can involve cloning procedures and recombinant expression.Co-domain sequences (e.g., sequences of the variable light and variable heavy chains of an intact antibody or sequences of the variable heavy chain of a single-domain antibody) can be obtained through 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, which is then transfected into cells and the binding agent 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 two separate expression vectors. Proper assembly of an intact antibody requires co-transfection with separate expression vectors. In the case of a single-domain antibody, a single expression vector (expressing the heavy chain) is sufficient.

[0814] Monospecific binding agents encompass binding agents that are specific to a single epitope of a given antigen.

[0815] An exemplary embodiment of a monospecific binding agent includes a binding agent comprising one antigen-binding fragment of a single-domain antibody. Another exemplary embodiment of a monospecific binding agent includes a binding agent comprising antigen-binding fragments of more than one single-domain antibody, but said antigen-binding fragments having the same CDR and frame region. Another exemplary embodiment of the binder includes a binder comprising an antigen-binding fragment containing more than one single-domain antibody, but the variable regions have the same CDR and different frame regions. A further exemplary embodiment of a monospecific binder includes a binder comprising an antigen-binding fragment containing a single-domain antibody, wherein one or more CDRs of the antigen-binding fragment have different amino acid sequences (e.g., conserved substitutions in one or more CDRs) without affecting its ability to bind the same antigen or epitope.

[0816] Multispecific binders encompass binders that are specific to more than one epitope (epitope of the same antigen or different antigens) or more than one antigen. For example, a multispecific polypeptide chain or binder may therefore have more than one variable region of a single-domain antibody, wherein at least two bind to different antigens or epitopes.

[0817] The binders of this disclosure may therefore be bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific, 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.

[0818] Other exemplary non-limiting embodiments of multispecific binders include multispecific binders with two antigen-binding fragments having different specificities. Still other exemplary non-limiting embodiments of multispecific binders include binders having more than two antigen-binding fragments that bind two different antigens, proteins, or two different epitopes on the same antigen or protein. Specification 67 / 226 pages 75 CN 121419995 A

[0819] In some embodiments, the binders of this disclosure are monovalent.

[0820] In some embodiments, the binders of this disclosure are multivalent.

[0821] Exemplary non-limiting embodiments of multivalent binders include binders composed of multivalent polypeptide chains. Other non-limiting exemplary embodiments of multivalent binders include binders composed of more than one monovalent polypeptide chain.

[0822] According to this disclosure, bispecific binders may be divalent or multivalent, depending on the number of variable regions they contain. Exemplary non-limiting embodiments of bispecific binders include binders comprising two identical bispecific polypeptide chains forming a dimer.

[0823] This document provides exemplary and non-limiting embodiments of bispecific binders.

[0824] In some embodiments, the bispecific binder comprises a TROP2-binding domain and an antigen-binding domain of Formula II (see PCT / CA2020 / 051753, filed December 18, 2020, and disclosed on June 24, 2021, under WO2021 / 119832A1, the entire contents of which are incorporated herein by reference).

[0825] In other embodiments, the binder comprises a polypeptide chain comprising, from N-terminus to C-terminus: a) an antigen-binding domain of a single-domain antibody, b) a linker, c) a dimerization domain, d) a linker, and e) an antigen-binding domain of a single-domain antibody; at least one of the antigen-binding domains a) or e) is an antigen-binding domain that binds TROP2.

[0826] In some embodiments, the antigen-binding domain a) is different from the antigen-binding domain e).

[0827] In some embodiments, the antigen-binding domain of a) is the same as the antigen-binding domain of e).

[0828] In some embodiments, the adapter of b) is the same as the adapter of d).

[0829] In some embodiments, the adapter of b) is different from the adapter of d).

[0830] In one exemplary embodiment, the antigen-binding domain of a) comprises the antigen-binding domain of a single-domain antibody that specifically binds to human TROP2.

[0831] In another exemplary embodiment, the antigen-binding domain of a) comprises the antigen-binding domain of a single-domain antibody that specifically binds to CD47 (e.g., human CD47).

[0832] In one exemplary embodiment, the antigen-binding domain of e) comprises an antigen-binding domain of a single-domain antibody that specifically binds to human TROP2.

[0833] In one exemplary embodiment, the antigen-binding domain of e) comprises an antigen-binding domain of a single-domain antibody that specifically binds to CD47 (e.g., human CD47).

[0834] In some embodiments, the bispecific binder comprises two polypeptide chains. The two polypeptide chains are non-covalently assembled via amino acid residues of the CH2 and / or CH3 domains contained therein to obtain a bispecific and tetravalent binder.

[0835] In one exemplary embodiment, the binder or antigen-binding domain of this disclosure may be able to inhibit the growth of tumor cells expressing human TROP2 and CD47.

[0836] In an exemplary embodiment, the binder comprises one or more antigen-binding domains capable of binding CD47.

[0837] In another exemplary embodiment, the binder comprises one or more antigen-binding domains capable of binding TROP2 and one or more antigen-binding domains capable of binding CD47.

[0838] In one exemplary embodiment, the antigen-binding domain capable of binding to CD47 comprises:

[0839] a. CDRH1 having the amino acid sequence shown in SEQ ID NO:156, CDRH2 having the amino acid sequence shown in SEQ ID NO:157, and CDRH3 having the amino acid sequence shown in SEQ ID NO:158; or

[0840] b. CDRH1 having the amino acid sequence shown in SEQ ID NO:159, CDRH2 having the amino acid sequence shown in SEQ ID NO:160, and CDRH3 having the amino acid sequence shown in SEQ ID NO:161. Specification 68 / 226 pages 76 CN 121419995 A

[0841] In one exemplary embodiment, the antigen-binding domain capable of binding to CD47 comprises the amino acid sequence shown in SEQ ID NO:155.

[0842] In one exemplary embodiment, the binder is capable of binding to CD47 and TROP2 and comprises the amino acid sequence of at least one antigen-binding domain of at least one single-domain antibody disclosed herein, as shown in SEQ ID NO:155.

[0843] In other exemplary embodiments, the binder may comprise at least one antigen-binding domain capable of binding CD47 and at least one antigen-binding domain capable of binding TROP2, and it comprises SEQ ID NO:22-26, SEQ ID NO:73-114, SEQ ID NO:221-224, SEQ ID NO:231, SEQ ID NO:232, SEQ ID NO:155, ...NO:239, SEQ ID NO:240, SEQ ID NO:247, SEQ ID NO:304, SEQ ID NO:295, SEQ ID NO:296, SEQ ID NO:297, SEQ ID NO:311, SEQ ID NO:318, SEQ ID NO:325, SEQ ID NO:332, SEQ ID NO:339, SEQ ID NO:346, SEQ ID NO:353, SEQ ID NO:362, SEQ ID NO:363, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:370, SEQ ID NO:371, 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, 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 The amino acid sequence of the antigen-binding domain of at least one single-domain antibody shown in any one of SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:496, SEQ ID NO:502, or SEQ ID NO:503.

[0844] In other exemplary embodiments, the binder may comprise at least one antigen-binding domain capable of binding CD47.The conjugate comprises a domain and at least one antigen-binding domain capable of binding TROP2, and contains the amino acid sequence of a humanized version of the single-domain antibody disclosed herein.

[0845] In other exemplary embodiments, the conjugate may comprise at least one antigen-binding domain capable of binding PD-1 and at least one antigen-binding domain capable of binding TROP2.

[0846] In other exemplary embodiments, the conjugate may comprise at least one antigen-binding domain capable of binding TROP2, at least one antigen-binding domain capable of binding CD47, and at least one antigen-binding domain capable of binding PD-1.

[0847] In an exemplary embodiment, the antigen-binding domain capable of binding PD-1 comprises:

[0848] a. CDRH1 having the amino acid sequence shown in SEQ ID NO:532, CDRH2 having the amino acid sequence shown in SEQ ID NO:535, and CDRH3 having the amino acid sequence shown in SEQ ID NO:534, or

[0849] b. CDRH1 having the amino acid sequence shown in SEQ ID NO:535, CDRH2 having the amino acid sequence shown in SEQ ID NO:536, and CDRH3 having the amino acid sequence shown in SEQ ID NO:537.

[0850] In an exemplary embodiment, the antigen-binding domain capable of binding PD-1 comprises the amino acid sequence shown in SEQ ID NO:538.

[0851] In an exemplary embodiment, the binder is capable of binding PD-1 and TROP2 and comprises the amino acid sequence shown 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. Specification 69 / 226 pages 77 CN 121419995 A

[0852] In some cases, the humanized version thereof has replaced the amino acid residues of its frame region with those amino acid residues of the corresponding human or humanized frame region. In some cases, the humanized version has replaced its frame region with those shown in SEQ ID NO: 211. In other cases, the humanized version has replaced its frame region with those shown in SEQ ID NO: 213.

[0853] In some embodiments, the frame region of the humanized version has been replaced with the frame region shown in any one of SEQ ID NO: 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.

[0854] In some embodiments, the humanized version of the frame region has been replaced with SEQ ID NO:272, 273, 274, 275,The frame region shown in any one of 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, or 294.

[0855] In another exemplary embodiment, the binder is capable of binding to CD47 and TROP2 and comprises an amino acid sequence as shown in SEQ ID NO:150 or SEQ ID NO:151.

[0856] Variations

[0857] This disclosure also covers variations of the sequences disclosed herein.

[0858] The binders of this disclosure therefore cover antibody variants (e.g., single-domain antibody variants), variants of antibody-like molecules, variants of protein scaffolds, and variants of immune cell modulators. Furthermore, the binders of this disclosure may comprise an antigen-binding fragment or antigen-binding domain of an antibody variant, such as an antigen-binding fragment or antigen-binding domain of a single-domain antibody variant.

[0859] This disclosure more specifically covers variants of the single-domain antibodies disclosed herein and binders comprising one or more antigen-binding domains of the single-domain antibody variants. Variants of the binders disclosed herein may have the same form as the binders disclosed herein. Therefore, variants of the binders covered by this disclosure may be multispecific and / or multivalent. Variants covered by this disclosure include those that may contain one or more amino acid residues inserted at one or more positions, one or more amino acid residues deleted at one or more positions, or one or more amino acid residues substituted at one or more positions (conservative or non-conservative substitution).

[0860] Such variations may occur in the antigen-binding domain or other elements of the binder, such as dimerizing domains, linkers, or spacers.

[0861] For example, naturally occurring residues are grouped according to common side-chain characteristics. Conservative substitution can be performed by replacing an amino acid from one of the following groups (Groups 1 to 6) with another amino acid from the same group. Non-conservative substitution would require replacing a member of one of these groups with a member of another group.

[0862] (Group 1) Hydrophobic: Leucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile)

[0863] (Group 2) Neutral-hydrophilic: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln)

[0864] (Group 3) Acidic: Aspartic acid (Asp), Glutamic acid (Glu)

[0865] (Group 4) Basic: Histidine (His), Lysine (Lys), Arginine (Arg)

[0866] (Group 5) Residues affecting chain orientation: Glycine (Gly), Proline (Pro); and

[0867] Group 6) Aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe)

[0868] Other exemplary embodiments of conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". If such substitutions result in undesirable properties, more substantial changes may be introduced, named "Exemplary Substitutions" in Table 2, or as further described below in reference to amino acid categories in the specification on page 78 of 70 / 226 of CN 121419995 A, and the products are screened.

[0869] Those skilled in the art will recognize that certain amino acids are less positively charged, neutral, negatively charged or have a reduced charge compared to other amino acids. Amino acids can be classified according to the net charge indicated by the isoelectric point of the amino acid. The isoelectric point is the pH value at which the average net charge of an amino acid molecule is zero. When pH > pI, the amino acid has a net negative charge, and when pH < pI, the amino acid has a net positive charge. In some embodiments, the measured pI value of the antibody is between about 3 and 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 between them. In some embodiments, the measured pI value of the antibody is between about 4 and 7 (e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0) and any value between them. Exemplary isoelectric points of amino acids are shown in Table 2 below. Amino acids that typically have positively charged side chains include, for example, Arginine (R), Histidine (H) and Lysine (K). Amino acids that have negatively charged side chains include, for example, Aspartic acid (D) and Glutamic acid (E). Amino acids with polar properties include, for example, Serine (S), Threonine (T), Asparagine (N), Glutamine (Q) 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).

[0870] In some embodiments, the isoelectric point of the antibody is modified via amino acid substitution. See, for example, US20110076275. In some embodiments, modifying the isoelectric point of the polypeptide constituting the antibody results in a change in the antibody half-life.

[0871] Table 2. Exemplary Amino Acid Substitutions

[0872] Specification page 79 of 71 / 226 of CN 121419995 A

[0873] Generally, the degree of similarity and identity between variable chains is used herein with 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) uses the default settings, namely the blastp program, BLOSUM62 matrix (open vacancy 11 and extended vacancy penalty 1; vacancy x decrease 50, expectation 10.0, word length 3) and activated filter assay.

[0874] Therefore, the identity percentage will indicate amino acids that are identical to and may occupy the same or similar positions as the original peptide.

[0875] The similarity percentage will indicate identical amino acids and amino acids that have been substituted by conserved amino acid substitutions at the same or similar positions as the original peptide.

[0876] Therefore, variations of this disclosure may contain a sequence that is at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the original or reference sequence or a portion of the original sequence.

[0877] In one exemplary embodiment, mutations in one or more CDRs, variable regions, or constant regions can be made by amino acid substitution. Exemplary embodiments of substitution include conserved amino acid substitutions or non-conserved amino acid substitutions. Exemplary substitutions are provided in Table 2. Other exemplary substitutions are provided in Table 13A. Still other exemplary substitutions are provided in Table 13B.

[0878] In some embodiments, a polypeptide chain having an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99% identical or less than 100% identical to a given amino acid sequence may have amino acid substitutions, additions, or deletions that are typically located outside the complementarity-determining region.

[0879] 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 still other embodiments, the variant may have at least 90% sequence identity with the sequence disclosed herein. In a further embodiment, 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.

[0880] In some embodiments, the variation may occur in one or more CDRs. The mutation may, for example, occur in CDR amino acid residues that do not involve interaction with TROP2. In one exemplary embodiment, 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 considered.

[0881] In other embodiments, changes can occur in the frame region. Mutations can occur, for example, in frame amino acid residues that do not involve interaction with TROP2. In one exemplary embodiment, amino acid residues of FR1 and FR3 and / or FR4 can be mutated. In other exemplary embodiments, amino acid residues of FR2 can be mutated. FR2 mutations that improve affinity for TROP2 are particularly considered.

[0882] In other embodiments, changes can occur in the constant region. In one exemplary embodiment, the constant region can be mutated to modulate ADCC activity. In another exemplary embodiment, the constant region can be mutated to modulate ADCP activity. In another exemplary embodiment, the constant region can be mutated to improve ADCP activity. In yet another exemplary embodiment, the constant region can be mutated to improve internalization. In yet another exemplary embodiment, the constant region can be mutated to improve stability.

[0883] This disclosure covers exemplary and non-limiting embodiments of mutations in constant regions (e.g., Fc regions) that modify one or more effector functions, exemplary embodiments of which are provided in Table A (List of Mutations, 72 / 226 pages, 80 CN 121419995 A Antibodies (Basel). 2020 Nov 17;9(4):64, the entire contents of which are incorporated herein by reference).

[0884] Table A

[0885]

[0886] This document provides exemplary and non-limiting embodiments of single-domain antibody variants. This disclosure also covers binders comprising one or more antigen-binding domains of one or more single-domain antibody variants.

[0887] Exemplary variants of this disclosure may include peptides comprising variable region sequences or heavy chain sequences from Tables 1A, 1B, 15A to 15K, 16, 18, or 19, and having one or more variations selected from amino acid substitutions, deletions, and / or additions. The variation may include, for example, 1 to 5 amino acid substitutions, 1 to 5 amino acid deletions, and / or 1 to 5 amino acid insertions. In one exemplary embodiment, the deletion or addition is sequential. In another exemplary embodiment, the deletion or addition is discontinuous. In yet another exemplary embodiment, the deletion or addition is at the N-terminus. In a further 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 yet another exemplary embodiment, the deletion or addition is outside the Kabat CDR sequence. In yet another exemplary embodiment, the deletion or addition is at the Kabat CDR sequence.Outside the CDR3 sequence and / or outside the Kabat FR2 sequence.

[0888] As used herein, the term “1 to 5” includes any single numerical value, such as “1”, “2”, “3”, “4”, and “5”, and any range therein, 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”.

[0889] Variations of this disclosure may include at least one amino acid substitution compared to the sequence disclosed herein. Alternatively, variations of this disclosure may include at least one amino acid deletion compared to the sequence disclosed herein. Alternatively, variations of this disclosure may include at least one amino acid addition compared to the sequence disclosed herein.

[0890] In some embodiments, variations of this disclosure may include at least one amino acid substitution and at least one amino acid deletion compared to the sequence disclosed herein. In some embodiments, variants of this disclosure may include at least one amino acid substitution and at least one amino acid addition compared to the sequence disclosed herein. In some embodiments, variants of this disclosure may include at least one amino acid addition and at least one amino acid deletion compared to the sequence disclosed herein. In some embodiments, variants of this 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 sequence disclosed herein.

[0891] 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).

[0892] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequences shown in SEQ ID NO:143 to SEQ ID NO:146.

[0893] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:143.

[0894] SEQ ID NO:143 (KD002 common)

[0895] In one exemplary embodiment, each of X1a to X1r is independently any (e.g., naturally occurring) amino acid residue.

[0896] In another exemplary embodiment, each of X1a to X1r is independently the amino acid sequence shown in SEQ ID NO:23 or SEQ ID NO:143.

[0897] In yet another exemplary embodiment, each of X1a to X1r is independently an amino acid residue found at the corresponding position of any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 (SEQ ID NO:195), or a conserved amino acid substitution or a non-conserved amino acid substitution thereof.

[0898] In a further exemplary embodiment, each of X1a to X1r is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO:23 or SEQ ID NO:73-93.

[0899] In another exemplary embodiment, each of X1a to X1r is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 to SEQ ID NO:26.

[0900] In yet another exemplary embodiment, each of X1a to X1r is independently an amino acid residue found at the corresponding position of any of SEQ ID NO: 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.

[0901] In a further exemplary embodiment, each of X1a to X1r is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 73, 75, 76, 77, 78, 81, 82, 83, 85, 89 or 91.

[0902] In a further exemplary embodiment, X1a is Q or E; X1b is Q, V or L; X1c is M or L; X1d is V or P; Specification 74 / 226 pages 82 CN 121419995 A X1e is G or Q; X1f is P, L or R; X1g is Y or V; X1h is D, N, E; X1i is P or A; X1j is S or T; X1k is A or S; X1l is L or Q; X1m is N or S; X1n is K or R; X1o is P or A; X1p is L or V; X1q is R or Y; and / or X1r is Q or M (SEQ ID NO: 196).

[0903] In another exemplary embodiment, X1a is Q or E; X1b is Q, V, or L; X1c is M or L; X1d is V or P; X1e isG or Q; X1f is P, L or R; X1g is Y; X1h is D; X1i is P or A; X1j is S or T; X1k is A or S; X1l is L or Q; X1m is N or S; X1n is K or R; X1o is P or A; X1p is L or V; X1q is R or Y; and / or X1r is Q or M (SEQ ID NO: 197).

[0904] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 211.

[0905]

[0906] In some embodiments, any one of X1a to X1r is as described herein (e.g., SEQ ID NO: 477, 478).

[0907] In other embodiments, any one of X1a to X1r is a corresponding amino acid residue of the variable region of the antibody heavy chain.

[0908] In yet another embodiment, any one of X1a to X1r is a corresponding amino acid residue of the sequence shown in any one of SEQ ID NO:248-271.

[0909] In yet another embodiment, any one of X1a to X1r is a corresponding amino acid residue of a human antibody heavy chain, for example, residues of a human germline antibody heavy chain.

[0910] According to this disclosure, [CDRH1], [CDRH2] and [CDRH3] in SEQ ID NO:211 correspond to the given variable region sequences disclosed herein and, in particular, the amino acid sequences of IMGT CDR1, CDR2 and CDR3 in Tables 15A to 15K, 16, 18 or 19.

[0911] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:144.

[0912] SEQ ID NO:144 (KD005 common)

[0913]

[0914] In one exemplary embodiment, each of X2a to X2p is independently any (e.g., naturally occurring) amino acid residue.

[0915] In another exemplary embodiment, each of X2a to X2p is independently an amino acid residue found at the corresponding position of any of SEQ ID NO:26 or SEQ ID NO:94-114 (SEQ ID NO:199), or a conserved or non-conserved amino acid substitution thereof.

[0916] In another exemplary embodiment, each of X2a to X2p is independently an amino acid residue found at the corresponding position of any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26 (SEQ ID NO:198), or a conserved or non-conserved amino acid substitution thereof.

[0917] In yet another exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 26 or SEQ ID NO: 94-114.

[0918] In a further exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 to SEQ ID NO: 26. (Page 75 / 226, CN 121419995 A)

[0919] In a further exemplary embodiment, each of X2a to X2p is independently an amino acid residue found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107 or 108 (SEQ ID NO: 209), or a conserved or non-conserved amino acid substitution thereof.

[0920] In another exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 98, 100, 101, 102, 103, 104, 105, 106, 107 or 108.

[0921] In another exemplary embodiment, each of X2a to X2p is independently an amino acid residue found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 100, 102, 103, 104, 105, 106, 108, 112, or 114 (SEQ ID NO: 210), or a conserved or non-conserved amino acid substitution thereof.

[0922] In a further exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 100, 102, 103, 104, 105, 106, 108, 112, or 114.

[0923] In a further exemplary embodiment, each of X2a to X2p is independently an amino acid residue found at the corresponding position in any of SEQ ID NO: 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 105, 112, 113, or 114 (SEQ ID NO: 210), or a conserved or non-conserved amino acid substitution thereof.

[0924] In another exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 105, 112, 113, or 114.

[0925] In another exemplary embodiment, each of X2a to X2p is independently the amino acid residue found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 102, or 105 (SEQ ID NO: 210), or a conserved or non-conserved amino acid substitution thereof.

[0926] In yet another exemplary embodiment, each of X2a to X2p is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO: 94, 95, 96, 97, 102, or 105.

[0927] In another exemplary embodiment, X2a is Q or E; X2b is Q, L or V; X2c is V or I; X2d is E or Q; X2e is A or P; X2f is P or L; X2g is Y, A or V; X2h is P or A; X2i is S or T; X2j is A or S; X2k is K or R; X2l is P or A; X2m is D or E; X2n is L or V; X2o is R or Y and / or X2p is Q or L (SEQ ID NO:199).

[0928] In yet another exemplary embodiment, X2a is Q or E; X2b is Q or L; X2c is V or I; X2d is E or Q; X2e is A or P; X2f is P or L; X2g is Y or A; X2h is P or A; X2i is S or T; X2j is A or S; X2k is K or R; X2l is P or A; X2m is D or E; X2n is L or V; X2o is R or Y and / or X2p is Q or L (SEQ ID NO:200).

[0929] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:213.

[0930]

[0931] In some embodiments, any one of X2a to X2p is as described herein, and X2q is L, X2r is A and / or X2s is W.

[0932] In other embodiments, any one of X2a to X2s is a corresponding amino acid residue of the variable region of the antibody heavy chain.

[0933] In yet another embodiment, any one of X2a to X2s is a corresponding amino acid residue of the sequence shown in any one of SEQ ID NO:272-294.

[0934] In yet another embodiment, any one of X2a to X2s is a corresponding amino acid residue of the human antibody heavy chain, for example, a residue of the human germline antibody heavy chain.

[0935] In some embodiments, X2a is Q or E; X2b is Q, L or V; X2c is V or I; X2d is E, Q, D or H; X2e is A or P; X2f is P or L; X2g is Y, A or V; X2h is P or A; X2i is S or T; X2j is A or S; X2k is K, R or T; X2l is P or A; X2m is D or E; X2n is L or V; X2o is R or Y; X2p is Q or L; X2q is L; X2r is A and / or X2s is W (SEQ ID NO:479).

[0936] In some embodiments, X2a is Q or E; X2b is Q, L or V; X2c is V or I; X2d is E, Q, D or H; X2e is A or P; X2f is P or L; X2g is Y, A, V or I; X2h is P or A; X2i is S or T; X2j is A or S; X2k is K, R or T; X2l is P or A; X2m is D or E; X2n is L or V; X2o is R or Y; X2p is Q or L; X2q is L or M; X2r is A or V and / or X2s is W or Q (SEQ ID NO: 480).

[0937] According to this disclosure, [CDRH1], [CDRH2] and [CDRH3] in SEQ ID NO:213 correspond to the given variable region sequences disclosed herein and, in particular, the amino acid sequences of IMGT CDR1, CDR2 and CDR3 in Tables 15A to 15K, 16, 18 or 19.

[0938] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:145.

[0939] SEQ ID NO:145 (common to the heavy chains KD001 to KD005)

[0940]

[0941] In one exemplary embodiment, each of X3a to X3y is independently any (e.g., naturally occurring) amino acid residue.

[0942] In another exemplary embodiment, each of X3a to X3y is independently an amino acid residue found at the corresponding position of any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26 (SEQ ID NO:201), or a conserved or non-conserved amino acid substitution thereof.

[0943] In yet another exemplary embodiment, each of X3a to X3y is independently the most frequently found amino acid residue in the group consisting of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 to SEQ ID NO:26.

[0944] In a further exemplary embodiment, X3a is M or L; X3b is Q, H or E; X3c is V, P or A; X3d is G or R; X3e is P or T; X3f is S, N or G; X3g is A, Y or S; X3h is N or T; X3i is A, S or G; X3j is D, G or S; X3k is G or missing; X3l is S or G; X3m is K, N or T; X3n is Y or D; X3o is I or T; X3p is T or M; X3q is L or Q; X3r is N or S; X3s is K or T; X3t is E or D; X3u is R or K; X3v is A or S; X3w is K or R; X3x is T or S; and / or X3y is Y or A (SEQ ID NO: 202).

[0945] In a further exemplary embodiment, X3a is M or L; X3b is Q, H or E; X3c is V, P or A; X3d is G or R; X3e is P or T; X3f is S, N or G; X3g is A, Y or S; X3h is N or T; X3i is A, S or G; X3j is D, G or S; X3k is G or missing; X3l is S or G; X3m is K, N or T; X3n is Y; X3o is I or T; X3p is T or M; X3q is L or Q; X3r is N or S; X3s is K or T; Specification 77 / 226 pages 85 CN 121419995 A X3t is E or D; X3u is R or K; X3v is A or S; X3w is R; X3x is S; and / or X3y is A (SEQ

[0946] Generally, the positions of the amino acid residues are as shown in SEQ ID NO:145 and are exemplified in Table 1C.

[0947]

[0948] According to this disclosure, a single-domain antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO:146.

[0949] SEQ ID NO:146 (common to KD001-KD005 and selected variable heavy chains) Specification 78 / 226 pages 86 CN 121419995 A

[0950]

[0951] In one exemplary embodiment, each of X4a to X5n is independently any (e.g., naturally occurring) amino acid residue.

[0952] In another exemplary embodiment, each of X4a to X5n is independently an amino acid residue found at the corresponding position in any of SEQ ID NO:22-26, SEQ ID NO:73-114 (SEQ ID NO:205), or a conserved or non-conserved amino acid substitution thereof.

[0953] In yet another exemplary embodiment, each of X4a to X5n is independently defined in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 20 ...An amino acid residue found at the corresponding position of any of SEQ ID NO:25 or SEQ ID NO:26 (SEQ ID NO:204), or a conserved or non-conserved amino acid substitution thereof.

[0954] In another exemplary embodiment, each of X4a to X5n is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO:22-26, SEQ ID NO:73-114.

[0955] In another exemplary embodiment, each of X4a to X5n is independently the amino acid residue most frequently found at the corresponding position of any of SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25 to SEQ ID NO:26.

[0956] In a further exemplary embodiment, X4a is Q or E, X4b is Q, L or V, X4c is M or L, X4d is V or I, X4e X4f is Q, H, or E; X4g is P, A, or V; X4h is P or T; X4i is G, S, or N; X4j is A or S; X4k is S or H; X4l is V or Y; X4m is G or Q; X4n is P, L, or R; X4o is E or V; X4p is Y, A, or R; X4q is T or N; X4r is A, S, or G; X4s is G, S, D, N, or E; X4t is missing or S; X4u is K or T; X4v X4w is Y, D or S, X4x is P or A, X4y is I or T, X4z is S or A, X5a is T or M, X5b is Q or L, X5c is S or N, X5d is K, R or T, X5e is P or A, X5f is E or D, X5g is V or L, X5h is Y or R, X5i is R or K, X5j is A or S, X5k is K or R, X5l is S or T, X5m is Y or A and / or X5n is Q, L or M (SEQ ID NO:205).

[0957] The antibodies in Tables 15B and 15C all have the same CDR1, CDR2 and CDR3 as the CDR of KD001 (KD008).

[0958] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequences shown in SEQ ID NO: 507, SEQ ID NO: 508, and SEQ ID NO: 509 (e.g., Tables 15B and 15C).

[0959] SEQ ID NO: 507, 508, 509 (common to KD 008 and KD 008 variants)

[0960] In an exemplary embodiment, each X from position 1 to 115 in SEQ ID NO: 507 is independently any (e.g., naturally occurring) amino acid residue.

[0961] 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; (See specification page 79 / 226, page 87, CN 121419995 A) 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 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).

[0962] In some embodiments, X at position 1 is E or Q; X at position 5 is Q or L; X at position 11 is L or M; X at position 12 is I or V; X at position 13 is Q, H, or D; X at position 14 is P or A; X at position 16 is G or D; X at position 23 is A or V; X at position 45 is P or L; X at position 60 is P or A; X at position 62 is S or T; X at position 74 is A or S; X at position 75 is K or N; X at position 77 is T or M; X at position 86 is R, K, or T; X at position 87 is P or A; X at position 88 is D or E; X at position 92 is L or V; X at position 93 is Y or D; X at position 110 is... It is W or Q and / or X at position 115 is L or Q (SEQ ID NO: 509).

[0963] In some embodiments, X at position 1 is E or Q; X at position 5 is Q or L; X at position 11 is L or M; X at position 12 is I or V; X at position 13 is Q, H, or D; X at position 14 is P or A; X at position 16 is D; X at position 23 is A or V; X at position 45 is P or L; X at position 60 is P or A; X at position 62 is S or T; X at position 74 is A or S; X at position 75 is N; X at position 77 is M; X at position 86 is R, K, or T; X at position 87 is P or A; X at position 88 is D or E; X at position 92 is L or V; X at position 93 is D; X at position 110 is W or Q and / or X at position 115 is L or Q (SEQ ID NO). NO: 509).

[0964] In some embodiments, X at position 11 is M or L; X at position 13 is H, D or Q; X at position 14 is P or A; X at position 16 is G or D; X at position 23 is A or V; X at position 75 is K or N; X at position 77 is T or M; X at position 86 is K or T; X at position 88 is D or E; X at position 93 is Y or D and / or X at position 110 is W or Q (SEQ ID NO: 510).

[0965] In some embodiments, X at position 1 is E or Q; X at position 5 is Q or L; X at position 11 is L or M; X at position 12 is I or V; X at position 13 is Q or H; X at position 45 is P or L; X at position 60 is P or A; X at position 62 is S or T; X at position 74 is A or S; X at position 86 is R or T; X at position 87 is A or P; X at position 88 is D or E; X at position 92 is L or V and / or X at position 115 is L or Q (SE ID NO: 511).

[0966] In other exemplary embodiments, any one of X at positions 1 to 115 of SEQ ID NO: 507 is independently an amino acid residue found at the corresponding position of any one of SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 469, SEQ ID NO: 470, SEQ ID NO: 471, SEQ ID NO: 472 or SEQ ID NO: 473, or a conserved or non-conserved amino acid substitution thereof.

[0967] In yet another exemplary embodiment, each of the X values ​​in positions 1 to 115 of SEQ ID NO: 507 is independently one of SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQ ID NO: 224, SEQ ID NO: 362, SEQ ID NO: 363, SEQ ID NO: 364, SEQ ID NO: 365, SEQ ID NO: 366, SEQ ID NO: 367, SEQ ID NO: 368, SEQ ID NO: 369, SEQ ID NO: 370, SEQ ID NO: 371, SEQ ID NO: 2 ...The amino acid residue most frequently found at the corresponding position of any one of SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, or SEQ ID NO:473.

[0968] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequence shown in SEQ ID NO:512 or SEQ ID NO: 80 / 226 pages 88 CN 121419995 A 513 (e.g., Table 15D).

[0969]

[0970] In one exemplary embodiment, X from SEQ ID NO:512 at position 16, at position 69, or at position 77 is each independently any (e.g., naturally occurring) amino acid residue.

[0971] In some embodiments, X at position 16 is R or G; X at position 69 is T or I; and / or X at position 77 is M or T (SEQ ID NO:513).

[0972] In yet another embodiment, any X in SEQ ID NO:512 at position 16 or position 69 is a conserved or non-conserved amino acid substitution of the amino acid residue found at the corresponding position in SEQ ID NO:25 or SEQ ID NO:304.

[0973] The antibodies in Tables 15E, 15F, 15G, 15H and 15I all have the same CDR1 and CDR3, and there are variations in CDR2 (Figures 45A-45B).

[0974] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequences shown in SEQ ID NO:539, SEQ ID NO:540 or SEQ ID NO:541 (e.g., Tables 15E-F).

[0975]

[0976] In one exemplary embodiment, X at position 11, position 13, position 14, position 23, position 48, position 75, position 77, position 85, position 88, position 92, or position 110 is any amino acid residue, and X at position 53 is A or G (SEQ ID NO: 539).

[0977] In yet another embodiment, X at position 11, position 13, position 14, position 23, position 48, position 75, position 77, position 85, position 88, position 92, or position 110 is 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: 539.A conservative or non-conservative amino acid substitution is found at the corresponding position of any of 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 or SEQ ID NO:387, and X at position 53 is A or G (SEQ ID NO:539).

[0978] 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.

[0979] In other embodiments, X at position 11 is M or L; X at position 13 is H or Q; X at position 14 is P or A; X at position 23 is A or V; X at position 48 is V or G; X at position 53 is A or G; X at position 75 is K or N; X at position 77 is T or M; X at position 85 is R or L; X at position 88 is E or D; X at position 92 is L or V; and / or X at position 110 is W or Q. Specification 81 / 226 pages 89 CN 121419995 A

[0980] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequences shown in SEQ ID NO:514, SEQ ID NO:515, or SEQ ID NO:516 (e.g., Table 15E).

[0981]

[0982] In one exemplary embodiment, X at positions 11 to 110 in SEQ ID NO:514 is each independently any (e.g., naturally occurring) amino acid residue.

[0983] 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).

[0984] In some embodiments, X at position 11 is M or L; X at position 13 is H or Q; X at position 14 is P or A; X at position 23 is A or V; X at position 48 is V or G; X at position 75 is K or N; X at position 77 is T or M; X at position 85 is R or L; X at position 88 is E or D; X at position 92 is L or V; and / or X at position 110 is W or Q (SEQ ID NO: 516).

[0985] In other exemplary embodiments, X in any of positions 11 to 110 in SEQ ID NO:514 is each independently an amino acid residue found at the corresponding position of any 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.

[0986] In yet another exemplary embodiment, X in any one of positions 11 to 110 in SEQ ID NO:514 is each independently the amino acid residue most frequently found at the corresponding position of 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.

[0987] In one aspect of this disclosure, single-domain antibody variants include those having the amino acid sequences shown in SEQ ID NO:517, SEQ ID NO:518, or SEQ ID NO:519 (e.g., Table 15F).

[0988]

[0989] In one exemplary embodiment, in SEQ IDIn NO:517, X at pos...

Claims

1. A binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds trophoblast cell surface antigen-2 (TROP2), and further comprising: a. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:225, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequences shown 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-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:227, or; b. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:228, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown 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-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:

230.

2. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises the amino acid sequence shown 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 shown in SEQ ID NO:

540.

4. The 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 shown 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 an amino acid sequence shown in any one of SEQ ID NO: 514-516.

6. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence shown in any one of SEQ ID NO: 517-519.

7. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence shown in any one of SEQ ID NO: 520-522.

8. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence shown in any one of SEQ ID NO: 523-525.

9. The binder according to claim 1, wherein at least one of the one or more antigen-binding domains comprises an amino acid sequence shown in any one of SEQ ID NO:526-528.

10. The binder according to any one of claims 1 to 9, wherein at least one of the one or more antigen-binding domains comprises 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 ... The amino acid sequences shown in NO:397 or SEQ ID NO:398 are at least 80% identical.

11. The binder according to any one of claims 1 to 10, wherein at least one of the one or more antigen-binding domains comprises 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 ... The amino acid sequences shown in NO:397 or SEQ ID NO:398 are at least 90% identical.

12. A binder comprising one or more antigen-binding domains, wherein at least one of the one or more antigen-binding domains binds trophoblast cell surface antigen-2 (TROP2), and further comprising: a. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:488, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:

490. b. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:485, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:486, SEQ ID NO:492 or SEQ ID NO:495, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:

487. c. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:500, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:

501. d. Heavy chain complementarity-determining region 1 (CDRH1) having the amino acid sequence shown in SEQ ID NO:485, heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:498 or SEQ ID NO:486, and heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:

499. e. CDRH1 having the amino acid sequence shown in SEQ ID NO:305, CDRH2 having the amino acid sequence shown in SEQ ID NO:306, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:307; f. CDRH1 having the amino acid sequence shown in SEQ ID NO:308, CDRH2 having the amino acid sequence shown in SEQ ID NO:309, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:310; g. CDRH1 having the amino acid sequence shown in SEQ ID NO:312, CDRH2 having the amino acid sequence shown in SEQ ID NO:313 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:314; h. CDRH1 having the amino acid sequence shown in SEQ ID NO:315, CDRH2 having the amino acid sequence shown in SEQ ID NO:316 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:317; i. CDRH1 having the amino acid sequence shown in SEQ ID NO:326, CDRH2 having the amino acid sequence shown in SEQ ID NO:327, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:328; j. CDRH1 having the amino acid sequence shown in SEQ ID NO:329, CDRH2 having the amino acid sequence shown in SEQ ID NO:330, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:331; k. CDRH1 having the amino acid sequence shown in SEQ ID NO:333, CDRH2 having the amino acid sequence shown in SEQ ID NO:334 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:335; l. CDRH1 having the amino acid sequence shown in SEQ ID NO:336, CDRH2 having the amino acid sequence shown in SEQ ID NO:337 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:338; m. CDRH1 having the amino acid sequence shown in SEQ ID NO:340, CDRH2 having the amino acid sequence shown in SEQ ID NO:341, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:342; n. CDRH1 having the amino acid sequence shown in SEQ ID NO:343, CDRH2 having the amino acid sequence shown in SEQ ID NO:344 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:345; o. CDRH1 having the amino acid sequence shown in SEQ ID NO:347, CDRH2 having the amino acid sequence shown in SEQ ID NO:348, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:349; p. CDRH1 having the amino acid sequence shown in SEQ ID NO:350, CDRH2 having the amino acid sequence shown in SEQ ID NO:351 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:352; q. CDRH1 having the amino acid sequence shown in SEQ ID NO:399, CDRH2 having the amino acid sequence shown in SEQ ID NO:400, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:401; r. CDRH1 having the amino acid sequence shown in SEQ ID NO:402, CDRH2 having the amino acid sequence shown in SEQ ID NO:403 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:404; s. CDRH1 having the amino acid sequence shown in SEQ ID NO:406, CDRH2 having the amino acid sequence shown in SEQ ID NO:407 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:408; t. CDRH1 having the amino acid sequence shown in SEQ ID NO:409, CDRH2 having the amino acid sequence shown in SEQ ID NO:410 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:411; u. CDRH1 having the amino acid sequence shown in SEQ ID NO:413, CDRH2 having the amino acid sequence shown in SEQ ID NO:414 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:415; v. CDRH1 having the amino acid sequence shown in SEQ ID NO:416, CDRH2 having the amino acid sequence shown in SEQ ID NO:417 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:418; w. CDRH1 having the amino acid sequence shown in SEQ ID NO:420, CDRH2 having the amino acid sequence shown in SEQ ID NO:421 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:422; x. CDRH1 having the amino acid sequence shown in SEQ ID NO:423, CDRH2 having the amino acid sequence shown in SEQ ID NO:424, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:425; y. CDRH1 having the amino acid sequence shown in SEQ ID NO:427, CDRH2 having the amino acid sequence shown in SEQ ID NO:428, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:429; z. CDRH1 having the amino acid sequence shown in SEQ ID NO:430, CDRH2 having the amino acid sequence shown in SEQ ID NO:431, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:432; aa. CDRH1 having the amino acid sequence shown in SEQ ID NO:434, CDRH2 having the amino acid sequence shown in SEQ ID NO:435 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:436; bb. CDRH1 having the amino acid sequence shown in SEQ ID NO:437, CDRH2 having the amino acid sequence shown in SEQ ID NO:438 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:439; cc. CDRH1 having the amino acid sequence shown in SEQ ID NO:441, CDRH2 having the amino acid sequence shown in SEQ ID NO:442 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:443; dd. CDRH1 having the amino acid sequence shown in SEQ ID NO:444, CDRH2 having the amino acid sequence shown in SEQ ID NO:445 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:446; ee. CDRH1 having the amino acid sequence shown in SEQ ID NO:448, CDRH2 having the amino acid sequence shown in SEQ ID NO:449 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:450; ff. CDRH1 having the amino acid sequence shown in SEQ ID NO:451, CDRH2 having the amino acid sequence shown in SEQ ID NO:452 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:453; gg. CDRH1 having the amino acid sequence shown in SEQ ID NO:455, CDRH2 having the amino acid sequence shown in SEQ ID NO:456 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:457; hh. CDRH1 having the amino acid sequence shown in SEQ ID NO:458, CDRH2 having the amino acid sequence shown in SEQ ID NO:459 and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:460; ii. CDRH1 having the amino acid sequence shown in SEQ ID NO:462, CDRH2 having the amino acid sequence shown in SEQ ID NO:463, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:464; jj. CDRH1 having the amino acid sequence shown in SEQ ID NO:465, CDRH2 having the amino acid sequence shown in SEQ ID NO:466, and / or CDRH3 having the amino acid sequence shown in SEQ ID NO:

467.

13. The binder of 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 shown in SEQ ID NO:488, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:490, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in any one of SEQ ID NO:493, SEQ ID NO:496, or SEQ ID NO:

491.

14. The binder of 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 shown in SEQ ID NO: 500, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO: 489, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO: 501, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown in either SEQ ID NO: 503 or SEQ ID NO:

502.

15. The binder of 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 shown in SEQ ID NO:308, a heavy chain complementarity-determining region 2 (CDRH2) having the amino acid sequence shown in SEQ ID NO:309, and a heavy chain complementarity-determining region 3 (CDRH3) having the amino acid sequence shown in SEQ ID NO:310, and an amino acid sequence that is at least 80% identical to the amino acid sequence shown 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 backbone, the backbone comprising the amino acid sequence shown 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 backbone, the backbone comprising the amino acid sequence shown in SEQ ID NO:213, SEQ ID NO:479 or SEQ ID NO:

480.

20. 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 backbone, the backbone comprising 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, SEQ ID NO:266, SEQ ID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270 or SEQ ID NO:

249. The amino acid sequence shown in NO:271, or an amino acid sequence containing one to five amino acid substitutions, additions, and / or deletions.

21. 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 backbone, the backbone comprising 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:285, SEQ ID NO:286, 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:

276. The amino acid sequence shown in NO:476, or an amino acid sequence containing one to five amino acid substitutions, additions, and / or deletions thereof.

22. The binder according to any one of the preceding claims, wherein the heavy chain variable region comprises a frame region 2 (FR2), the frame region 2 comprising the sequence shown 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 trophoblast cell surface antigen-2 (TROP2).and comprises an amino acid sequence shown in SEQ ID NO:373, SEQ ID NO:493, SEQ ID NO:503, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, SEQ ID NO:224, SEQ ID NO:363, SEQ ID NO:364, SEQ ID NO:365, SEQ ID NO:366, SEQ ID NO:367, SEQ ID NO:368, SEQ ID NO:369, SEQ ID NO:370, SEQ ID NO:371, SEQ ID NO:469, SEQ ID NO:470, SEQ ID NO:471, SEQ ID NO:472, SEQ ID NO:473, SEQ ID NO:304, 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: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, 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 of claim 24, wherein the binder comprises a dimerizing domain.

25. The binder according to any one of the preceding claims, 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 the preceding claims, 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 Camelidae VH or VHH.

28. The binder according to any one of the preceding claims, wherein the binder comprises two or more antigen-binding domains.

29. The binder according to any one of the preceding claims, wherein the binder is single-specific.

30. The binder according to any one of claims 1 to 28, wherein the binder is bispecific.

31. The binder according to any one of claims 1 to 28, wherein the binder is multispecific.

32. The binder according to claim 30 or 31, wherein the binder comprises one or more antigen-binding domains that bind CD47 and / or one or more antigen-binding domains that bind PD-1.

33. The binder according to any one of claims 1 to 32, wherein the binder comprises two polypeptide chains, and wherein each polypeptide chain comprises, from N-terminus to C-terminus: a) one or more antigen-binding domains, b) optional linkers, and c) dimerization domains.

34. The binder according to any one of claims 1 to 32, wherein the binder comprises two polypeptide chains, and wherein each polypeptide chain comprises 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 Where m is 0, 1, 2 or an integer greater than 2; Where n is 0, 1, 2 or an integer greater than 2; Where m and n are not both 0; Where Ab a Ab d Each represents an antigen-binding domain, wherein at least one of the antigen-binding domains is antigen-binding domain 1 (ABD1), antigen-binding domain 2 (ABD2), or antigen-binding domain 3 (ABD3). Where X or Y is present or absent independently, and contains an amino acid sequence; Where L b L c Each independently contains one or more connectors; and Where DD represents the dimerization domain.

35. The binder according to claim 24, 33 or 34, wherein the dimerizing domain comprises the CH3 domain of the antibody or a portion thereof.

36. The binder of claim 35, wherein the dimerizing 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 the heavy chain of human IgG1, human IgG2, human IgG3 or human IgG4.

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 shown in SEQ ID NO:212 or SEQ ID NO:

506.

39. The binder according to any one of the preceding claims, wherein the binder comprises at least two antigen-binding domains, and wherein the at least two antigen-binding domains are the same or different.

40. The binder according to any one of the preceding claims, wherein the binder comprises an Fc or Fc portion capable of binding to an Fc receptor on monocytes and / or macrophages.

41. The binder according to any one of the preceding claims, wherein the one or more antigen-binding domains are arranged in tandem.

42. The binding agent according to any one of the preceding claims, wherein the binding agent comprises a single-domain antibody.

43. The binding agent according to any one of the preceding claims, wherein the binding agent is a single-domain antibody.

44. The binder according to any one of the preceding claims, wherein the binder is bare.

45. The binder according to any one of the preceding claims, wherein the binder is conjugated to the treatment portion.

46. ​​The binder according to any one of the preceding claims, wherein the binder is conjugated to the detectable portion.

47. The binder according to any one of the preceding claims, wherein the one or more antigen-binding domains or the binder specifically binds to human TROP2.

48. The binding agent according to any one of the preceding claims, wherein the binding agent is a single-domain antibody comprising two identical heavy chain variable regions or two identical heavy chains.

49. A pharmaceutical composition comprising the binder or single-domain antibody as described in any of the preceding claims, and a pharmaceutically acceptable carrier.

50. A nucleic acid or nucleic acid genome encoding a binding agent or single-domain antibody as described in any of the preceding claims.

51. A vector comprising the nucleic acid of claim 50.

52. A cell expressing the binding agent or single-domain antibody as described in any of the preceding claims.

53. A cell comprising the nucleic acid of claim 50 or the vector of claim 51.

54. A kit comprising the binding agent or single-domain antibody as described in any of the preceding claims.

55. A kit comprising the nucleic acid of claim 50, the vector of claim 51, or the cells of claim 52 or 53.

56. A method of treating a symptom or disease, comprising administering a binder or single-domain antibody as described in any of the preceding claims, or a pharmaceutical composition comprising a binder or single-domain antibody as described in any of the preceding claims.

57. The method of claim 56, wherein the condition or disease is associated with the expression or overexpression of TROP2.

58. The method of claim 57, wherein the condition 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, rectal cancer, pancreatic cancer, glioblastoma, cervical cancer, colorectal cancer, gastric cancer, ovarian cancer, thyroid cancer, stomach cancer, bladder cancer, uterine cancer, esophageal cancer, hematologic malignancy, or head and neck cancer.

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