Anti-Nectin-4 antibody, its antibody-drug conjugate and applications

Abstract

The present invention discloses an anti-Nectin-4 antibody, its antibody-drug conjugate and applications, and the anti-Nectin-4 antibody and its antibody-drug conjugate can be used to treat diseases.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine and relates to an anti-Nectin-4 antibody, its antibody-drug conjugate and applications. [Background technology]

[0002] Nectin-4 is a calcium-independent cell adhesion molecule (CAM) of the immunoglobulin superfamily (IgSF), also known as poliovirus receptor-like protein 4 (PVRL4) or poliovirus receptor-related (PRR4). Nectin-4 is a single-pass transmembrane protein consisting of an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain contains three highly glycosylated domains: two C2 domains proximal to the cell membrane and one V domain distal to the cell membrane. Nectin-4 binds to H protein via its N-terminal V domain, thereby achieving viral infection of cells. Nectin-4 is involved in the formation and maintenance of adherens junctions through its interaction with cadherins. Nectin-4 mediates Ca2+-independent adhesion and promotes anchorage-independent growth by driving cell-cell adhesion and activation of the matrix-independent integrin protein β4 / SHP-2 / c-Src.

[0003] Nectin-4 is expressed during fetal development, and unlike the widespread expression of other nectins in adult tissues, its expression in adult tissues is significantly reduced. Multiple research groups have confirmed that Nectin-4 is re-expressed as a tumor-associated antigen of carcinogenic factors in various tumors, including breast cancer and bladder cancer. Nectin-4 is weakly to moderately expressed in normal adult tissues, such as the stratum corneum, skin appendages (sweat glands and hair follicles), bladder transitional epithelium, salivary glands, esophagus, mammary glands, and stomach, and relatively weakly expressed in the larynx, pituitary gland, placenta, testis, ureter, and uterus. This target has relatively good specificity and can mediate endocytosis of antibodies, making it a potential therapeutic target for ADC drugs. Summary of the Invention

[0004] The present application provides anti-Nectin-4 antibodies and drug conjugates thereof.

[0005] One or more embodiments of the present application provide an antibody or antigen-binding unit, which specifically binds to Nectin-4 and (a) a VH CDR1 comprising the amino acid sequence shown in SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 5; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7; It includes one or more of the following.

[0006] In one or more embodiments, the antibody or antigen binding unit comprises a VL CDR3 set forth in SEQ ID NO:7, and optionally further comprises one or more of a VH CDR1 set forth in SEQ ID NO:2, a VH CDR2 set forth in SEQ ID NO:3, a VH CDR3 set forth in SEQ ID NO:4, a VL CDR1 set forth in SEQ ID NO:5, and a VL CDR2 set forth in SEQ ID NO:6.

[0007] In one or more embodiments, the antibody or antigen-binding unit comprises a VL CDR1 set forth in SEQ ID NO:5, a VL CDR2 set forth in SEQ ID NO:6, and a VL CDR3 set forth in SEQ ID NO:7.

[0008] In one or more embodiments, the antibody or antigen-binding unit comprises a VH CDR1 set forth in SEQ ID NO: 2, a VH CDR2 set forth in SEQ ID NO: 3, a VH CDR3 set forth in SEQ ID NO: 4, a VL CDR1 set forth in SEQ ID NO: 5, a VL CDR2 set forth in SEQ ID NO: 6, and a VL CDR3 set forth in SEQ ID NO: 7.

[0009] In one or more embodiments, the antibody or antigen-binding unit comprises a heavy chain variable region and / or a light chain variable region. In one or more embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 8. In one or more embodiments, the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 10. In one or more embodiments, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:10.

[0010] In one or more embodiments, the antibody or antigen-binding unit further comprises a heavy chain constant region and / or a light chain constant region. In one or more embodiments, the heavy chain constant region is selected from the IgG1, IgG2, IgG3, or IgG4 type. In one or more embodiments, the light chain constant region is a λ or κ chain constant region.

[0011] In one or more embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 9, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 9. In one or more embodiments, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 11. In one or more embodiments, the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 9, and the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0012] In one or more embodiments, the antibody comprises a heavy chain and a light chain, hi one or more embodiments, the antibody comprises two heavy chains with identical sequences and two light chains with identical sequences.

[0013] In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 12. In one or more embodiments, the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 14. In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 12, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having one or more conservative amino acid substitutions compared to the amino acid sequence set forth in SEQ ID NO: 14. In one or more embodiments, the heavy chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 12, and the light chain of the antibody comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0014] One or more embodiments of the present application provide a biomaterial, comprising: (1) a nucleic acid encoding the antibody or antigen-binding unit or a portion thereof; (2) A vector, host cell, or microorganism containing (1), (3) the expression product, suspension, or supernatant of (2) above; The present invention provides a biomaterial selected from the group consisting of:

[0015] In one or more embodiments, the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 12 is as set forth in SEQ ID NO: 13. In one or more embodiments, the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 14 is as set forth in SEQ ID NO: 15. [Table 1-1] [Table 1-2] [Table 1-3]

[0016] One or more embodiments of the present application provide a method for producing the antibody or antigen-binding unit of the present application, comprising culturing the host cell of the present application to express the antibody or antigen-binding unit of the present application, and isolating the antibody or antigen-binding unit from the host cell.

[0017] One or more embodiments of the present application provide an antibody-drug conjugate comprising the above-described antibody or antigen-binding unit, or a pharmaceutically acceptable salt or solvate thereof, coupled to a drug (e.g., a drug described herein) via a linker.

[0018] In one or more embodiments, the linker is a degradable linker.

[0019] One or more embodiments of the present application provide an antibody drug conjugate having the structure of Formula IA, Formula IB, Formula IC, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: [ka] Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (eg, a drug described herein).

[0020] One or more embodiments of the present application provide an antibody drug conjugate having the structure of formula IA or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (e.g., a drug described herein); M is [ka] where * is connected to Abu, ** is connected to B, and R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -Arylene-(CH2) r -, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m(CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which each R m are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; B is [ka] where * is connected to M, ** is connected to L, and *** is connected to G, L is -(AA) i -(FF) f - in which AA is an amino acid or polypeptide and i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or each AA is independently Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, and each FF is independently selected from the amino acid or peptide sequences Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly. [ka] Of these, each R Fare independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, wherein * is linked to AA, ** is linked to D, z is 0, 1, 2, 3 or 4, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G is [ka] wherein n is 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10.

[0021] In one or more embodiments, R is —(CH) r - and r is 1 or 5.

[0022] In one or more embodiments, AA is Val-Cit and i is 1.

[0023] In one or more embodiments, each FF independently [ka] where * is connected to AA and ** is connected to D.

[0024] In one or more embodiments, FF is [ka] and f is 1, in which * is connected to AA and ** is connected to D.

[0025] In one or more embodiments, L is [ka] where * is connected to B and ** is connected to D.

[0026] In one or more embodiments, L is [ka] where * is connected to B and ** is connected to D.

[0027] In one or more embodiments, G is [ka] where n is 4 to 12.

[0028] In one or more embodiments, n is 4-8.

[0029] In one or more embodiments, n is 4.

[0030] In one or more embodiments, n is 8.

[0031] In one or more embodiments, p is 2-8.

[0032] In one or more embodiments, p is 4-8.

[0033] In one or more embodiments, p is 6-8.

[0034] In one or more embodiments, p is 7-8.

[0035] One or more embodiments of the present application provide an antibody drug conjugate having the structure of formula IB or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (e.g., a drug described herein); M is [ka] where * is connected to Abu, ** is connected to L, and R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -arylene-(CH2)r-, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which each R m are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; L is -(AA) i -(FF) fwherein AA is an amino acid or polypeptide, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and each AA is independently selected from Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, C and each FF is independently selected from the amino acid or peptide sequences of Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly. [ka] Of these, each R F are independently a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, wherein * is linked to AA, ** is linked to D, z is 0, 1, 2, 3 or 4, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p is 1 to 10.

[0036] In one or more embodiments, R is —(CH) r - and r is 1 or 5.

[0037] In one or more embodiments, AA is Val-Cit and i is 1.

[0038] In one or more embodiments, each FF independently [ka] where * is connected to AA and ** is connected to D.

[0039] In one or more embodiments, FF is [ka] and f is 1, in which * is connected to AA and ** is connected to D.

[0040] In one or more embodiments, L is [ka] where * is connected to M and ** is connected to D.

[0041] In one or more embodiments, L is [ka] where * is connected to M and ** is connected to D.

[0042] In one or more embodiments, p is 2-8.

[0043] In one or more embodiments, p is 4-8.

[0044] In one or more embodiments, p is 6-8.

[0045] In one or more embodiments, p is 7-8.

[0046] One or more embodiments of the present application provide an antibody drug conjugate having the structure of formula IC or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (e.g., a drug described herein); M is [ka] where * is connected to Abu, ** is connected to V, and R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -arylene-(CH2)r-, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which each R m are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; V is [ka] wherein * is connected to M and ** is connected to -NH-CH-; p is 1 to 10.

[0047] In one or more embodiments, R is —(CH) r - and r is 1 or 5.

[0048] In one or more embodiments, V is [ka] wherein * is connected to M and ** is connected to -NH-CH2-.

[0049] In one or more embodiments, p is 2-8.

[0050] In one or more embodiments, p is 4-8.

[0051] In one or more embodiments, p is 6-8.

[0052] In one or more embodiments, p is 7-8.

[0053] One or more embodiments of the present application provide an antibody-drug conjugate having a structure of formula IA-1 or IA-2 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the above formulas IA-1 and IA-2 are as follows: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -arylene-(CH2)r-, -(CH2) r-(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2) r -, among which each R m are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; D is a drug (e.g., a drug described herein); n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10.

[0054] In one or more embodiments, R is —(CH 2 ) r —, where r is 1 or 5.

[0055] In one or more embodiments, n is 4-12.

[0056] In one or more embodiments, n is 4-8.

[0057] In one or more embodiments, n is 4.

[0058] In one or more embodiments, n is 8.

[0059] In one or more embodiments, p is 2-8.

[0060] In one or more embodiments, p is 4-8.

[0061] In one or more embodiments, p is 6-8.

[0062] In one or more embodiments, p is 7-8.

[0063] One or more embodiments of the present application provide an antibody-drug conjugate having a structure of formula IA-3 or IA-4 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the above formulas IA-3 and IA-4 are as follows: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (e.g., a drug described herein); n is an integer from 1 to 24, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24; p is 1 to 10.

[0064] In one or more embodiments, n is 4-12.

[0065] In one or more embodiments, n is 4-8.

[0066] In one or more embodiments, n is 4.

[0067] In one or more embodiments, n is 8.

[0068] In one or more embodiments, p is 2-8.

[0069] In one or more embodiments, p is 4-8.

[0070] In one or more embodiments, p is 6-8.

[0071] In one or more embodiments, p is 7-8.

[0072] In one or more embodiments, there is provided an antibody drug conjugate having the structure of formula IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, or IA-11, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein the above formulas IA-5, IA-6, IA-7, IA-8, IA-9, IA-10, IA-11 are as follows: [ka] [ka] [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, D is a drug (e.g., a drug described herein); p is 1 to 10.

[0073] In one or more embodiments, p is 2-8.

[0074] In one or more embodiments, p is 4-8.

[0075] In one or more embodiments, p is 6-8.

[0076] In one or more embodiments, p is 7-8.

[0077] In one or more embodiments, the drug in the antibody drug conjugates described herein is a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease.

[0078] In one or more embodiments, the drug is an anti-cancer drug.

[0079] In one or more embodiments, the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor.

[0080] In one or more embodiments, the drug is a tubulin inhibitor, and the tubulin inhibitor is selected from dolastatin, auristatins, and maytansines.

[0081] In one or more embodiments, the drug is an auristatin and is selected from monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and auristatin F (AF).

[0082] In one or more embodiments, the drug is a DNA damaging agent selected from the group consisting of calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine).

[0083] In one or more embodiments, the drug is a DNA topoisomerase inhibitor or a salt thereof, and is selected from the group consisting of irinotecan, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivatives SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolinone, 2-cyano-3-(3,4-dihydroxybenzoyl) ... and N-[2-(dimethylamino)ethyl]-4-acridinecarboxamide dihydrochloride, ...

[0084] In one or more embodiments, the DNA topoisomerase inhibitor is camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, or exatecan.

[0085] In one or more embodiments, the drug is [ka] and among them X 1 and X 2 are each independently H, hydroxy groups, C1 to C6 alkyl group, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups, or cyano groups; a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, nitro group, cyano group, mercapto group, alkylthio groups, an amino group, an amino group substituted by an amino group-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino group moiety by an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino group moiety with an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocycle, the heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocycle, wherein the heterocyclyl is optionally substituted with a C1-C6 alkyl group or a C1-C6 alkoxy group, and the amino group is optionally substituted with an amino-protecting group, halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, wherein the nitrogen atom or the amino group moiety in the heterocyclyl moiety is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino group, the nitrogen atom or amino moiety of which is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X 3 is a C1-C6 alkyl group, X 4 is H, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q -CH3, -(CH2) q -(C3-C8 carbocyclyl)-CH3, -(C3-C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3-C8 heterocyclyl)-CH3, -(C3-C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3, or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, wherein each R n are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; ** is the connection point, y is 0, 1 or 2; Y is O, S or CR 1D R 2D Among them, R 1D and R 2D are each independently H or a C1-C6 alkyl group, s and t are each independently 0, 1 or 2, but are not both 0.

[0086] In one or more embodiments, X 4 is H or a C1 to C6 alkyl group.

[0087] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0088] In one or more embodiments, the C1-C6 alkyl group is —CH3.

[0089] In one or more embodiments, the halogen is F.

[0090] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0091] In one or more embodiments, the C1-C6 alkyl group is —CH3.

[0092] In one or more embodiments, the halogen is F.

[0093] In one or more embodiments, X1 and X 2 are -CH3, respectively.

[0094] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0095] In one or more embodiments, X 1 and X 2 are F, respectively.

[0096] In one or more embodiments, X 1 and X 2 are each independently —CH 3 , F, or —OH.

[0097] In one or more embodiments, X 1 and X 2 are each independently F or —CH3.

[0098] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0099] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0100] In one or more embodiments, the C1-C6 alkyl group is —CH3.

[0101] In one or more embodiments, the halogen is F.

[0102] In one or more embodiments, the drug is [ka] Among them, X 1 and X 2 are each independently a C1 to C6 alkyl group, halogen or -OH, and ** is a connection point.

[0103] In one or more embodiments, the C1-C6 alkyl group is —CH3.

[0104] In one or more embodiments, the halogen is F.

[0105] In one or more embodiments, X 1 and X 2 are -CH3, respectively.

[0106] In one or more embodiments, X 1 and X 2 are each independently F, Cl, Br or I.

[0107] In one or more embodiments, X 1 and X 2 are F, respectively.

[0108] In one or more embodiments, X 1 and X 2 are each independently —CH 3 , F, or —OH.

[0109] In one or more embodiments, X 1 and X 2 are each independently F or —CH3.

[0110] In one or more embodiments, X 1 is -CH3, and X 2 is F.

[0111] In one or more embodiments, the drug is [ka] where ** is a connection point, R 2 is H or a C1-C8 alkyl group, R 3 is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), R 4 is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), R 5 is H or a methyl group, Or, R 4 and R 5 are linked to form a carbocyclyl group and have the formula -(CR a R b ) j -, in which R a and R b are each independently H, a C1-C8 alkyl group, or a C3-C8 carbocyclyl group, and j is 2, 3, 4, 5, or 6; R 6 is H or a C1-C8 alkyl group, R 7 is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), Each R 8 are each H, OH, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, or O—(C1-C8 alkyl), R 9 is H or a C1-C8 alkyl group, and R 10 is -C(R8 )2-C(R 8 )2-aryl, -C(R 8 )2-C(R 8 )2-(C3-C8 heterocyclyl) or -C(R 8 )2-C(R 8 )2-(C3-C8 carbocyclyl).

[0112] In one or more embodiments, the drug is [ka] where ** is a connection point.

[0113] In one or more embodiments, there is provided an antibody drug conjugate having the structure of formula IA-12, IA-13, IA-14, IA-15, IA-16, IA-17, IA-18, IA-19, IA-20, IA-21, IA-22, IA-23, IA-24, or IA-25, or a stereoisomer thereof, wherein said formulas IA-12, IA-13, IA-14, IA-15, IA-16, IA-17, IA-18, IA-19, IA-20, IA-21, IA-22, IA-23, IA-24, IA-25 are as follows: [ka] [ka] [ka] [ka] [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, p is 1 to 10.

[0114] In one or more embodiments, p is 2-8.

[0115] In one or more embodiments, p is 4-8.

[0116] In one or more embodiments, p is 6-8.

[0117] In one or more embodiments, p is 7-8.

[0118] In one or more embodiments, there is provided an antibody drug conjugate having the structure of formula IB-1 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein said formula IB-1 is as follows: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, p is 1 to 10.

[0119] In one or more embodiments, p is 2-8.

[0120] In one or more embodiments, p is 4-8.

[0121] In one or more embodiments, p is 6-8.

[0122] In one or more embodiments, p is 7-8.

[0123] In one or more embodiments, there is provided an antibody drug conjugate having the structure of Formula IC-1 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein said Formula IC-1 is as follows: [ka] Eventually Abu is the anti-Nectin-4 antibody or antigen-binding unit of the present application, p is 1 to 10.

[0124] In one or more embodiments, p is 2-8.

[0125] In one or more embodiments, p is 4-8.

[0126] In one or more embodiments, p is 6-8.

[0127] In one or more embodiments, p is 7-8.

[0128] In one or more embodiments, there is provided a pharmaceutical composition comprising the present anti-Nectin-4 antibody or antigen-binding unit, the present biomaterial, or the present antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, and / or additive. In one or more embodiments, the pharmaceutical composition optionally further comprises another drug.

[0129] One or more embodiments provide a pharmaceutical composition comprising an antibody or antigen-binding unit, antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, and / or additive. In one or more embodiments, the pharmaceutical composition optionally further comprises another anti-cancer drug. The pharmaceutical composition may be administered by any convenient route, for example, by infusion or bolus injection, may be absorbed through epithelial or mucocutaneous (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other bioactive agents. Thus, the pharmaceutical composition may be administered subcutaneously, intravenously, orally, rectally, parenterally, intracerebrally, vaginally, intraperitoneally, topically (e.g., via powder, ointment, drops, or transdermal patch), buccally, or as an oral or nasal spray.

[0130] In some embodiments, the composition is prepared by conventional steps as a pharmaceutical composition suitable for intravenous injection into humans. Compositions for intravenous administration are usually solutions in sterile isotonic buffer. The composition may further include a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site. Generally, the active ingredients are provided singly or in admixture in unit dosage form, for example, in a hermetically sealed container (e.g., an ampoule or sachet) indicating the amount of active agent in the form of a dry lyophilized powder or water-free concentrate. When the composition is administered by injection, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be used to allow the active ingredients to be mixed prior to administration.

[0131] In one or more embodiments, there is provided use of the anti-Nectin-4 antibody or antigen-binding unit, biomaterial, or antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof described herein, or the pharmaceutical composition in the manufacture of a drug for treating a disease. In one or more embodiments, the drug is further used in combination with another drug.

[0132] In one or more embodiments, there is provided a use of an antibody or antigen-binding unit described herein, a biomaterial of the present application, an antibody-drug conjugate of the present application or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition of the present application in the treatment of a disease, in one or more embodiments, the use includes combination with other anti-cancer drugs.

[0133] One or more embodiments provide a method of treating a disease, the method comprising administering to a patient in need thereof an effective amount of an antibody or antigen-binding unit, antibody-drug conjugate, or pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition described herein. The effective amount refers to the amount of active compound or drug that produces the biological or pharmaceutical response, including treatment of the disease, in a tissue, system, animal, individual, or human that a researcher, veterinarian, physician, or other clinician is seeking. Typically, a suitable dosage range may be about 0.1 to 100 milligrams per kilogram, and the administration frequency may be, for example, once a month. The administration form may be intravenous infusion, intravenous bolus injection, subcutaneous injection, intramuscular injection, or the like. In one or more embodiments, the method further comprises administering to the patient another anti-cancer drug.

[0134] In one or more embodiments, the disease is a disease associated with Nectin-4 expression or overexpression. In one or more embodiments, the disease is a disease associated with abnormal Nectin-4 expression. In one or more embodiments, the disease is a cancer or tumor. In one or more embodiments, the disease is a tumor that expresses or overexpresses Nectin-4. In one or more embodiments, the disease is a cancer that expresses or overexpresses Nectin-4. In one or more embodiments, the disease is a solid tumor or a blood cancer. In one or more embodiments, the disease is selected from breast cancer (e.g., triple-negative breast cancer (TNBC), localized, metastatic TNBC), pancreatic cancer, bladder cancer, urothelial cancer, melanoma, lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma, or lung adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer (e.g., squamous cell carcinoma of the cervix), ovarian cancer, choriocarcinoma, skin cancer, esophageal cancer (e.g., esophageal adenocarcinoma), gastric cancer, uterine cancer (e.g., endometrial cancer), gallbladder cancer, liver cancer, hepatocellular carcinoma, urethral cancer, renal pelvis cancer, ureter cancer, colorectal cancer, colon cancer, and prostate cancer.

[0135] In one or more embodiments, the antibody drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, is an antibody drug conjugate of Formula IA, Formula IB, Formula IC of the present application, or a pharmaceutically acceptable salt or solvate thereof.

[0136] Pharmaceutically acceptable salts include those produced by antibody drug conjugates and a variety of organic and inorganic counterions well known in the art; merely exemplary salts include organic or inorganic salts such as sodium, potassium, calcium, magnesium, ammonium, isopropylamine, trimethylamine, diethylamino, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethyl, piperidine, polyamine resins, and tetraalkylammonium salts when the molecule contains an acidic functional group; and organic or inorganic acid salts such as hydrochloride, hydrobromide, tartrate, methanesulfonate, acetate, maleate, and oxalate when the molecule contains a basic functional group. Other non-limiting examples of acids include sulfuric acid, nitric acid, phosphoric acid, propionic acid, glycolic acid, pyruvic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc. Solvates include hydrates.

[0137] One or more embodiments include an antibody or antigen-binding unit, an antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition described herein; containers, and The present invention provides a product comprising a package insert, label, or instruction indicating that the antibody or antigen-binding unit, antibody-drug conjugate, or pharmaceutically acceptable salt or solvate thereof, or pharmaceutical composition is used to treat a disease. [Brief explanation of the drawings]

[0138] [Figure 1] Binding curves of antibodies ASG-AM and ASG-22 to T47D cells. [Figure 2A] Figure 2 shows the growth inhibitory effect of ADC on OVCAR-3 cells, T-47D cells, and MDA-MB-468 cells. Figure 2A shows the growth inhibitory effect of ADC on OVCAR-3 cells. [Figure 2B] Figure 2B shows the growth inhibitory effect of ADC on OVCAR-3 cells, T-47D cells, and MDA-MB-468 cells. Figure 2B shows the growth inhibitory effect of ADC on T-47D cells. [Figure 2C] Figure 2C shows the growth inhibitory effect of ADCs on OVCAR-3 cells, T-47D cells, and MDA-MB-468 cells. Figure 2C shows the growth inhibitory effect of ASG-AM-MMAE on MDA-MB-468 cells. [Figure 3] Tumor-suppressive activity of ADC in a human breast cancer MDA-MB-468 subcutaneously transplanted tumor model in nude mice. [Figure 4] Tumor suppressive activity of ADC in a female NOD / SCID mouse animal model subcutaneously xenografted with human choriocarcinoma JEG-3 cell line. DETAILED DESCRIPTION OF THE INVENTION

[0139] Unless otherwise defined, scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0140] definition It should be noted that the term "one" entity refers to one or more of that entity, e.g., "one antibody" should be understood as one or more antibodies, and therefore the terms "one" (or "one"), "one or more" and "at least one" may be used interchangeably herein.

[0141] As used herein, the terms "comprising" or "including" mean that the antibody, composition, method, etc. includes the recited elements, such as components or steps, but does not exclude others. "Consisting essentially of" means that the antibody, composition, method, etc. excludes other elements that materially affect the characteristics of the combination, but does not exclude elements that do not materially affect the antibody, composition, method, etc. "Consisting of" means excluding elements not specifically recited.

[0142] The term "antibody," as used herein, refers to immunoglobulin (Ig) molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. Antibodies include, but are not limited to, monoclonal antibodies, chimeric antibodies, dAbs (domain antibodies), single-chain antibodies, Fab, Fab- and F(ab')2 fragments, Fv and Fab expression libraries.

[0143] The term "antibody" encompasses a wide variety of biochemically distinguishable polypeptides. The antibodies, antigen-binding units or derivatives disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., class Fab, class Fab', and class F(ab')2), and class single-chain Fvs (scFv).

[0144] The term "monoclonal antibody" (mAb) refers to a population of antibody molecules that contain only one type of antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity-determining regions (CDRs) of a monoclonal antibody are the same in all molecules of the population. MAbs contain an antigen-binding site capable of immunoreacting with a specific epitope of an antigen.

[0145] The term "single-chain antibody" (scFv) refers to an antibody in which the heavy chain variable region (VH) and light chain variable region (VL) of the antibody are linked by a linker of 15 to 20 amino acids. The linker may be glycine-rich to increase flexibility, or serine- or threonine-rich to increase solubility, and may be linked to the N-terminus of the VH and the C-terminus of the VL, or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are generally known in the art, for example, as described in U.S. Patent No. 5,892,019.

[0146] Those skilled in the art will understand that heavy chain classes include gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε), and further include several subclasses (e.g., γ1 to γ4). The nature of the chain determines the "type" of an antibody, whether it is IgG, IgM, IgA, IgD, or IgE. For example, immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, and IgG5, have already been well characterized, and their associated functional specificities are known. All immunoglobulin types are within the scope of the present invention. In one or more embodiments, the immunoglobulin molecule type is IgG. Two heavy chains and two light chains are connected by disulfide bonds in a "Y" configuration, with the light chains starting at the mouth of the "Y" and surrounding the heavy chains through the variable region. Light chains can be classified as kappa (κ) or lambda (λ). Each heavy chain can be linked to a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are linked via covalent bonds, and the "tails" of the two heavy chains are linked via covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. The kappa light chain variable region of an immunoglobulin is V κ and the lambda light chain variable region of the immunoglobulin is V λ is.

[0147] The antibody light chain variable region (VL) and heavy chain variable region (VH) determine antigen recognition and specificity. The light chain constant region (CL) and heavy chain constant region (CH) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement fixation. By convention, the numbering of constant regions increases with increasing distance from the antigen-binding site or amino terminus of the antibody. The N-terminal portion is the variable region, and the C-terminal portion is the constant region; the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0148] In naturally occurring antibodies, assuming that the antibody exhibits its three-dimensional configuration in an aqueous environment, the six "complementarity-determining regions" or "CDRs" present in each antigen-binding domain are short, noncontiguous amino acid sequences that specifically bind to antigens, forming the antigen-binding domain. The remaining amino acids in the antigen-binding domain, called "framework" ("FR") regions, exhibit relatively little intermolecular variability. Most of the framework regions adopt a β-sheet conformation, forming a cyclic structure to which the CDRs are connected, or in some cases, forming part of the β-sheet structure. Thus, the framework regions form a stent, positioning the CDRs in the correct orientation through interchain noncovalent interactions. An antigen-binding domain with CDRs at specific positions forms a surface complementary to the epitope on the antigen, and this complementary surface promotes noncovalent binding of the antibody to its antigen epitope. Typically, an antibody molecule has three CDRs in each heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3, respectively. Typically, in order of position, a heavy chain variable region comprises VH FR1, VH CDR1, VH FR2, VH CDR2, VH FR3, VH CDR3, and VH FR4, and a light chain variable region comprises VL FR1, VL CDR1, VL FR2, VL CDR2, VL FR3, VL CDR3, and VL FR4. For a given heavy or light chain variable region, one skilled in the art can identify the amino acids comprising the CDR and framework regions by known methods (see Kabat, E., et al., USDapartment of Health and Human Services, Sequences of Proteins of Immunological Interest, (1983) and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0149] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor antibody CDR or shared framework can be mutagenized by substitution, insertion, and / or deletion of at least one amino acid residue, such that the CDR or framework residue at that site does not correspond to the donor antibody or shared framework. Typically, at least 80%, at least 85%, or even at least 90% or at least 95% of the humanized antibody residues correspond to those residues in the parental FR and CDR sequences. As used herein, the term "shared framework" refers to the framework region in a shared immunoglobulin sequence. As used herein, the term "shared immunoglobulin sequence" refers to the sequence formed by the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). Within an immunoglobulin family, each position in the shared sequence is occupied by the amino acid that occurs most frequently at that position within the family. If two amino acids occur equally frequently, the shared sequence may include either one.

[0150] Where a term used and / or accepted in the art has two or more definitions, the definition of the term used herein includes all of these meanings unless otherwise expressly indicated to the contrary. A specific example is the use of the term "complementarity-determining region" ("CDR") to describe the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides. This particular region is described in Kabat et al., U.S. Dept. of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) and Chothia et al., J. Mol. Biol. 196:901-917 (1987), all of which are incorporated herein by reference. Kabat et al. further defined a numbering system that can be applied to any antibody variable region sequence. One skilled in the art can apply the "Kabat numbering" system to any variable region sequence without relying on any experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed in Kabat et al., US Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU or Chothia numbering systems.

[0151] The antibodies disclosed herein may be derived from any animal, including, but not limited to, fish, birds, and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, camel, llama, horse, or chicken. In other embodiments, the variable region may be derived from a chondricthoid (e.g., shark).

[0152] A "heavy chain constant region" comprises at least one of a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment. The heavy chain constant region of an antibody may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody may comprise a CH1 structural domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In other embodiments, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and a hinge region derived in part from an IgG3 molecule. In other embodiments, a portion of the heavy chain may comprise a chimeric hinge region derived in part from an IgG1 molecule and a chimeric hinge region derived in part from an IgG4 molecule.

[0153] A "light chain constant region" comprises a portion of an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain. A "light chain-heavy chain pair" refers to an assembly of a light chain and a heavy chain that can form a dimer via disulfide bonding between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0154] A "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The thiol group of a cysteine can form a disulfide bond or cross-link with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond.

[0155] A "chimeric antibody" refers to any antibody whose variable regions are obtained or derived from a first species and whose constant regions (which may be complete, partial, or modified) are derived from a second species. In some embodiments, the variable regions are non-human (e.g., murine or primate) and the constant regions are human.

[0156] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or fragment thereof or a T-cell receptor. Epitope determinants usually consist of chemically active surface groupings of molecules (e.g., amino acids or sugar side chains) and usually have specific three-dimensional structural characteristics and specific charge characteristics.

[0157] As used herein, the terms "specifically bind" or "immunoreact" refer to a noncovalent interaction that occurs between an immunoglobulin molecule and one or more antigenic determinants of its target antigen. The strength or affinity of an immunological binding interaction can be expressed as the equilibrium dissociation constant (KD) of the interaction, with a smaller KD representing a higher affinity. The immunobinding properties of a selected polypeptide may be quantified by methods well known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect this rate equally in both directions. Thus, both the "on" rate constant (k) and the "off" rate constant (koff) can be determined by calculating the actual association and dissociation rates versus the concentrations (see Malmqvist, M., Nature 361:186-87 (1993)). The ratio k / k can eliminate all parameters unrelated to affinity and is equal to the equilibrium dissociation constant, K (see Davies et al. (1990) Annual Rev Biochem 59:439-473). Specific binding can be measured by radioligand binding assays, surface plasmon resonance (SPR), flow cytometry binding assays, or similar assays known to those skilled in the art.

[0158] As used herein, the term "isolated" in reference to a cell, nucleic acid, polypeptide, etc., refers to a molecule, such as DNA or RNA, that has been isolated from one or more other components of the cell's natural environment. As used herein, the term "isolated" also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or cell culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Furthermore, "isolated nucleic acid" includes nucleic acid fragments that are not naturally occurring and is not naturally occurring. As used herein, the term "isolated" also refers to a cell or polypeptide that has been isolated from other cellular proteins or tissues. Isolated polypeptides are meant to include purified polypeptides and recombinant polypeptides. Isolated polypeptides, etc., are generally prepared by at least one purification step. In one or more embodiments, the purity of the isolated nucleic acid, polypeptide, etc. is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values (inclusive), or any value therein.

[0159] The term "code," when applied to a polynucleotide, refers to a polynucleotide that is said to "encode" a polypeptide, and which, either in its natural state or when manipulated by methods known to those of skill in the art, is capable of producing that polypeptide and / or fragments thereof by transcription and / or translation.

[0160] The term "recombinant," in reference to a polypeptide or polynucleotide, refers to a form of a polypeptide or polynucleotide that does not occur in nature, a non-limiting example being one that can be combined to produce a polynucleotide or polypeptide that does not normally occur.

[0161] "Amino acid" refers to an organic compound containing both an amino group and a carboxy group, such as α-amino acids and β-amino acids, which may be encoded by nucleic acids either as such or in precursor form. A single amino acid is encoded by a nucleic acid consisting of three nucleotides (a so-called codon or base triplet). Each amino acid is encoded by at least one codon. The encoding of the same amino acid by different codons is called the "degeneracy of the genetic code." Amino acids include natural amino acids and unnatural amino acids.

[0162] As used herein, the 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd ed., edited by E.S. Golub and D.R. Gren, Sinauer Associates, Sunderland Mass. (1991)). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids (such as α- and α-disubstituted amino acids), N-alkyl amino acids, lactic acid, and other unconventional amino acids may be suitable components of the polypeptides of the present disclosure. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysyl, σ-N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention. Conventional (or naturally occurring) amino acids include alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), valine (Val, V), and the like.

[0163] The term "polypeptide" is intended to include a singular "polypeptide" and plural "polypeptides" and refers to a molecule consisting of amino acid monomers linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids and does not refer to a specific length of the product. Thus, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term for referring to two or more amino acid chains, and the term "polypeptide" may be used in place of or interchangeably with any one of the above terms. The term "polypeptide" is also intended to refer to products that have been modified after expression of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / closing groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides may be derived from natural biological sources or produced by recombinant technology, but need not be translated from a designated nucleic acid sequence and may be produced by any method, including chemical synthesis.

[0164] When applied to polypeptides, the term "essentially the same" means that two peptide sequences share at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, and most preferably at least 99% sequence identity when optimally aligned using default gap weights, such as with the GAP or BESTFIT programs.

[0165] A polynucleotide consists of a specific sequence of four bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, if the polynucleotide is RNA, thymine is replaced by uracil (U). A "polynucleotide sequence" may refer to the characters of a polynucleotide molecule, which may be entered into a database in a computer having a central processing unit and used in bioinformatics applications, such as functional genomics and homology searching.

[0166] The terms "polynucleotide," "polynucleotide(s)," and "oligonucleotide" may be used interchangeably and refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are genes or gene fragments (e.g., probes, primers, ESTs, or SAGE labels), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA, RNA, nucleic acid probes, and primers. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If such modifications are present, modifications to the nucleotide structure can be made before or after assembly of the polynucleotide. The sequence of nucleotides may be interrupted by non-nucleotide components. Polynucleotides may be further modified after polymerization, such as by conjugation with a labeling component. The term refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide embodiment of the present disclosure includes a double-stranded form and each of two complementarily capable single-stranded forms that are known or predicted to constitute the double-stranded form.

[0167] A polynucleotide or polynucleotide sequence (or polypeptide or antibody sequence) having a certain percentage (e.g., 90%, 95%, 98%, or 99%) of "identity or sequence identity" with another sequence refers to that percentage of bases (or amino acids) that are the same in the two sequences being compared when the sequences are aligned. The alignment and percentage identity or sequence identity can be determined by visual inspection or by software programs known in the art, such as those described in Current Protocols in Molecular Biology, Ausubel et al. eds. (2007). Preferably, the alignment is performed using default parameters. One such alignment program is BLAST using default parameters, including BLASTN and BLASTP, both of which use the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sortby=HIGHSCORE; Databases=non-redundant; GenBank+EMBL+DDBJ+PDB+GenBankCDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalent polynucleotides are those polynucleotides that have the percentage identity specified above and encode polypeptides having the same or similar biological activity.

[0168] Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are all encompassed by the present disclosure, provided that the amino acid sequence identity is maintained at least 90%, e.g., at least 92%, 95%, 98%, or 99%. In some embodiments, the changes are conservative amino acid substitutions. Conservative amino acid substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are broadly classified as follows: (1) acidic amino acids, which are aspartate and glutamate; (2) basic amino acids, which are lysine, arginine, and histidine; (3) nonpolar amino acids, which are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; and (4) uncharged polar amino acids, which are glycine, asparagine, glutamine, cysteine, serine, threonine, and tyrosine. Other families of amino acids include (i) serine and threonine in the aliphatic-hydroxyl family, (ii) asparagine and glutamine in the amide-containing family, (iii) alanine, valine, leucine, and isoleucine in the aliphatic family, and (iv) phenylalanine, tryptophan, and tyrosine in the aromatic family. In some embodiments, conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. For example, individual substitutions of leucine with isoleucine or valine, aspartic acid with glutamate, and threonine with serine, or similar substitutions of one amino acid with another structurally related amino acid, can be reasonably predicted without significantly affecting the binding or properties of the resulting molecule, particularly if the substitution does not involve an amino acid within a binding site. Whether an amino acid change produces a functional peptide can be readily determined by measuring the specific activity of the polypeptide derivative. Such assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily produced by one skilled in the art.

[0169] In some embodiments, amino acid substitutions have the effect of (1) reducing the protein's susceptibility to hydrolysis, (2) reducing its susceptibility to oxidation, (3) altering its binding affinity for forming protein complexes, (4) altering its binding affinity, and (5) conferring or improving other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins whose sequences differ from those of naturally occurring peptide sequences. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the naturally occurring sequence (preferably in the portion of the polypeptide other than the domains that form intermolecular contacts). Conservative amino acid substitutions should not significantly alter the structural characteristics of the parent sequence (e.g., the substituted amino acids should not tend to disrupt the helical structure present in the parent sequence or other types of secondary structure that characterize the parent sequence). Examples of artificially recognized secondary and tertiary structures of polypeptides are described in Proteins, Structures and Molecular Principles (edited by Creighton, W.H. Freeman and Company, New York (1984)), Introduction to Protein Structure (edited by C. Branden and J. Tooze, Garland Publishing, New York, NY (1991)), and Thornton et al. Nature 354:105 (1991).

[0170] The number of amino acids in the conservative amino acid substitutions in VL and VH may be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, or about 15 conservative amino acid substitutions, or a range between any two of these values (including the end values), or any value therein. The number of amino acids in the conservative amino acid substitutions in the heavy chain constant region, light chain constant region, heavy chain, or light chain can be about 1, about 2, about 3, about 4, about 5, about 6, about 8, about 9, about 10, about 11, about 13, about 14, about 15, about 18, about 19, about 22, about 24, about 25, about 29, about 31, about 35, about 38, about 41, or about 45 conservative amino acid substitutions, or a range between any two of these values (inclusive), or any value therein.

[0171] As used herein, the term "reagent" refers to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

[0172] As used herein, the term "label" or "labeled" refers to the incorporation of a detectable label, such as by incorporation of a radiolabeled amino acid or by attachment to a polypeptide of a biotin moiety that is detectable by labeled avidin (e.g., streptavidin that contains a fluorescent label or has enzymatic activity that is detectable by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131Examples of suitable reporters include, but are not limited to, fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In some embodiments, labels are linked via spacer arms of various lengths to reduce potential steric hindrance. The terms "pharmaceutical agent" or "drug" refer to a compound or composition capable of eliciting a desired therapeutic effect when properly administered to a patient.

[0173] "About" refers to the normal error range of the corresponding numerical value, which would be readily known to one of ordinary skill in the art. In some embodiments, "about" as referred to herein refers to the stated numerical value and a range of ±10%, ±5%, or ±1% thereof.

[0174] The half maximum effective concentration (EC 50 ” (concentration for 50% of maximal effect, EC 50 ) refers to the concentration that can produce 50% of the maximum effect.

[0175] "I C 50 " represents the 50% inhibitory concentration, i.e., the concentration of drug or inhibitor required to inhibit a designated biological process by half.

[0176] "Treatment" refers to therapeutic and prophylactic or preventative treatment, the purpose of which is to prevent, alleviate, ameliorate, or halt an undesirable physiological change or disorder, such as the progression of a disease, and includes, but is not limited to, detectable or undetectable results such as alleviation of symptoms, lessening of the extent of the disease, stabilization of the disease state (i.e., not worsening), delaying or alleviating the progression of the disease, improvement, palliative, reduction, or elimination (partial or complete) of the disease state, or an increase in expected survival time if not receiving treatment. Patients in need of treatment include those already suffering from a condition or disorder, those susceptible to a condition or disorder, or those in need of prevention of the condition or disorder, and those who may or may be expected to benefit from administering an antibody or pharmaceutical composition disclosed by the present invention for detection, diagnostic processes, and / or treatment.

[0177] The term "cancer" is meant or intended to describe the physiological condition in mammals typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, or leukemia. More specific examples of such cancers include, but are not limited to, colon cancer, lung cancer, ovarian cancer, uterine cancer, endometrial cancer, salivary gland cancer, peritoneal cancer, fallopian tube cancer, pancreatic cancer, thyroid cancer, head and neck squamous cell carcinoma, nasopharyngeal cancer, laryngeal cancer, lung adenocarcinoma, lung squamous cell carcinoma, liver cancer, hepatocellular carcinoma, gastrointestinal cancer, glioblastoma, breast cancer, brain cancer, kidney cancer, renal cell carcinoma, colon cancer, rectal cancer, prostate cancer, vulvar cancer, testicular cancer, squamous cell carcinoma, small cell lung cancer, cervical cancer, bladder cancer, retinoblastoma, glioblastoma, mesothelioma, oral epithelioid carcinoma, choriocarcinoma, and head and neck cancer.

[0178] The terms "overexpression" and "overexpressed" may interchangeably refer to a gene that is typically transcribed or translated at a detectably higher level in certain cells, such as cancer cells, compared to normal cells. Overexpression can also include protein or RNA overexpression (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability, and altered proteolysis), as well as localized overexpression (increased nuclear localization) and enhanced functional activity due to altered protein transport mode, e.g., enzymatic hydrolysis of a substrate. Compared to normal or control cells, overexpression can be 1%, 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the anti-Nectin-4 antibodies and antibody-drug conjugates of the present invention are used to treat solid tumors that express or overexpress Nectin-4.

[0179] As used herein, the term "tumor that overexpresses Nectin-4" refers to a tumor (including benign tumors and cancers) that overexpresses Nectin-4. In some examples, Nectin-4 expression in a tumor sample above background levels in immune tissue (e.g., as measured by immunohistochemical staining) indicates that the tumor is a tumor that overexpresses Nectin-4. Methods for detecting Nectin-4 expression in tumors are known in the art, for example, immunohistochemical assays.

[0180] As used herein, the terms "administration," "dosing," and "application" are used interchangeably and refer to the delivery of a substance (e.g., an anti-Nectin-4 antibody or ADC) to achieve a therapeutic objective (e.g., treatment of a disease associated with Nectin-4 expression or overexpression). Administration methods may be parenteral, enteral, and topical. Parenteral administration is typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0181] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a drug, e.g., an antibody or ADC, sufficient to reduce or ameliorate the severity and / or duration of a condition (e.g., cancer) or one or more symptoms thereof, prevent progression of the condition, cause regression of the condition, prevent the recurrence, progression, onset, or development of one or more symptoms associated with the condition, detect the condition, or enhance or improve the prophylactic or therapeutic effects of another therapy (e.g., a prophylactic or therapeutic agent). For example, an effective amount of an antibody can inhibit tumor growth (e.g., inhibit an increase in tumor volume), reduce tumor growth (e.g., reduce tumor volume), reduce the number of cancer cells, and / or alleviate to some extent one or more symptoms associated with cancer. For example, an effective amount can improve disease-free survival (DFS), improve overall survival (OS), or reduce the likelihood of recurrence.

[0182] The terms "patient" and "subject" are used interchangeably and refer to any mammal, including but not limited to, humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, etc., in need of diagnosis, prognosis, or treatment.

[0183] As used herein, the term "in need of" refers to a patient being identified as needing a particular method or treatment. In some embodiments, this can be identified by any diagnostic method. The patient may be in need of any of the methods and treatments described herein.

[0184] As used herein, the term "tumor-treating agent" refers to an agent that has the functional property of inhibiting the development or progression of tumors in humans, particularly malignant (cancerous) lesions such as carcinomas, sarcomas, lymphomas, or leukemias. Inhibition of metastasis is often a property of anti-tumor drugs.

[0185] The term "antibody drug conjugate" or "ADC" refers to a binding protein (e.g., an antibody or antigen-binding unit) linked to one or more chemical drugs, which may optionally be therapeutic or cytotoxic agents. In a preferred embodiment, an ADC comprises an antibody, a drug (e.g., a cytotoxic drug), and a linker by which the drug can be attached or coupled to the antibody. Non-limiting examples of drugs that may be included in an ADC include antimitotic agents, antitumor antibiotics, immunomodulatory agents, gene therapy vectors, alkylating agents, antiangiogenic agents, antimetabolites, boron-containing agents, chemoprotective agents, hormones, antihormonal agents, corticosteroids, photoactive therapeutic agents, oligonucleotides, radionuclide agents, topoisomerase inhibitors, kinase inhibitors (e.g., TEC-family kinase inhibitors and serine / threonine kinase inhibitors), and radiosensitizers.

[0186] The terms "antibody drug conjugate" and "ADC" are used interchangeably. The terms "anti-Nectin-4 antibody drug conjugate" and "anti-Nectin-4 ADC" are used interchangeably and refer to an ADC comprising an antibody that specifically binds to Nectin-4, wherein the antibody is coupled to one or more drugs. In one or more embodiments, the anti-Nectin-4 ADC comprises an antibody coupled to exatecan. In one or more embodiments, the anti-Nectin-4 antibody or ADC binds to Nectin-4 (e.g., human Nectin-4).

[0187] The term "drug-antibody conjugation ratio" or "DAR" refers to the quantity of drug (e.g., exatecan) in an ADC attached to an antibody. The DAR of an ADC can range from 1 to 10, although higher loadings (e.g., 20) are possible depending on the number of linkage sites on the antibody. The term DAR can be used when referring to the quantity of drug loaded onto a single antibody, or alternatively, when referring to the average or mean DAR of a set of ADCs. In some embodiments, the value is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. When considering the average number of small molecule drugs conjugated, i.e., the average number of drugs conjugated to an antibody, or what is referred to as the average drug-antibody conjugation ratio, the value is selected from about 0 to about 10, or from about 2 to about 8. In some embodiments, the drug-antibody conjugation ratio is about 3 to about 6. In other embodiments, the drug-antibody conjugation ratio is about 6 to about 8, or from about 7 to about 8. The DAR value can be represented herein by p. The DAR value of an ADC can be measured using ultraviolet-visible absorption spectroscopy (UV-Vis), high-performance liquid chromatography-hydrophobic chromatography (HPLC-HIC), high-performance liquid chromatography-reverse-phase chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. These techniques are described in Ouyang, J. Methods Mol Biol, 2013, 1045: pp. 275-83.

[0188] Each substituent is defined as follows: In some cases, the number of carbon atoms in a substituent (e.g., alkyl, alkenyl, alkynyl, alkoxy, aminoalkoxy, aminoalkyl, aminoalkylamino, alkylamino, heterocyclyl, heterocyclylamino, and aryl) is indicated by the prefix "Cx-Cy" or "Cx-y," where x is the minimum number of carbon atoms and y is the maximum number of carbon atoms. Thus, for example, a "C1-C6 alkyl group" refers to an alkyl group containing 1 to 6 carbon atoms. When a substituent is described as being "substituted with," a hydrogen atom on the carbon or nitrogen is replaced with a non-hydrogen group. For example, a substituted alkyl substituent is an alkyl substituent in which at least one hydrogen atom on the alkyl group is replaced with a non-hydrogen group. For purposes of illustration, a monofluoroalkyl group is an alkyl group substituted with one fluorine group, and a difluoroalkyl group is an alkyl group substituted with two fluorine groups. When there are more than one substitutions on a substituent, it should be understood that each substitution may be the same or different (unless otherwise specified). When a substituent is described as being "optionally substituted with...", the substituent may be (1) unsubstituted or (2) substituted. Possible substituents include a hydroxy group, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, a C1-C6 aminoalkoxy group, a halogen atom, a nitro group, a cyano group, a mercapto group, an alkylthio group, an amino group, a C1-C6 aminoalkyl group, a C1-C6 aminoalkylamino group, a C1-C6 alkyl group linked to a heterocycle, a C1-C6 alkylamino group linked to a heterocycle, a heterocyclyl group, a heterocyclyl group substituted by an amino group, a heterocyclylamino group, a carbamoyl group, morpholin-1-yl, piperidin-1-yl, -(CH2) q -CH3, -(CHR n ) q -CH3, C3-C8 carbocyclyl group, -O-(CH2) q -CH3, arylene-CH3, -(CH2) q -Arylene-CH3, -Arylene-(CH2) q-CH3, -(CH2) q -(C3-C8 carbocyclyl)-CH3, -(C3-C8 carbocyclyl)-(CH2) q -CH3, C3-C8 heterocyclyl group, -(CH2) q -(C3-C8 heterocyclyl)-CH3, -(C3-C8 heterocyclyl)-(CH2) q -CH3, -(CH2) q C(O)NR n (CH2) q -CH3, -(CH2CH2O) q -CH3, -(CH2CH2O) q -CH2-CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH3, -(CH2) q C(O)NR n (CH2CH2O) q -CH2-CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH3, -(CH2CH2O) q C(O)NR n (CH2CH2O) q -CH2-CH3, or -(CH2CH2O) q C(O)NR n (CH2) q -CH3, of which each R n is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0189] An "alkyl group" refers to a saturated aliphatic hydrocarbon group, and the term includes straight-chain and branched-chain hydrocarbon groups. For example, a C1-C20 alkyl group, such as a C1-C6 alkyl group. A C1-C20 alkyl group refers to an alkyl group having 1 to 20 carbon atoms, such as an alkyl group having 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms, 10 carbon atoms, 11 carbon atoms, 12 carbon atoms, 13 carbon atoms, 14 carbon atoms, 15 carbon atoms, 16 carbon atoms, 17 carbon atoms, 18 carbon atoms, 19 carbon atoms, or 20 carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, and the like. The alkyl group may be unsubstituted or substituted with one or more substituents, including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carbonyl, carboxy, aryl, heteroaryl, amino, halogen, sulfonyl, sulfinyl, phosphonyl, and the like.

[0190] "Alkoxy group" refers to a group formed by replacing at least one carbon atom in an alkyl group with an oxygen atom. Non-limiting examples include methoxy group, ethoxy group, n-propoxy group, isopropoxy group, n-butoxy group, sec-butoxy group, tert-butoxy group, n-pentyloxy group, n-hexyloxy group, cyclopropoxy group, and cyclobutoxy group. The definition of the alkyl group is the same as that of the "alkyl group" above.

[0191] The term "alkenyl group" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group containing 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) carbon-carbon double bonds and consisting of 2 to 20 carbon atoms; it is preferably an alkenyl group having 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, more preferably an alkenyl group having 2 to 8 carbon atoms, and even more preferably an alkenyl group having 2 to 6 carbon atoms. Non-limiting examples include vinyl, propen-2-yl, buten-2-yl, buten-2-yl, penten-2-yl, penten-4-yl, hexen-2-yl, hexen-3-yl, hepten-2-yl, hepten-3-yl, hepten-4-yl, octen-3-yl, nonen-3-yl, decen-4-yl, and undecen-3-yl. The alkenyl groups may optionally be further substituted with one or more substituents.

[0192] The term "alkynyl group" refers to a straight-chain or branched-chain unsaturated aliphatic hydrocarbon group containing 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) carbon-carbon triple bonds and consisting of 2 to 20 carbon atoms; it is preferably an alkynyl group having 2 to 12 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) carbon atoms, more preferably an alkynyl group having 2 to 8 carbon atoms, and even more preferably an alkynyl group having 2 to 6 carbon atoms. Non-limiting examples include ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-2-yl, butyn-3-yl, 3,3-dimethylbutyn-2-yl, pentyn-1-yl, pentyn-2-yl, hexyn-1-yl, 1-heptyn-1-yl, heptyn-3-yl, heptyn-4-yl, octyn-3-yl, nonyn-3-yl, decyn-4-yl, undecyn-3-yl, and dodecyn-4-yl. The alkynyl groups may be optionally further substituted with one or more substituents.

[0193] "Carbocyclyl group" refers to a stable non-aromatic monocyclic or polycyclic hydrocarbon radical, consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems having 3 to 15 carbon atoms, e.g., 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) carbon atoms, and which is saturated or unsaturated and connected to the remainder of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. Polycyclic radicals include, for example, adamantyl, norbornyl, decahydronaphthyl, and the like. As specifically described herein, a carbocyclyl group may be optionally substituted with one or more substituents independently selected from an alkyl group, a halogen, a haloalkyl group, a cyano group, a nitro group, an oxo group, an aryl group, an aralkyl group, a carbocyclyl group, a carbocyclylalkyl group, a heterocyclyl group, a heterocyclylalkyl group, a heteroaryl group, and a heteroarylalkyl group.

[0194] An "aryl group" refers to an all-carbon monocyclic or all-carbon fused ring system having a completely conjugated π-electron system, typically having 5 to 14 carbon atoms, e.g., 6, 10, 12, or 14 carbon atoms. An aryl group can be unsubstituted or substituted with one or more substituents, including, but not limited to, alkyl, alkoxy, cyano, hydroxy, carboxy, aryl, aralkyl, amino, halogen, sulfonyl, sulfinyl, and phosphonyl groups. Examples of unsubstituted aryl groups include, but are not limited to, phenyl, naphthyl, and anthracenyl groups.

[0195] The term "heteroaryl group" refers to a substituted or unsubstituted aromatic ring, which may be a 3- to 8-membered (e.g., 3, 4, 5, 6, 7, or 8-membered) monocyclic, a 5- to 12-membered (e.g., 5, 6, 7, 8, 9, 10, 11, or 12-membered) bicyclic, or a 10- to 15-membered (e.g., 10, 11, 12, 13, 14, or 15-membered) tricyclic ring system, and which contains 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) heteroatoms selected from N, O, or S, and is preferably a 5- to 8-membered heteroaryl group, in which 1 to 4 (e.g., 1, 2, 3, or 4) optionally substituted N or S atoms in the ring of the heteroaryl group may be oxidized to various oxidation states. Heteroaryl groups may be linked to heteroatoms or carbon atoms, and may be bridged or spirocyclic rings, with non-limiting examples including cyclopyridyl, furanyl, thienyl, pyranyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyridazinyl, imidazolyl, piperidinylbenzimidazolyl, benzopyridyl, and pyrrolopyridyl groups. Heteroaryl groups are optionally further substituted with one or more substituents.

[0196] "Cycloalkyl group" refers to a saturated cyclic hydrocarbon group, the ring of which may be a 3- to 10-membered (e.g., 3, 4, 5, 6, 7, 8, 9, 10-membered) monocyclic, a 4- to 12-membered (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12-membered) bicyclic, or a 10- to 20-membered (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20-membered) polycyclic ring system, preferably having 3 to 10 carbon atoms, more preferably 3 to 8 carbon atoms. Non-limiting examples of "cycloalkyl groups" include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 1,5-cyclooctadienyl, 1,4-cyclohexadienyl, and cycloheptatrienyl. When a cycloalkyl group is substituted, it may optionally be further substituted with one or more substituents.

[0197] The term "heterocycloalkyl group" refers to a substituted or unsubstituted saturated non-aromatic ring group, which may be a 3- to 8-membered (e.g., 3-, 4-, 5-, 6-, 7-, or 8-membered) monocyclic, a 4- to 12-membered (e.g., 4-, 5-, 6-, 7-, or 8-membered) bicyclic, or a 10- to 15-membered (e.g., 10-, 11-, 12-, 13-, 14-, or 15-membered) tricyclic ring system, and which contains 1, 2, or 3 heteroatoms selected from N, O, or S, and is preferably a 3- to 8-membered heterocyclyl group. The 1, 2, or 3 N or S atoms optionally substituted within the ring of the "heterocycloalkyl group" may be oxidized to various oxidation states, and the "heterocycloalkyl group" may be linked to a heteroatom or carbon atom, and the "heterocycloalkyl group" may be a bridged ring or a spiro ring. Non-limiting examples of "heterocycloalkyl groups" include epoxyethyl, aziridinyl, oxetanyl, azetidinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3-dioxanyl, azepanyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, 1,3-dithianyl, tetrahydrofuranyl, tetrahydropyrrolyl, tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydropyranyl, azabicyclo[3.2.1]octanyl, azabicyclo[5.2.0]nonanyl, oxatricyclo[5.3.1.1]dodecyl, azaadamantyl, and oxaspiro[3.3]heptanyl.

[0198] A "heterocyclyl group" refers to a stable 3- to 18-membered aromatic or non-aromatic ring substituent, which consists of two to eight (e.g., 2, 3, 4, 5, 6, 7, or 8) carbon atoms and one to six (one, two, three, four, five, or six) heteroatoms selected from nitrogen, oxygen, and sulfur. Unless stated otherwise in the specification, a heterocyclyl group may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include fused or bridged ring systems, and the nitrogen, carbon, or sulfur atoms in the heterocyclyl group may be optionally oxidized, the nitrogen atoms may be optionally quaternized, and the heterocyclyl group may be partially or fully saturated. Illustrative examples of such heterocyclyl groups are dioxolanyl, dioxinyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidinyl, 4-pyridin ... These include, but are not limited to, nonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, 1,2,4-thiadiazol-5(4H)-yl, tetrahydrofuranyl, trioxanyl, trithianyl, triazinanyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. As specifically described herein, heterocyclyl groups may be optionally substituted with one or more substituents selected from alkyl groups, alkenyl groups, halogens, haloalkyl groups, cyano groups, oxo, thioxo, nitro groups, aryl groups, aralkyl groups, cyclic hydrocarbon groups, cyclic hydrocarbon alkyl groups, optionally substituted heterocyclyl groups, optionally substituted heterocyclylalkyl groups, optionally substituted heteroaryl groups, and optionally substituted heteroarylalkyl groups.

[0199] An "alkoxy group" refers to a group of the formula -O-(alkyl), where alkyl is an alkyl group as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, and tert-butoxy. Alkoxy groups may be substituted or unsubstituted.

[0200] "Halogen" refers to fluorine (F), chlorine (Cl), bromine (Br), or iodine (I).

[0201] An "amino group" refers to -NH2.

[0202] A "cyano group" refers to -CN.

[0203] A "nitro group" refers to -NO2.

[0204] A "hydroxy group" refers to -OH.

[0205] A "carboxy group" refers to -COOH.

[0206] A "mercapto group" refers to -SH.

[0207] A "carbonyl group" refers to C=O.

[0208] When the above-mentioned "alkyl group", "alkoxy group", "alkenyl group", "alkynyl group", "aryl group", "heteroaryl group", "carbocyclyl group", "carbocyclyl", "heterocyclyl group", "heterocycle", "cycloalkyl group", "heterocycloalkyl group" or "heterocyclyl group" is substituted, it may optionally further include F, Cl, Br, I, hydroxy group, mercapto group, nitro group, cyano group, amino group, C 1-6 Alkylamino group, =O, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyl group, C 2-6 Alkynyl group, -NR q4R q5 , =NR q6 , -C(=O)OC 1-6 Alkyl group, -OC(=O)C 1-6 Alkyl group, -C(=O)NR q4 R q5 , C 3-8 Cycloalkyl groups, C 3-8 Heterocycloalkyl groups, C 6-10 Aryl group, C 5-10 Heteroaryl group, -C(=O)OC 6-10 Aryl group, -OC(=O)C 6-10 Aryl group, -OC(=O)C 5-10 Heteroaryl group, -C(=O)OC 5-10 Heteroaryl group, -OC(=O)C 3-8 Heterocycloalkyl groups, -C(=O)OC 3-8 Heterocycloalkyl groups, -OC(=O)C 3-8 Cycloalkyl groups, -C(=O)OC 3-8 Cycloalkyl groups, -NHC(=O)C 3-8 Heterocycloalkyl groups, -NHC(=O)C 6-10 Aryl group, -NHC(=O)C 5-10 Heteroaryl group, -NHC(=O)C 3-8 Cycloalkyl groups, -NHC(=O)C 3-8 Heterocycloalkyl groups, -NHC(=O)C 2-6 Alkenyl group or -NHC(=O)C 2-6 alkynyl groups, and among these, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 2-6 Alkenyl group, C 2-6 Alkynyl group, C 3-8 Cycloalkyl groups, C 3-8 Heterocycloalkyl groups, C 6-10 Aryl group, C 5-10 Heteroaryl group, -NHC(=O)C 6-10 Aryl group, -NHC(=O)C 5-10 Heteroaryl group, -NHC(=O)C 3-8Heterocycloalkyl group or -NHC(=O)C 3-8 The cycloalkyl group may optionally further include OH, F, Cl, Br, I, C 1-6 Alkyl group, C 1-6 Alkoxy group, -NR q4 R q5 or ═O, and R q1 is C 1-6 Alkyl group, C 1-6 Alkoxy group or C 6-10 aryl groups, R q2 , R q3 is H or C 1-6 alkyl groups, among which R q4 , R q5 is H, C 1-6 Alkyl group, -NH(C=NR q1 )NR q2 R q3 , -S(=O)2NR q2 R q3 , -C(=O)R q1 or -C(=O)NR q2 R q3 Among them, the above C 1-6 The alkyl group may optionally further comprise OH, F, Cl, Br, I, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 6-10 Aryl group, C 5-10 Heteroaryl groups, C 3-8 Cycloalkyl group or C 3-8 substituted by one or more substituents selected from heterocycloalkyl groups, or R q4 and R q5 forms one 3- to 8-membered heterocycle together with the N atom, and the heterocycle may contain one or more heteroatoms selected from N, O, and S.

[0209] "Pharmaceutically acceptable salt" or "a pharmaceutically acceptable salt thereof" refers to a salt in which a compound retains the biological effectiveness and properties of the free acid or free base, and which is obtained by reacting the free acid with a non-toxic inorganic or organic base, or a salt obtained by reacting the free base with a non-toxic inorganic or organic acid.

[0210] A "pharmaceutical composition" refers to a mixture of one or more compounds, pharmaceutically acceptable salts or prodrugs thereof, and other chemical components, where "other chemical components" refers to pharmaceutically acceptable carriers, excipients, and / or one or more other therapeutic agents.

[0211] The term "carrier" refers to a material that does not significantly irritate the body and does not eliminate the biological activity and properties of the administered compound, i.e., a diluent, adjuvant, excipient, or carrier that can be administered to a patient together with an active ingredient. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including petroleum and oils of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solution, aqueous glucose solution, and glycerin solution may also be used as liquid carriers, particularly for injectable solutions. An "excipient" refers to an inert substance added to a pharmaceutical composition to facilitate administration of a compound. Non-limiting examples include calcium carbonate, calcium phosphate, starch, cellulose derivatives (including microcrystalline cellulose), gelatin, vegetable oil, polyethylene glycols, diluents, granulating agents, lubricants, adhesives, and disintegrants. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition may be prepared as a suppository with conventional adhesives and carriers such as triglycerides. Oral formulations may include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference. Such compositions contain a clinically effective dose of an antibody or antigen-binding fragment, preferably in purified form, combined with an appropriate amount of carrier to provide a dosage form suitable for the patient. The formulation should be adapted to the mode of administration. The parent formulation may be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0212] "Stereoisomer" refers to an isomer resulting from different arrangements of atoms in a molecule in space, and includes cis-trans isomers, enantiomers, and conformational isomers.

[0213] "Optionally" or "optionally" or "selectively" or "optionally" means that the subsequently described event or condition may, but need not, occur, and the description includes cases where the event or condition occurs and cases where it does not occur. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that the alkyl group may, but need not, be present, and the description includes cases where the heterocyclyl group is substituted with an alkyl group and cases where the heterocyclyl group is not substituted with an alkyl group.

[0214] Anti-Nectin-4 antibody The present invention provides antibodies or antigen-binding units. In one or more embodiments, the antibodies or antigen-binding units of the present invention can specifically bind to Nectin-4 (e.g., human Nectin-4). In one or more embodiments, the antibodies or antigen-binding units of the present invention have one or more of the following properties: in vitro binding to Nectin-4 (e.g., human Nectin-4), binding to Nectin-4-expressing cells, high affinity, and endocytosis.

[0215] In one or more embodiments, the antigen-binding unit of the antibody is a Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, or single domain antibody.

[0216] In one or more embodiments, the antibody may be a monoclonal antibody. The binding specificity of the antibodies or antigen-binding units disclosed by the present invention can be detected by in vitro experiments such as co-immunoprecipitation, radioimmunoassay (RIA), surface plasmon resonance, flow cytometry (Facs) or enzyme-linked immunosorbent assay (ELISA).

[0217] The present invention also includes antibodies that bind to the same epitope as the antibodies described herein. For example, the antibodies of the present invention specifically bind to an epitope comprising one or more amino acid residues on human Nectin-4.

[0218] In one or more embodiments, the antibody comprises a heavy chain constant region, such as an IgG, IgA, IgE, IgM, or IgD constant region. In one or more embodiments, the antibody or antigen-binding unit comprises an immunoglobulin heavy chain constant domain selected from a human IgG constant domain, a human IgA constant domain, a human IgE constant domain, a human IgM constant domain, and a human IgD constant domain. In one or more embodiments, the antibody or antigen-binding unit comprises an IgG1 heavy chain constant region, an IgG2 heavy chain constant region, an IgG3 heavy chain constant region, or an IgG4 heavy chain constant region. In one or more embodiments, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. In one or more embodiments, the antibody or antigen-binding unit comprises a light chain constant region, such as a κ light chain constant region or a λ light chain constant region.

[0219] In one or more embodiments, the antibodies or antigen-binding units of the invention are linked to a detectable reagent, such as by incorporation of a radiolabeled amino acid or by attachment to the polypeptide of a biotin group moiety that is detectable by labeled avidin (e.g., streptavidin containing a fluorescent label or having enzymatic activity detectable by optical or calorimetric methods). In some cases, the marker or label may be therapeutic. A variety of methods for labeling polypeptides and glycoproteins are known and available in the art. Examples of markers for polypeptides include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131Examples of suitable secondary reporter genes include, but are not limited to, fluorescent markers (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent labels, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pair sequences, secondary antibody binding sites, metal binding domains, epitope tags). In one or more embodiments, the labels are linked via spacer arms of various lengths to reduce potential steric hindrance.

[0220] In one or more embodiments, the antibody or antigen-binding unit of the present invention can be used to detect the presence of Nectin-4 (e.g., human Nectin-4) in a sample. In one or more embodiments, the antibody comprises a detectable reagent. The antibody is a polyclonal antibody, or more preferably, a monoclonal antibody. An intact antibody or antigen-binding unit (e.g., Fab, scFv, or F(ab')2) can be used. The above detection methods can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detecting analyte mRNA include northern hybridization and in situ hybridization, in vitro techniques for detecting analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, and immunofluorescence, and in vitro techniques for detecting analyte genomic DNA include Southern hybridization. In addition, in vivo techniques for detecting analyte protein include introducing a labeled anti-analyte protein antibody into a patient's body. For example, the antibody can be labeled with a radioactive label whose presence and location in the patient can then be detected by standard imaging techniques.

[0221] In one or more embodiments, Nectin-4 can be detected in biological samples as part of a clinical trial procedure, for example, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (e.g., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase. Examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylfluorescein, dansyl chloride, or phycoerythrin. An example of a luminescent material includes luminol. Examples of bioluminescent materials include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 Contains H.

[0222] In one or more embodiments, the antibody or antigen-binding unit of the invention comprises a VH CDR1 set forth in SEQ ID NO:2, a VH CDR2 set forth in SEQ ID NO:3, and a VH CDR3 set forth in SEQ ID NO:4.

[0223] In one or more embodiments, the antibody or antigen-binding unit of the invention comprises a VL CDR1 set forth in SEQ ID NO:5, a VL CDR2 set forth in SEQ ID NO:6, and a VL CDR3 set forth in SEQ ID NO:7.

[0224] In one or more embodiments, the antibody or antigen-binding unit of the invention comprises a VH CDR1 set forth in SEQ ID NO:2, a VH CDR2 set forth in SEQ ID NO:3, a VH CDR3 set forth in SEQ ID NO:4, a VL CDR1 set forth in SEQ ID NO:5, a VL CDR2 set forth in SEQ ID NO:6, and a VL CDR3 set forth in SEQ ID NO:7.

[0225] In one or more embodiments, the antibody or antigen-binding unit of the invention has a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having suitable sequence identity, such as at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 8, and / or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having suitable sequence identity, such as at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 10. In one or more embodiments, at least the CDRs of these amino acid sequences with sequence identity are unchanged.

[0226] In one or more embodiments, the antibody or antigen-binding unit of the invention comprises a heavy chain variable region set forth in SEQ ID NO:8 and a light chain variable region set forth in SEQ ID NO:10.

[0227] In one or more embodiments, the antibody or antigen-binding unit of the invention further comprises a heavy chain constant region comprising the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO:9.

[0228] In one or more embodiments, the antibody or antigen-binding unit of the invention further comprises a light chain constant region comprising the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 11.

[0229] In one or more embodiments, the heavy chain of the antibody of the invention comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 12.

[0230] In one or more embodiments, the light chain of the antibody of the invention comprises the amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence having suitable sequence identity, such as at least 80% sequence identity, or at least 90% sequence identity, or at least 95% sequence identity, or at least 99% sequence identity, compared to the amino acid sequence set forth in SEQ ID NO: 14.

[0231] In one or more embodiments, the heavy chain of an antibody or antigen-binding unit of the invention, or a fragment thereof, further comprises a signal peptide such as, for example, MELGLCWVFLVAILEGVQC (SEQ ID NO: 18), whose DNA sequence is atggagctgggcctgtgttgggtgtttctggtggccatcctggagggcgtgcagtgc (SEQ ID NO: 19).

[0232] In one or more embodiments, the light chain of an antibody or antigen-binding unit of the invention, or a fragment thereof, further comprises a signal peptide, such as, for example, MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 20), whose DNA sequence is atggacatgagagtgcctgcccagctgctgggcctgctgctgctgtggttccctggcagcagatgc (SEQ ID NO: 16).

[0233] In one or more embodiments, the antibodies or antigen-binding units of the invention have a binding dissociation equilibrium constant (KD) for binding to Nectin-4 of about 1 μM or less. In one or more embodiments, the antibodies or antigen-binding units of the invention have a KD for binding to Nectin-4 of about 100 nM to about 1 pM or less. In one or more embodiments, the antibodies or antigen-binding units of the invention have a KD for binding to Nectin-4 of about 10 nM to about 1 pM or less. In one or more embodiments, the antibodies or antigen-binding units of the invention have a KD for binding to Nectin-4 of about 10 nM to about 1 nM or less.

[0234] In one or more embodiments, the antibodies or antigen-binding units of the invention have a KD for binding to human Nectin-4 (e.g., the antigen Nectin-4-His shown in SEQ ID NO: 1) of between about 100 nM and about 1 pM or less. In one or more embodiments, the antibodies or antigen-binding units of the invention have a KD for binding to human Nectin-4 of between about 10 nM and about 1 pM or less, or between about 10 nM and about 1 nM or less.

[0235] Antibody-drug conjugates The antibodies or antigen-binding units of the present invention can be coupled to drugs to form anti-Nectin-4 antibody-drug conjugates (anti-Nectin-4 ADCs). Because antibody-drug conjugates (ADCs) can selectively deliver one or more drugs to target tissues (e.g., tumors that express or overexpress Nectin-4), antibody-drug conjugates (ADCs) can improve the therapeutic efficacy of antibodies in treating diseases (e.g., cancer). In one or more embodiments, the antibody-drug conjugates (ADCs) of the present invention comprise an anti-Nectin-4 antibody or antigen-binding unit described herein and at least one drug (e.g., exatecan). The ADCs of the present invention have one or more of the following properties: in vitro binding to Nectin-4 (e.g., human Nectin-4), binding to Nectin-4-expressing cells, high affinity, endocytosis, and reduction or inhibition of cancer cell or tumor growth.

[0236] The antigen binding units of the present invention can be coupled to the drugs described herein. In one or more embodiments, the antigen binding units described herein are coupled to the drugs via a linker to form anti-Nectin-4 ADCs.

[0237] The anti-Nectin-4 ADCs of the present invention comprise an antibody or antigen-binding unit that specifically binds to Nectin-4 (e.g., human Nectin-4) linked to one or more drugs. The specificity of the ADC can be determined by the specificity of the antibody (e.g., anti-Nectin-4 antibody) or its antigen-binding unit. In one or more embodiments, the anti-Nectin-4 antibody is linked to one or more drugs (e.g., DNA topoisomerase inhibitors), and the drugs (e.g., DNA topoisomerase inhibitors) are delivered to cells that express or overexpress Nectin-4, particularly cancer cells that express or overexpress Nectin-4. In one or more embodiments, the anti-Nectin-4 antibody-drug conjugate comprises an anti-Nectin-4 antibody coupled to a drug (e.g., exatecan) via a linker. The anti-Nectin-4 antibodies or antigen-binding units described herein provide the ADC with the ability to bind to Nectin-4, thereby enabling the delivery of a drug attached to the antibody to cells that express or overexpress anti-Nectin-4, particularly cancer cells that express or overexpress Nectin-4.

[0238] In one or more embodiments, the ADC has the structure shown in formula IA, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof. [ka] wherein Abu, M, B, G, L, D, and p are as defined herein.

[0239] In one or more embodiments, Abu is the antibody ASG-AM.

[0240] In one or more embodiments, the ADC is ASG-AM-ExaD8 or ASG-AM-ExaD4, or a pharmaceutically acceptable salt or solvate thereof.

[0241] In one or more embodiments, the ADC has the structure shown in formula IB, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof. [ka] wherein Abu, M, L, D, and p are as defined herein.

[0242] In one or more embodiments, Abu is antibody ASG-22 or ASG-AM.

[0243] In one or more embodiments, the ADC is ASG-22-MMAE or ASG-AM-MMAE, or a pharmaceutically acceptable salt or solvate thereof.

[0244] In one or more embodiments, the ADC has the structure shown in formula IC, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof. [ka] wherein Abu, M, V, D, and p are as defined herein.

[0245] In one or more embodiments, Abu is the antibody ASG-AM.

[0246] In one or more embodiments, the ADC is ASG-AM-Dxd, or a pharmaceutically acceptable salt or solvate thereof.

[0247] Pharmaceutical Composition The antibodies or antigen-binding units described in the present invention, and antibody-drug conjugates to which the antibodies or antigen-binding units described in the present invention are coupled, can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations for preparing such compositions, as well as guidance on the selection of components, are known in the art.

[0248] Such compositions generally comprise an antibody or antigen-binding unit or antibody-drug conjugate and a pharmaceutically acceptable carrier. In one or more embodiments, the antigen-binding unit is the smallest inhibitory fragment that specifically binds to the target protein, such as a peptide based on the variable region sequence of an antibody and retaining the ability to bind to the target protein sequence. In one or more embodiments, the pharmaceutical composition further comprises an anti-cancer agent (e.g., an immune checkpoint inhibitor).

[0249] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, stabilizers, buffers, dispersion media, coatings, antibacterial agents, isotonic and absorption delaying agents, and the like, compatible with drug administration. Suitable carriers are described in Remington's Pharmaceutical Sciences. Such carriers or diluents optionally include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin.

[0250] In one or more embodiments, formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0251] The pharmaceutical composition may be prepared in dosage unit form, which is easy to administer and has uniform dosage. As used herein, dosage unit form refers to a physically separable unit suitable as a unitary dose for use in a patient to be treated, each unit containing a predetermined amount of one or more of the above-mentioned antibodies or antigen-binding units or antibody-drug conjugates, which are calculated to be combined with the required drug carrier to produce the desired therapeutic effect.

[0252] The pharmaceutical compositions can be placed in a container or dispenser and packaged together with instructions for administration.

[0253] The pharmaceutical compositions of the present invention may also contain other active ingredients depending on the specific condition requiring treatment, preferably with complementary activities that do not adversely affect each other. In one or more embodiments, the composition may include an agent that enhances its function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth inhibitory agent. Such active ingredients are present in appropriate combinations in amounts effective for the intended purpose.

[0254] The compositions of the present invention may be formulated in a neutral or salt form.

[0255] Treatment Methods and Uses The antibodies or antigen-binding units or ADCs described herein can be used in a variety of applications, including, but not limited to, therapeutic methods such as treating tumors. In one or more embodiments, the antibodies or antigen-binding units or ADCs can be used to inhibit tumor growth, reduce tumor volume, and / or reduce the tumorigenicity of tumors. Methods of use can be in vitro, ex vivo, or in vivo.

[0256] In one or more embodiments, the method for inhibiting tumor growth comprises contacting a tumor in vitro with an antibody, antigen-binding unit, or ADC, or a pharmaceutical composition comprising them. For example, an immortalized cell line or cancer cells expressing Nectin-4 are cultured in a medium supplemented with an antibody, antigen-binding unit, or ADC, or a pharmaceutical composition comprising them. In one or more embodiments, tumor cells are isolated from a patient sample and cultured in a medium containing an antibody, antigen-binding unit, or ADC, or a pharmaceutical composition comprising them. In one or more embodiments, the method for inhibiting tumor growth comprises contacting a tumor or tumor cells in vivo with an antibody, antigen-binding unit, or ADC, or a pharmaceutical composition comprising them.

[0257] The antibodies, antigen-binding units, or ADCs provided by the present invention, or pharmaceutical compositions containing them, can be used for the diagnosis, prognosis, monitoring, treatment, alleviation, and / or prevention of diseases and conditions associated with abnormal Nectin-4 expression in patients. When the presence of diseases and conditions associated with abnormal Nectin-4 expression in a patient is identified using conventional methods, the antibodies, antigen-binding units, or ADCs described in the present invention, or pharmaceutical compositions containing them, can be administered. In one or more embodiments, the level of Nectin-4 expression is detected by immunohistochemistry (IHC), flow cytometry, nucleic acid hybridization, or the like.

[0258] In one or more embodiments, the present invention relates to a method for treating a disease associated with Nectin-4 as a therapeutic target, which method is a method for ameliorating, alleviating, inhibiting, treating, or preventing any disease or condition associated with abnormal expression of Nectin-4 (e.g., overexpression of Nectin-4), and relates to a method for treating a tumor (including benign tumors and cancer) in a patient, a method for alleviating the symptoms of a tumor (including benign tumors and cancer) in a patient, or a method for preventing recurrence of a tumor (including benign tumors and cancer) in a patient, which method comprises administering to the patient an effective amount of an antibody or antigen-binding unit or ADC described herein.

[0259] In one or more embodiments, the present invention provides a method for preventing, treating, or ameliorating a disease, the method comprising administering to a patient in need thereof an effective amount of an antibody, antigen-binding unit, or ADC described herein, or a pharmaceutical composition comprising the same. In one or more embodiments, the present invention provides use of the antibody, antigen-binding unit, or ADC in the manufacture of a medicament for preventing, treating, or ameliorating a disease. In one or more embodiments, the disease is a disease associated with Nectin-4 expression. In one or more embodiments, the disease is a disease associated with abnormal Nectin-4 expression. In one or more embodiments, the disease is a disease associated with Nectin-4 overexpression. In one or more embodiments, the disease is a tumor expressing Nectin-4. In one or more embodiments, the disease is a tumor overexpressing Nectin-4. In one or more embodiments, the disease is a cancer overexpressing Nectin-4. In one or more embodiments, the disease is a cancer overexpressing human Nectin-4.

[0260] In one or more embodiments, the present invention provides a method of treating tumors (including benign tumors and cancers) comprising administering to a patient in need thereof an effective amount of an antibody or antigen-binding unit or ADC described herein, or a pharmaceutical composition comprising same. Examples of cancers include, but are not limited to, solid tumors, hematological cancers, and metastatic lesions. Specific examples of such cancers include, but are not limited to, breast cancer (e.g., triple-negative breast cancer (TNBC), localized, metastatic TNBC), pancreatic cancer, bladder cancer, urothelial cancer, melanoma, lung cancer (e.g., non-small cell lung cancer, squamous cell carcinoma, or lung adenocarcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), cervical cancer (e.g., squamous cell carcinoma of the cervix), ovarian cancer, choriocarcinoma, skin cancer, esophageal cancer (e.g., esophageal adenocarcinoma), gastric cancer, uterine cancer (e.g., endometrial cancer), gallbladder cancer, liver cancer, hepatocellular carcinoma, urethral cancer, renal pelvis cancer, ureter cancer, colorectal cancer, colon cancer, and prostate cancer. In one or more embodiments, the cancer is metastatic or advanced cancer. In one or more embodiments, the patient is a patient with increased Nectin-4 expression levels. In one or more embodiments, the patient is human. The antibodies, antigen-binding units, or ADCs of the invention can be administered to human patients for therapeutic purposes. Alternatively, the antibodies, antigen-binding units, or ADCs of the invention can be administered to non-human mammals expressing Nectin-4 (for veterinary purposes or as animal models of human disease). These animal models of human disease can be used to assess the therapeutic efficacy of the antibodies, antigen-binding units, or ADCs of the invention (e.g., dose testing and time courses of administration).

[0261] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody or derivative (e.g., ADC or pharmaceutical composition) used, the patient's age and weight, general health, sex, diet, and administration time, metabolic rate, drug combination, and the severity of the particular disease being treated. These factors are within the skill of a medical professional. The dosage will further depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The dosage used can be determined by pharmacological and pharmacokinetic principles well known in the art. In one or more embodiments, the effective dosage ranges from about 0.1 mg / kg to about 100 mg / kg, and the administration frequency may be, for example, once a month. For example, the administration form may be intravenous infusion, intravenous bolus injection, subcutaneous injection, intramuscular injection, etc. It should be noted that dosage values can vary depending on the type and severity of the condition to be alleviated. It is further understood that for any particular patient, specific dosage regimens may be adjusted according to the patient's needs and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the compositions as required.

[0262] Methods of administration of antibodies or antigen-binding units or ADCs include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, spinal epidural, and oral. Administration may be systemic or local. The antibodies or antigen-binding units or ADCs of the present invention may need to be introduced into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be assisted by connecting the catheter to a reservoir (which may be an Ommaya reservoir) via an intraventricular catheter. Pulmonary administration may also be used, for example, using an inhaler or nebulizer, and by using atomized formulations.

[0263] A variety of known delivery systems can be used to administer the antibodies or antigen-binding units of the invention, or polynucleotides or ADCs encoding them, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compounds, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), or construction of the nucleic acid as part of a retroviral or other vector.

[0264] Combination therapy In one or more embodiments, antibodies or antigen-binding units or ADCs of the invention can be combined with other therapeutic or prophylactic regimens, including the combined or combined use of one or more antibodies or antigen-binding units or ADCs of the invention with one or more other therapeutic agents or methods. In the case of combined treatment, the antibodies or antigen-binding units or ADCs can be administered simultaneously with or separately from the other therapeutic agents. In the case of separate administration, the antibodies or antigen-binding units or ADCs of the invention can be administered before or after the administration of the other therapeutic agents.

[0265] In one or more embodiments, when an antibody or antigen-binding unit or ADC of the invention is administered to a patient, the antibody or antigen-binding unit or ADC or pharmaceutical composition or immunoconjugate disclosed herein may also be co-administered to the patient with one or more other therapies, e.g., treatment methods and / or other preparations (e.g., therapeutic agents).

[0266] In one or more embodiments, the antibodies, antigen-binding units, or ADCs of the invention can be used in combination with immune checkpoint inhibitors. In one or more embodiments, the antibodies, antigen-binding units, or ADCs of the invention can be used in combination with other therapeutic or prophylactic regimens, such as radiation therapy.

[0267] Such combination therapies include combined administration (wherein two or more formulations are in the same preparation or in separate preparations) and separate administration, in which an antibody or antigen-binding unit or ADC of the invention can be administered before, during, and / or after administration of another therapy, e.g., a therapeutic method and / or agent. The antibody or antigen-binding unit or ADC and / or the other therapy, e.g., a therapeutic agent or method, can be administered during active disease or during remission or less active disease. The antibody or antigen-binding unit or ADC can be administered before the other treatment, simultaneously with the other treatment, after treatment, or during disease remission.

[0268] Antibody production method One or more CDRs of an antibody of the present invention can be inserted into framework regions using conventional recombinant DNA techniques. The framework regions may be naturally occurring or consensus framework regions, but human framework regions are preferred (see Chothia et al., J. Mol. Biol. 278:457-479 (1998) which lists a series of human framework regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of a target antigen produced by the combination of the framework regions and CDRs. One or more amino acid substitutions can be made within the framework regions, and amino acid substitutions that can improve binding of the antibody to its antigen can be selected. Also, antibody molecules lacking one or more interchain disulfide bonds can be produced in this manner by substituting or deleting cysteine residues in one or more variable regions involved in interchain disulfide bond formation. Other modifications made to polynucleotides within the skill of the art are also encompassed by the present invention.

[0269] Antibodies can be produced using conventional recombinant DNA techniques. Antibody-producing vectors, cell lines, and the like can be selected, constructed, and cultured using techniques known to those skilled in the art. All of these techniques are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including any supplementary material, are incorporated herein by reference.

[0270] An antibody (e.g., an anti-Nectin-4 antibody) or its antigen-binding unit gene can be inserted into an expression vector by standard methods (e.g., ligation of the antibody gene fragment into complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). The expression vector may be a plasmid, retrovirus, YAC, EBV-induced adduct, etc. To express an antibody (e.g., an anti-Nectin-4 antibody), DNA encoding the full-length light and heavy chains can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. "Operably linked" means that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their expected function of regulating the transcription and translation of the antibody gene.

[0271] The J region of the heavy and light chain consensus sequences may be used to design oligonucleotides to be used as primers for linking V region fragments to C region fragments after introducing useful restriction sites into the J region. The cDNA of the C region may be modified by site-directed mutagenesis to place restriction sites at analogous positions in the human sequence. In one or more embodiments, an expression vector already carries antibody constant region sequences prior to insertion of the light or heavy chain gene sequences related to the antibody. For example, one method for converting the VH and VL sequences related to an anti-Nectin-4 antibody into full-length antibody genes is to insert them into expression vectors already encoding heavy and light chain constant regions, respectively, such that the VH segment is operably linked to the CH segment in the vector and the VL segment is operably linked to the CL segment in the vector.

[0272] The recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody heavy and light chains by a host cell. Alternatively, the antibody heavy and light chain genes can be cloned into a vector encoding a signal peptide that facilitates secretion of the antibody heavy and light chains by a host cell, such that the signal peptide is linked in-frame to the amino terminus of the antibody heavy and light chain genes. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein), e.g., MELGLCWVFLVAILEGVQC (SEQ ID NO: 18), MDMRVPAQLLGLLLLWFPGSRC (SEQ ID NO: 20).

[0273] In addition to the antibody heavy and light chain genes, the recombinant expression vector can also carry regulatory sequences that control the expression of the antibody chain genes in a host cell. Regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, 1990. Those skilled in the art will recognize that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of host cell to be transformed, the desired expression level of protein, and the like. Suitable regulatory sequences for use in mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter / enhancer), simian virus 40 (SV40) (such as the SV40 promoter / enhancer), adenovirus (such as the adenovirus major processive promoter (AdMLP)), and polyoma. For further description of viral regulatory elements and sequences thereof, see, e.g., U.S. Patents 5,168,062, 4,510,245, and 4,968,615.

[0274] In addition to the antibody chain genes and regulatory sequences, recombinant expression vectors can carry other sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, commonly selected marker genes confer resistance to drugs (e.g., G418, hygromycin, or methotrexate) on the host cells into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection / amplification), the neo gene (for G418 selection), and the GS gene. For expression of the heavy and light chains, expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. In one or more embodiments, the inserted gene fragment must contain a selectable marker gene. Common selectable markers include screening genes such as dihydrofolate reductase, glutamine synthetase, neomycin resistance, and hygromycin resistance, which facilitate the screening and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells lacking the gene and cultured in a selective medium. Successfully transfected cells grow in large quantities and produce the desired target protein. The antibody is then purified by one or more purification steps. Purification can be carried out using conventional methods, such as first centrifuging the cell suspension, collecting the supernatant, and centrifuging it again to further remove impurities. Methods such as protein A affinity columns and ion exchange columns can be used to purify antibody proteins.

[0275] The antibodies of the present invention (e.g., anti-Nectin-4 antibodies) can be produced by recombinantly expressing antibody heavy and light chain genes in host cells. For example, host cells can be transfected with one or more recombinant expression vectors carrying heavy and light chain DNA fragments encoding the antibody, thereby expressing the heavy and light chains in the host cells, and the expressed antibody can be secreted into the culture medium in which the host cells are cultured, from which the antibody can be recovered. "Transfection" refers to various techniques commonly used to introduce foreign DNA into eukaryotic host cells, such as electroporation, lipofection, calcium phosphate precipitation, and DEAE-dextran transfection. Standard recombinant DNA methods for obtaining antibody heavy and light chain genes, incorporating these genes into expression vectors, and introducing the vectors into host cells are known in the art and are described, for example, in U.S. Patent No. 4,816,397. The DNAs expressing the antibody heavy and light chains can be incorporated into the same vector or different vectors. If they are incorporated into different vectors, the antibody heavy chain-expressing vector and the antibody light chain-expressing vector can be transfected into host cells in an appropriate ratio (e.g., Tihomir S. Dodev et al., "A Tool Kit for Rapid Cloning and Expression of Recombinant Antibodies," Scientific Reports, Volume 4, Article Number: 5885 (2014)). In one or more embodiments, the antibody expression vector contains at least one promoter element, an antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements may include an enhancer, a Kozak sequence, and RNA splicing donor and acceptor sites on both sides of the inserted sequence. Highly efficient transcription can be achieved using the early and late promoters of SV40, long terminal repeats from retroviruses such as RSV, HTLV-1, and HIV-1, and the early promoter of cytomegalovirus. Other cellular promoters, such as the actin promoter, can also be used.Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, pLXSN, pLNCX, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), pSVL, pMSG, pRSVcat, pSV2dhfr, pBC12MI, pCS2, or pCHO1.0, etc.

[0276] Antibodies of the present invention (e.g., anti-Nectin-4 antibodies) can be expressed in eukaryotic host cells. In some embodiments, antibodies are expressed in eukaryotic cells, such as mammalian host cells. Exemplary host cells for expressing antibodies of the present invention include Chinese hamster ovary cells (CHO cells) or modified CHO cells such as CHO-S, CHO-dhfr, CHO / DG44, or ExpiCHO, NSO bone marrow cells, COS cells, Cos1 cells, Cos7 cells, SP2 cells, CV1 cells, mouse L cells, human embryonic kidney cells HEK293, or modified HEK293 cells such as HEK293T, HEK293F, or HEK293E cells. After introducing a recombinant expression vector encoding an antibody chain gene into the host cells, the host cells are cultured in culture medium to produce the antibody, either for expression in the host cells or for secretion into the culture medium. The antibody can be recovered from the culture medium using standard protein purification methods.

[0277] Antibody-producing cell lines can be selected, constructed, and cultured by techniques known to those of skill in the art, all of which are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including any supplements, are incorporated by reference in their entirety.

[0278] For recombinant expression of an antibody of the present invention (e.g., an anti-Nectin-4 antibody), host cells can be co-transfected with two recombinant expression vectors: a first recombinant expression vector encoding the antibody heavy chain and a second recombinant expression vector encoding the antibody light chain. The two recombinant expression vectors can contain the same selectable marker, or they can each contain a different selectable marker. Alternatively, host cells can be transfected with recombinant expression vectors encoding the antibody heavy and light chains.

[0279] The antibodies of the present invention (e.g., anti-Nectin-4 antibodies) can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd Edition, 1984, The Pierce Chemical Co., Rockford, Ill.). Variant antibodies can also be produced using cell-free platforms (see, e.g., Chu et al., Biochemia No. 2, 2001 (Roche Molecular Biologicals) and Murray et al., 2013, Current Opinion in Chemical Biology, 17:420-426).

[0280] Antibodies (e.g., anti-Nectin-4 antibodies) produced by recombinant expression can be purified by any method known in the art for purifying immunoglobulin molecules, such as chromatography (e.g., ion exchange, affinity chromatography, and fractional column chromatography), centrifugation, differential solubility, or any other standard protein purification technique. For example, affinity chromatography with protein A or protein G primarily provides the IgG fraction in immune serum. Alternatively, specific antigens targeted by immunoglobulins or their epitopes can be immobilized on a column to purify immune-specific antibodies by immunoaffinity chromatography. The antibodies of the present invention (e.g., anti-Nectin-4 antibodies) can be fused to heterologous polypeptide sequences known in the art to facilitate purification. For details on immunoglobulin purification, see D. Wilkinson's article (The Scientist, The Scientist, Inc., Philadelphia, Pa., Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

[0281] Furthermore, mutations, including but not limited to site-directed mutagenesis and PCR-mediated mutagenesis, which result in amino acid substitutions, can be introduced into the nucleotide sequence encoding the antibody of the present invention using standard techniques known to those skilled in the art. Variants (including derivatives) encode fewer than 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, or 2 amino acid substitutions relative to the original heavy chain variable region (VH CDR1, VH CDR2, VH CDR3) and light chain variable region (VL CDR1, VL CDR2, VL CDR3). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. In one or more embodiments, the substitutions described herein are conservative amino acid substitutions.

[0282] Synthesis method The present invention further provides methods for producing drug conjugates, such as antibody-drug conjugates, and intermediates. The drug conjugates, such as antibody-drug conjugates, and intermediates of the present invention can be produced by known formulations and methods. In some embodiments, the production method is as follows. [ka]

[0283] In step 1, a compound of general formula 1-1 and a compound of general formula 1-1' are reacted under basic conditions to give a compound of general formula 1-2.

[0284] In step 2, a compound of general formula 1-2 and a compound of general formula (AA) i -(FF 1 ) fis reacted in the presence of a condensing agent under basic conditions to give a compound of general formula 1-3.

[0285] In step 3, the amino-protecting group W1 of the compound of general formula 1-3 is removed to give a compound of general formula 1-4.

[0286] In step 4, a compound of general formula 1-4 and a compound of general formula 1-5 are reacted under basic conditions to give a compound of general formula 1-6.

[0287] In step 5, a compound of general formula 1-6 and bis(4-nitrophenyl) carbonate are reacted under basic conditions to give a compound of general formula 1-7.

[0288] [ka]

[0289] In step 1, a compound of general formula 1 and a compound of general formula 1′ are reacted under basic conditions to give a compound of general formula 2.

[0290] In step 2, a compound of general formula 2 and a compound of general formula (AA) i -(FF 1 ) f is reacted in the presence of a condensing agent under basic conditions to give a compound of general formula 3.

[0291] In step 3, the amino protecting group W1 of the compound of general formula 3 is removed to give a compound of general formula 4.

[0292] In step 4, a compound of general formula 4 and a compound of general formula 5 are reacted under basic conditions to give a compound of general formula 6.

[0293] In step 5, a compound of general formula 6 and bis(4-nitrophenyl) carbonate are reacted under basic conditions to give a compound of general formula 7.

[0294] Eventually W1 is an amino protecting group such as 9-fluorenylmethoxycarbonyl, and W2 is a carboxylic acid active ester such as a succinimidyl ester.

[0295] wherein B, G, n, R, AA, i, and f are as defined herein; M' is [ka] wherein * is connected to B and R is -(CH2) r -, -(CHR m ) r -, C3-C8 carbocyclyl group, -O-(CH2) r -, arylene group, -(CH2) r -Arylene-, -Arylene-(CH2) r -, -(CH2) r -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH2) r -, C3-C8 heterocyclyl group, -(CH2) r -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH2) r -, -(CH2) r C(O)NR m (CH2) r -, -(CH2CH2O) r -, -(CH2CH2O) r -CH2-, -(CH2) r C(O)NR m (CH2CH2O) r -, -(CH2) r C(O)NR m (CH2CH2O) r -CH2-, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -, -(CH2CH2O) r C(O)NR m (CH2CH2O) r -CH2- and -(CH2CH2O) r C(O)NR m (CH2)r -, among which each R m are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or R is —(CH2) r or r is 1 or 5; L' is -(AA) i -(FF') f wherein AA is an amino acid or polypeptide and i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; or each AA is independently Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, P or AA is Val-Cit, i is 1, and each FF' is independently selected from the amino acid or peptide sequences of heLa-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly; [ka] Among them, R F is a C1-C6 alkyl group, a C1-C6 alkoxy group, -NO2 or halogen, in which * is connected to AA, or z is 0 or R F is F, or z is 0, 1, 2, 3, or 4, or z is 1 or 2, or each F-F' is independently [ka] [ka] wherein * is linked to AA and f is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or FF' is p-aminobenzyl (4-nitrophenyl) carbonate or p-aminobenzyl alcohol and f is 1, or L' is [ka] where * is connected to B, or L' is [ka] where * is connected to B, Each FF 1 are independently [ka] Of these, each R F are independently a C1-C6 alkyl group, a C1-C6 alkoxy group, —NO2 or halogen, z is 0, 1, 2, 3 or 4, in which * is linked to AA; Each FF 2 are independently [ka] Of these, each R F are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO2 or halogen, and z is 0, 1, 2, 3 or 4, in which * is linked to AA.

[0296] The basic conditions can be provided using reagents including organic bases and inorganic bases, including but not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide, and inorganic bases, including but not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

[0297] The condensing agent may be O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate. ester, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate. [ka]

[0298] The compound of general formula 1-7 and D are reacted in the presence of a condensing agent under basic conditions to give the compound of general formula 1-8. [ka]

[0299] The compound of general formula 7 and D are reacted in the presence of a condensing agent under basic conditions to give the compound of general formula 8.

[0300] Among them, M', B, L, D, G, n, R, AA, FF, FF 2 , i, f are as defined herein.

[0301] The basic conditions can be provided using reagents including organic bases and inorganic bases, including but not limited to triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium tert-butoxide, or potassium tert-butoxide, and inorganic bases, including but not limited to sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

[0302] The condensing agent may be O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate. ester, 1-hydroxybenzotriazole, 1-hydroxy-7-azabenzotriazole, O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate. [ka]

[0303] Compounds of general formula 1-8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 1-9. [ka]

[0304] Compounds of general formula 8 and Abu are coupled under mildly acidic conditions to give compounds of general formula 9.

[0305] wherein Abu, M, B, L, D, G, n, R, AA, FF, I, and f are as described in formula IA.

[0306] The weakly acidic conditions can be provided using reagents including organic acids and inorganic acids, where the organic acids include, but are not limited to, acetic acid, benzoic acid, tartaric acid, oxalic acid, malic acid, citric acid, ascorbic acid, citric acid, salicylic acid, caffeic acid, sorbic acid, quinic acid, oleanolic acid, succinic acid, chlorogenic acid, formic acid, and propionic acid, and the inorganic acids include, but are not limited to, carbonic acid, nitrous acid, acetic acid, hypochlorous acid, hydrofluoric acid, sulfurous acid, hydrosulfic acid, silicic acid, metasilicic acid, phosphoric acid, metaphosphoric acid, sodium bicarbonate, and sodium bisulfite.

[0307] The drug conjugates can be purified by conventional methods such as preparative high performance liquid chromatography (prep-HPLC).

[0308] The present application will be described below in conjunction with specific examples, but the contents of the present application are not limited to these.

[0309] Unless explicitly stated, all reagents and instruments used in the following methods are commonly used in the art and can be commercially obtained, and the methods used are conventional methods in the art. Those skilled in the art can easily carry out the above methods and obtain the corresponding results based on the contents described in the examples. [Example]

[0310] Example 1: Human Nectin-4 extracellular antigen Nectin-4-His expression Human Nectin-4 extracellular antigen Nectin-4-His was produced according to conventional methods: the DNA sequence encoding the antigen Nectin-4-His (the signal peptide sequence of the human Nectin-4 extracellular sequence (sequence derived from Q96NY8) was replaced with the albumin signal peptide, and an 8x HIS tag (HHHHHHHH) was added to the C-terminus to construct Nectin-4-His; its amino acid sequence is shown in SEQ ID NO: 1) was cloned into an expression vector, transiently transfected into eukaryotic HEK293F cells or stably transfected into CHO cells, and stable cell lines were selected, purified, and expressed to obtain the antigen Nectin-4-His.

[0311] MKWVTFISLLFLFSSAYSGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASHHHHHHHH (SEQ ID NO: 1, the underlined portion is the albumin signal peptide).

[0312] The resulting proteins were sequenced and analyzed, and the results were consistent with expectations.

[0313] Example 2: Anti-Nectin-4 antibody and its production The heavy chain of the anti-Nectin-4 antibody ASG-AM is shown in SEQ ID NO: 12, and the light chain is shown in SEQ ID NO: 14. To facilitate expression in host cells, an N-terminal signal peptide is added to each of the heavy and light chains, of which the heavy chain signal peptide is shown in SEQ ID NO: 18 and its DNA sequence is shown in SEQ ID NO: 19, and the light chain signal peptide is shown in SEQ ID NO: 20 and its DNA sequence is shown in SEQ ID NO: 16. The amino acid sequences of the heavy and light chains of the control antibody ASG-22 are shown in Table 2.

[0314] The DNA sequences encoding the antibody heavy and light chains were cloned into an expression vector to construct a recombinant expression vector, which was then transiently transfected into HEK293F cells, cultured, and purified to yield the antibody, which was confirmed to be consistent with the expected antibody. [Table 2]

[0315] Example 3: Affinity measurement of antibody ASG-AM - SPR The affinity of candidate antibodies (ASG-22, ASG-AM) for the human Nectin-4 extracellular antigen was detected using surface plasmon resonance (SPR) technology (BIAcore: GE Healthcare, T200).

[0316] Capture was performed using a Protein A chip (Cat. #29127556, GE Healthcare). A 5 μg / mL antibody dilution was passed through the experimental channels (Fc2, Fc4) at a flow rate of 10 μL / min for 10 seconds (s). The flow rate was then adjusted to 30 μL / min. Different concentrations of the antigen Nectin-4-His prepared in Example 1 (0 nM, 1.23 nM, 3.7 nM, 11.1 nM, 33.3 nM, 100 nM) were sequentially added and passed simultaneously over the experimental channels (Fc2, Fc4) and the reference channels (Fc1, Fc3). The binding time was 120 s, and the dissociation time was 180 s. Finally, the chip was regenerated with Glycine 1.5. Fitting analysis was performed using BiaEvaluation 3.2. The fitting model was a 1:1 model. The fitting results are shown in Table 3 below.

[0317] The antibody ASG-AM clearly had improved affinity compared to the control antibody ASG-22. [Table 3]

[0318] Example 4: Affinity measurement of antibody ASG-AM - BLI The affinity of candidate antibodies (ASG-22, ASG-AM) for the human Nectin-4 extracellular antigen was detected using Bio-Layer Interferometry (BLI) technology on the Octet platform (Fertebio, Octet QKe).

[0319] Antibody loading was performed using a Protein A biosensor (Fortebio, 18-5010). The antibody concentration was 20 μg / mL, the loading time was 60 s, and the Nectin-4-His antigen concentrations were set at 200 nM, 100 nM, 50 nM, 25 nM, and 0 nM. The antigen binding time was 180 s, and the dissociation time was 200 s. Finally, the data was processed and analyzed using Acquisition 8.2 to obtain the affinity constants (shown in Table 4). The results showed that antibody ASG-AM had significantly improved affinity compared to the control antibody ASG-22. [Table 4]

[0320] Example 5: Antibody ASG-AM Cell Binding Curve The binding activity of antibody ASG-AM to T-47D cells (human breast ductal carcinoma cells, derived from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu 87) was detected by FACS and compared with that of the control antibody ASG-22. The EC binding activity of the candidate antibody to the cells was 50 The four-parameter method was used to analyze the antibody activity. The method is briefly described as follows: T-47D cells were digested with pancreatin, and candidate antibodies were diluted three-fold starting at 100 nM. The antibodies were incubated with T-47D cells at 4°C for 0.5 to 1 hour. After that, a PE-labeled anti-human Fc secondary antibody (Invitrogen, 12-4998-82) was added. The signal intensity of the red fluorescence value was detected using a flow cytometer and plotted using Graphpad Prism to determine the EC. 50 Calculate the binding curve and EC 50 The results are shown in Figure 1. The results showed that the binding curves of antibody ASG-AM and control antibody ASG-22 to T-47D cells were essentially identical.

[0321] Example 6: Endocytosis experiments To reflect the endocytosis efficiency, the relative amount of antibody remaining on the cell surface after a certain period of incubation at 37°C was detected by FACS. The method is briefly described as follows: T-47D cells (human breast ductal carcinoma cells, derived from the Cell Bank of the Chinese Academy of Sciences, catalog number TCHu 87) were used, and the antibody to be measured was adjusted to 10 μg / mL. The cells (density 0.5 × 10 6 The cells were incubated with 0.2% N3Na (0.2% NaCl) for 1 hour on ice, washed three times with pre-chilled PBS, and then incubated at 37°C for 0, 2, and 4 hours. Endocytosis was stopped by adding 0.2% N3Na. The cells were washed three times with PBS, and then a PE-labeled anti-human Fc secondary antibody (Invitrogen 12-4998-82) was added and incubated at 4°C for 30 minutes. Red fluorescent signals were detected using a flow cytometer. The endocytosis results for each antibody are shown in Table 5 below. The results showed no significant difference in the endocytosis efficiency between the two antibodies. [Table 5]

[0322] Example 7: Steps for the synthesis of compound (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (D-1)

[0323] 1. Synthesis of N,N'-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide [ka]

[0324] Under a nitrogen atmosphere, potassium tert-butoxide in tetrahydrofuran (42 mL, 1 M) was added to a dry reaction flask and stirred. The temperature was then lowered to 0-5 °C. N-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (CAS No.: 143655-49-6, 5 g, 21 mmol) dissolved in tetrahydrofuran (25 mL) was slowly added dropwise to the reaction flask. Then, tert-butyl nitrite (4.32 g, 2 eq) was added dropwise (temperature controlled at 0-5 °C). The mixture was then heated to 15-20 °C and stirred for 2 h. After the reaction was complete, the temperature was lowered to 0-5 °C. Acetic acid (25 mL) and acetic anhydride (25 mL) were added dropwise (temperature controlled at 10 °C or less). The mixture was then stirred for 20 min. The mixture was maintained at 5-10°C and zinc powder (8 eq) was added in proportion to the amount of N-(3,4-difluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide. The mixture was stirred at 20-25°C for 1 h. The solid was filtered, rinsed with ethyl acetate (50 mL), cooled to 0-5°C, washed three times with 15% aqueous NaCO3 (50 mL), extracted with ethyl acetate (25 mL), and the combined organic phases were washed with saturated aqueous NaCl. Ethyl acetate (10 mL) was added, stirred at 40°C for 30 min, gradually cooled to 0-5°C, and stirred for 2 h. The solid was filtered and washed with ethyl acetate / petroleum ether (1 / 2, 10 mL). The mixture was dried in vacuo to give a gray powder (2.1 g, 33.7%). LC-MS: [M+H]+ = 297.

[0325] 2. Synthesis of N-(8-amino-5,6-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide [ka]

[0326] N,N'-(3,4-Difluoro-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (500 mg, 1.68 mmol) was added to 2 M ethanolic hydrochloric acid (5 mL) and stirred at 50 °C for 4 h. After confirming the complete reaction, water (7.5 mL) was added, the temperature was lowered to 0-5 °C, and triethylamine (1.03 g) was added dropwise and stirred for 3 h. The mixture was filtered and washed with 40% cold aqueous ethanol (3 mL) and water (3 mL). The mixture was dried in vacuo to give a gray powder (320 mg, 74.6%). LC-MS: [M+H]+ = 255.

[0327] 3. Synthesis of N-((9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide [ka]

[0328] Under a nitrogen atmosphere, N-(8-amino-5,6-difluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1.1 g, 1 eq), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1.15 g, 1 eq), and toluene (50 mL) were added to a reaction flask and heated to reflux. After stirring for 1 h, pyridinium 4-toluenesulfonate (100 mg) was added and the mixture was refluxed for 20 h. The mixture was then cooled to room temperature and stirred for 1 h. After filtration, the solid was washed with acetone (10 mL) and cold ethanol (5 mL), respectively. After drying under vacuum, a gray-brown powder (1.1 g, 53%) was obtained. LC-MS: [M+H]+ = 482.

[0329] 4. Synthesis of (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride [ka]

[0330] N-((9S)-9-Ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1.1 g, 2.28 mmol) and 6 M aqueous hydrochloric acid (44 mL) were added to the reaction flask and stirred under reflux for 4 h under a nitrogen atmosphere. The solvent was concentrated to remove the solid and purified by HPLC to give a white powder (200 mg, 18%). LC-MS: [M+H]+ = 440.

[0331] Example 8: Synthesis steps of 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl (4-nitrophenyl)carbonate (CB07) [ka]

[0332] 1) Synthesis of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-aza-hentriacontan-31-oic acid (CB01). [ka]

[0333] Under nitrogen gas protection at 0-5°C, 8.14 g of N2-fluorenylmethyloxycarbonyl-L-2,4-diaminobutyric acid (CB00-2) was dissolved in 40 mL of dimethylformamide (DMF), 10 g of 4,7,10,13,16,19,22,25-octaoxahexacosanoic acid-N-succinimidyl ester (CB00-3) and 10 mL of DMF were added, and 6.5 mL of DIPEA was added dropwise while maintaining the temperature at 0-5°C. 1 h after the addition was completed, the reaction was allowed to proceed with stirring at room temperature for 4 h. After the reaction was completed, the DMF was removed under reduced pressure, and 14.2 g of a pale yellow oily liquid, CB01, was obtained by silica gel column chromatography (eluting with dichloromethane and methanol in a volume ratio of 20:1).

[0334] 2) Synthesis of (9H-fluoren-9-yl)methyl [(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamate (CB02). [ka]

[0335] At room temperature under nitrogen gas protection, 11 g of (S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanoic acid (CB00-4) and 5.5 g of p-aminobenzyl alcohol (CB00-5) were dissolved in 400 mL of dichloromethane and 200 mL of methanol. With mechanical stirring, 17 g of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) was added in several portions and the reaction was allowed to proceed in the dark for 15 h. After completion of the reaction, the solvent was removed under reduced pressure to obtain a paste-like solid. This solid was purified by silica gel column chromatography (eluting with dichloromethane and methanol in a volume ratio of 20:1) to obtain 11.9 g of an off-white solid.

[0336] 3) Synthesis of (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CB03). [ka]

[0337] At room temperature under nitrogen gas protection, 11.9 g of [(S)-1-[[(S)-1-[[4-(hydroxymethyl)phenyl]amino]-1-oxo-5-ureidopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamate (9H-fluoren-9-yl)methyl (CB02) was added to 300 mL of acetonitrile, and 18 mL of piperidine was added dropwise with stirring. After the addition was complete, the reaction was allowed to proceed at room temperature for 2 h. After the reaction was complete, the solvent and piperidine were removed by vacuum distillation, and 7.5 g of a white solid, CB03, was obtained by silica gel column chromatography (eluting with dichloromethane and methanol in a volume ratio of 20:1).

[0338] 4) Synthesis of (9H-fluoren-9-yl)methyl ((30S,33S,36S)-41-amino-36-((4-(hydroxymethyl)phenyl)carbamoyl)-33-isopropyl-26,31,34,41-tetraoxo-2,5,8,11,14,17,20,23-octaoxa-27,32,35,40-tetraaz-30-yl)carbamate (CB04). [ka]

[0339] Under a nitrogen gas atmosphere at 0°C, 14.2 g of (S)-30-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-26-oxo-2,5,8,11,14,17,20,23-octaoxa-27-aza-hentriacontan-31-oic acid (CB01) was dissolved in 100 mL of DMF, and 11 g of O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) was added in several portions. The reaction was allowed to proceed with stirring for 30 minutes, after which 7.5 g of (S)-2-((S)-2-amino-3-methylbutanamido)-N-(4-(hydroxymethyl)phenyl)-5-ureidopentanamide (CB03) was added and the reaction was allowed to proceed for 2.5 hours while maintaining the temperature at 0°C. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 9.66 g of solid CB04 was obtained by silica gel column chromatography (elution solvent: dichloromethane and methanol in a volume ratio of 10:1).

[0340] 5) Synthesis of N-((S)-3-amino-4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB05). [ka]

[0341] At room temperature under nitrogen gas protection, 9.66 g of (9H-fluoren-9-yl)methyl ((30S,33S,36S)-41-amino-36-((4-(hydroxymethyl)phenyl)carbamoyl)-33-isopropyl-26,31,34,41-tetraoxo-2,5,8,11,14,17,20,23-octaoxa-27,32,35,40-tetraaz-30-yl)carbamate (CB04) was dissolved in 50 mL of DMF, 12 mL of diethylamine was added, and the mixture was stirred for 1.5 h. After completion of the reaction, the solvent was removed by vacuum distillation, and 7.7 g of a pale yellow solid, CB05, was obtained by silica gel column chromatography (eluting with dichloromethane and methanol in a volume ratio of 7.5:1).

[0342] 6) Synthesis of N-((S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-butanon-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB06). [ka]

[0343] 7.7 g of N-((S)-3-amino-4(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosan-26-amide (CB05) was dissolved in 40 mL of DMF, and maleimidoacetic acid succinimidyl ester (CB00-1) was added in several portions under a nitrogen atmosphere at 0-5 °C. The reaction was carried out for 4 h while maintaining the temperature at 0-5 °C. After completion of the reaction, the solvent was removed by vacuum distillation, and 9.5 g of a pale yellow solid, CB06, was obtained by silica gel column chromatography (eluting with dichloromethane and methanol in a volume ratio of 10:1).

[0344] 7) Synthesis of 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl(4-nitrophenyl)carbonate (CB07). [ka]

[0345] 9.5 g of N-((S)-3-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-4-(((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-butanon-2-yl)amino)-4-oxo)-2,5,8,11,14,17,20,23-octaoxahexacosane-26-amide (CB06) was dissolved in 50 mL of DMF, and 14.0 g of bis(4-nitrophenyl)carbonate ((PNP)CO) was added under a nitrogen atmosphere at 0°C. After dissolution, 8.2 mL of N,N-diisopropylethylamine (DIPEA) was added and the mixture was allowed to react for 4 hours while maintaining the temperature at 0°C. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and 2.6 g of a white solid CB07 was obtained by silica gel column chromatography (elution solvent: dichloromethane and methanol in a volume ratio of 8:1).

[0346] Example 9 Synthesis of 4-((18S,21S,24S)-18-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxa-15,20,23-triazapentacosan-25-amido)benzyl(4-nitrophenyl)carbonate (CB14) [ka]

[0347] CB14 was prepared by following the synthesis of CB07 in Example 8, except that 4,7,10,13,16,19,22,25-octaoxahexacosanoic acid-N-succinimidyl ester was replaced with 4,7,10,13-tetraoxatetradecanoic acid-N-succinimidyl ester, resulting in a white solid, CB14.

[0348] Example 10: Synthesis of intermediate (CB07-Exatecan) Synthesis of 4-(30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl (1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-carbamate. [ka]

[0349] 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl(4-nitrophenyl)carbonate (CB07) (2.6 g, 2.21 mmol) and N,N-dimethylformamide (23 mL) were added to reaction flask R1, and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. Simultaneously, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione mesylate (exatecan mesylate, 0.98 g, 1.84 mmol, Advanced ChemBlocks) was added to another reaction flask (R2) together with N,N-dimethylformamide (5 mL). Triethylamine (230 mg, 2.27 mmol) was added dropwise at 0-5 °C and stirred until completely dissolved. After the solution in reaction flask (R2) was added dropwise to reaction flask (R1), reaction flask (R2) was washed with N,N-dimethylformamide (2 mL), and the washings were added to reaction flask (R1). 1-Hydroxybenzotriazole (497 mg, 3.68 mmol) and pyridine (1.45 g, 18.4 mmol) were further weighed and added to reaction flask R1. The mixture was stirred at 0-5°C for 10 min, then warmed to room temperature and stirred for 5.5 h. After the reaction was complete, the solvent was removed by concentration under reduced pressure at 35°C. The product was purified by preparative high-performance liquid chromatography (prep-HPLC) and lyophilized to give a white powder (1.6 g, 59%). LC-MS: [½M+H]+ = 737.

[0350] Example 11: Synthesis of intermediate (CB07-D-1) Synthesis of 4-(30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl (1S,9S)-9-ethyl-4,5-difluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-carbamate [ka]

[0351] 4-((30S,33S,36S)-30-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-33-isopropyl-26,31,34-trioxo-36-(3-ureidopropyl)-2,5,8,11,14,17,20,23-octaoxa-27,32,35-triazaheptatriacontan-37-amido)benzyl(4-nitrophenyl)carbonate (CB07) (220 mg, 0.189 mmol) and N,N-dimethylformamide (5 mL) were added to reaction flask R1, and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. Simultaneously, (1S,9S)-1-amino-9-ethyl-4,5-difluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione hydrochloride (D-1) (90 mg, 0.189 mmol) was added to another reaction flask R2 together with N,N-dimethylformamide (5 mL), and three drops of triethylamine were added dropwise at 0-5 °C and stirred until completely dissolved. The solution in reaction flask R2 was added dropwise to reaction flask R1, and 1-hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) were further weighed and added to reaction flask R1. The mixture was stirred at 0-5°C for 10 min, then warmed to room temperature and stirred for 3 h. After the reaction was complete, the mixture was concentrated under reduced pressure to remove the solvent. The mixture was purified by preparative high-performance liquid chromatography (prep-HPLC) and lyophilized to give a white powder (55 mg, 20%). LC-MS: [1 / 2M+H]+ = 739.

[0352] Example 12: Synthesis of intermediate (CB14-Exatecan) Synthesis of 4-(18S,21S,24S)-18-(2-(2,5-dioxane-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxa-15,20,23-triazapentacosan-25-amido)-(1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1-carbamate [ka]

[0353] Similarly, 4-((18S,21S,24S)-18-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamido)-21-isopropyl-14,19,22-trioxo-24-(3-ureidopropyl)-2,5,8,11-tetraoxa-15,20,23-triazapentacosan-25-amido)benzyl(4-nitrophenyl)carbonate (190 mg, 0.19 mmol) and N,N-dimethylformamide (23 mL) were added to reaction flask R1, and the mixture was stirred under nitrogen gas protection and cooled to 0-5°C. Simultaneously, (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione mesylate (exatecan mesylate, 101 mg, 0.19 mmol, Advanced ChemBlocks) was added to another reaction flask R2 together with N,N-dimethylformamide (5 mL). Triethylamine (3 drops) was added dropwise at 0-5 °C and stirred until completely dissolved. After the solution in reaction flask R2 was added dropwise to reaction flask R1, reaction flask R2 was washed with N,N-dimethylformamide (1 mL), and the washings were added to reaction flask R1. 1-Hydroxybenzotriazole (60 mg, 0.44 mmol) and pyridine (0.5 mL) were further weighed and added to reaction flask R1. The mixture was stirred at 0-5°C for 10 min, then warmed to room temperature and stirred for 5.5 h. After the reaction was complete, the solvent was removed by concentration under reduced pressure at 35°C. The mixture was purified by preparative high-performance liquid chromatography (prep-HPLC) and lyophilized to obtain a white powder.

[0354] Example 13: Preparation of ASG-22-MMAE The structural formula of ASG-22-MMAE is as follows: [ka]

[0355] Antibody ASG-22 was adjusted to a concentration of 10 mg / mL with 10 mM succinic acid, 2.5 molar equivalents of TCEP were added, and the pH of the system was then adjusted to 8 with 1 M Tris base. The mixture was reduced by incubation at 25°C for 1.5 hours. TCEP was removed by ultrafiltration with 10 mM succinic acid. The sulfhydryl group concentration was determined by measuring absorbance, and the thiol group concentration was determined by reacting the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0356] The coupling reaction was terminated by adding 6 molar equivalents of MC-VC-PAB-MMAE and stirring at 25°C for 1 hour, followed by the addition of 0.1 M acetylcysteine to a final concentration of 2 mM and stirring for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter and then eluted with 10 mM succinic acid on Sephadex G-25 resin.

[0357] The DAR value was detected by employing reverse phase chromatography, and the DAR value of ASG-22-MMAE was 3.14.

[0358] DAR measurement: Experimental Method Sample treatment: Samples awaiting measurement were diluted to approximately 1 mg / mL with water, 0.5 M DTT (dithiothreitol) was added, the mixture was reduced in a 37°C water bath for 30 min, and centrifuged at 13,000 g for 5 min. The supernatant was then subjected to liquid chromatography analysis. Instrument conditions: The mobile phase was 0.1% DFA (difluoroacetic acid) ACN (acetonitrile) and 0.05% DFA HO, the flow rate was 0.5 mL / min, and the liquid chromatographic separation was performed using a Waters BioResolve chromatography column (2.1 × 100 mm, 2.7 μm), and the detection wavelength was 214 nm. Calculation of results: After reduction, the sample was separated by reversed-phase chromatography to obtain chromatographic peaks of different light-chain and heavy-chain coupled drugs. The area normalization method was used to calculate the concentration of each peak, and the drug-antibody binding ratio (DAR) was calculated based on the concentration of these peaks.

number

[0359] Wherein, L0 is the content of small molecular weight drugs without light chains, L1 is the content of one small molecular weight drug with a light chain, H0 is the content of small molecular weight drugs without heavy chains, H1 is the content of one small molecular weight drug with a heavy chain, H2 is the content of two small molecular weight drugs with heavy chains, and H3 is the content of three small molecular weight drugs with heavy chains.

[0360] Example 14: Preparation of ASG-AM-MMAE The structural formula of ASG-AM-MMAE is as follows: [ka]

[0361] The antibody ASG-AM was adjusted to a concentration of 10 mg / mL with 10 mM succinic acid, 2.5 molar equivalents of TCEP were added, and the pH of the system was then adjusted to 8 with 1 M Tris base. The mixture was then reduced by incubation at 25°C for 1.5 hours. TCEP was removed by ultrafiltration with 10 mM succinic acid. The sulfhydryl group concentration was determined by measuring absorbance, and the thiol group concentration was determined by reacting the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0362] The coupling reaction was terminated by adding 6 molar equivalents of MC-VC-PAB-MMAE and stirring at 25°C for 1 hour, followed by the addition of 0.1 M acetylcysteine to a final concentration of 2 mM and stirring for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter and then eluted with 10 mM succinic acid on Sephadex G-25 resin. The DAR value of ASG-AM-MMAE was 3.48.

[0363] Example 15: Preparation of ASG-AM-ExaD8 The structural formula of ASG-AM-ExaD8 is as follows: [ka]

[0364] The antibody ASG-AM was adjusted to a concentration of 18 mg / mL with 10 mM succinic acid, and 4.5 molar equivalents of TCEP (tris(2-carboxyethyl)phosphine) was added. The pH of the system was then adjusted to 8 with 1 M Tris base. The mixture was then reduced by incubation at 25°C for 1.5 hours. TCEP was then removed by ultrafiltration with 10 mM succinic acid. The sulfhydryl group concentration was determined by measuring absorbance, and the thiol group concentration was determined by reacting the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm. The coupling reaction was stopped by adding 12 molar equivalents of CB07-Exatecan and stirring at 25°C for 1 hour. 0.1 M acetylcysteine was added to a final concentration of 2 mM and stirring was continued for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter and then eluted with 10 mM succinic acid on Sephadex G-25 resin. The DAR of ASG-AM-ExaD8 was determined to be 7.59 by reverse phase chromatography.

[0365] Example 16: Preparation of ASG-AM-ExaD4 The structural formula of ASG-AM-ExaD4 is as follows: [ka]

[0366] The antibody ASG-AM was adjusted to a concentration of 18 mg / mL with 10 mM succinic acid, 2.5 molar equivalents of TCEP were added, and the pH of the system was then adjusted to 8 with 1 M Tris base. The mixture was reduced by incubation at 25°C for 1.5 hours. TCEP was removed by ultrafiltration with 10 mM succinic acid. The sulfhydryl group concentration was determined by measuring absorbance, and the thiol group concentration was determined by reacting the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0367] The coupling reaction was stopped by adding 6 molar equivalents of CB07-Exatecan and stirring at 25°C for 1 hour. 0.1 M acetylcysteine was added to a final concentration of 2 mM and stirring was continued for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter and then eluted with 10 mM succinic acid on Sephadex G-25 resin. The DAR of ASG-AM-ExaD4 was determined to be 4.03 by reverse phase chromatography.

[0368] Example 17: Preparation of ASG-AM-Dxd The structural formula of ASG-AM-Dxd is as follows: [ka]

[0369] The antibody ASG-AM was adjusted to a concentration of 18 mg / mL with 10 mM succinic acid, 4.5 molar equivalents of TCEP were added, and the pH of the system was then adjusted to 8 with 1 M Tris base. The mixture was reduced by incubation at 25°C for 1.5 hours. TCEP was removed by ultrafiltration with 10 mM succinic acid. The sulfhydryl group concentration was determined by measuring absorbance, and the thiol group concentration was determined by reacting the thiol group with DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich) and measuring the absorbance at 412 nm.

[0370] The coupling reaction was stopped by adding 12 molar equivalents of Deruxtecan (MCE, Cat. #HY-13631E) and stirring at 25°C for 1 hour. 0.1 M acetylcysteine was added to a final concentration of 2 mM, and stirring was continued for 15 minutes. The reaction mixture was filtered through a 0.22 micron filter and then loaded onto Sephadex G-25 resin and eluted with 10 mM succinic acid. The DAR of ASG-AM-Exd was determined to be 7.88 by reverse phase chromatography.

[0371] Example 18: Comparison of in vitro efficacy of ADCs This study was performed using T-47D, OVCAR-3, and MDA-MB-468 cells. Each cell was digested and seeded into a 96-well plate. T-47D, OVCAR-3, and MDA-MB-468 cells were seeded at a density of 6,000 cells per well and incubated in an incubator for 4 h. The test drug was then added, starting at 12.5 μg / mL and diluted four-fold for a total of nine gradients. After incubation in an incubator for 3 days, relative cell proliferation analysis was performed using the Cell Counting Reagent Kit-8 (CCK-8, Dojindo Laboratories, Japan). The test results (see Figure 2A-C) demonstrated that ASG-22-MMAE and ASG-AM-MMAE have similar in vitro efficacy.

[0372] Example 19: In vitro efficacy of ASG-AM-Dxd The in vitro efficacy of ASG-AM-Dxd was compared in multiple cells. The test steps were as in Example 18. After adding the drug, the incubation time was adjusted to 7 days. The EC values of the substances to be measured on different cells were then compared. 50 The results are shown in Table 6 below. [Table 6]

[0373] Example 20: Efficacy of ADC in MDA-MB-468 xenograft tumor model The in vivo antitumor activity of the ADC was evaluated using a human breast cancer MDA-MB-468 subcutaneously xenografted tumor model in nude mice.

[0374] MDA-MB-468 tumor cells were subcutaneously inoculated into female nude mice to establish an MDA-MB-468 nude mouse transplant tumor model. 18 days after inoculation, the average tumor volume was approximately 140 mm. 3 The animals were randomly assigned to six groups based on tumor volume, with six animals per group, and the day of grouping was designated Day 0. ASG-22-MMAE and ASG-AM-MMAE were administered at doses of 1 mg / kg and 5 mg / kg, respectively, and were administered to mice in each group via intravenous tail vein injection (IV) on Day 0, for a total of one dose. ASG-AM-Dxd was administered at a dose of 5 mg / kg via intravenous tail vein injection (IV) on Day 0, for a total of one dose. A vehicle control group (physiological saline) was also administered a single dose via intravenous tail vein on the day of grouping, for a total of one dose. The results are shown in Table 7 and Figure 3.

[0375] After 21 days of administration, the mean tumor volume in the vehicle control group was 382.85 ± 23.2 mm 3 The study was terminated on Day 21. Compared with the vehicle control group, the relative tumor growth rates (T / C) in the different doses of ASG-22-MMAE (1 mg / kg and 5 mg / kg) groups on Day 21 were 49.82% (TGI = 49.14%, P < 0.001) and 17.56% (TGI = 81.91%, P < 0.001), respectively.

[0376] In the different dose groups of ASG-AM-MMAE (1 mg / kg and 5 mg / kg), the relative tumor growth rates (T / C) were 53.76% (TGI=44.70%, P<0.001) and 21.51% (TGI=78.06%, P<0.001), respectively.

[0377] The relative tumor growth rate (T / C) in the ASG-AM-Dxd (5 mg / kg) dose group was 13.98% (TGI=85.71%, P<0.001).

[0378] In this study, on Day 21, animals in the vehicle control group had an average weight gain of 3.11%. Animals in the low-dose and high-dose ASG-22-MMAE groups, the low-dose and high-dose ASG-AM-MMAE groups, and the ASG-AM-Dxd group all had average weight gains of 1.03%, 2.42%, 3.93%, 2.98%, and 1.46%, respectively. There were no drug-related deaths in any of the studies, and no other drug-related toxic side effects were observed. [Table 7]

[0379] Example 21: Pharmacodynamic evaluation of JEG-3 cell line subcutaneous xenograft tumors Research purpose: Pharmacodynamic studies of the test drugs ASG-AM-ExaD4, ASG-AM-ExaD8 and ASG-AM-Dxd will be evaluated preclinically in a female NOD / SCID mouse animal model subcutaneously xenografted with human choriocarcinoma JEG-3 cell line.

[0380] Experimental design The animals were divided into the following five groups, with six animals per group, and administration began on the day of grouping. The experimental design is shown in detail in Table 8 below. [Table 8]

[0381] Test Method JEG-3 cells were cultured in MEM containing 10% fetal bovine serum and 0.01 mM NEAA. Exponentially growing JEG-3 cells were harvested and resuspended in PBS to the appropriate concentration for subcutaneous tumor inoculation in mice.

[0382] 1 x 10 7 JEG-3 cells were subcutaneously inoculated into the mice, and the cells were resuspended in a 1:1 mixture of PBS and Matrigel (0.2 mL / mouse). Tumor growth was monitored periodically, and tumors grew to an average volume of 129.56 mm. 3 When the tumors grew to 100 μg / ml, the mice were randomly divided into groups based on tumor size and body weight, and the administration began on Day 0.

[0383] Test results During the experimental period, no obvious abnormalities or weight loss were observed in the mice of any group. The tumor growth status of each treatment group and control group in the JEG-3 xenograft model is shown in Table 9 below and Figure 4. Each treatment group significantly suppressed tumor growth after a single administration, and ASG-AM-ExaD4 and ASG-AM-ExaD8 had significantly stronger efficacy than ASG-AM-Dxd. [Table 9]

Claims

1. an antibody or antigen binding unit that specifically binds to Nectin-4; (a) a VH CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 2; (b) a VH CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 3; (c) a VH CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 4; (d) a VL CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5; (e) a VL CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 6; (f) a VL CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 7; characterized in that it comprises one or more of: Antibody or antigen-binding unit.

2. 2. The antibody or antigen-binding unit of claim 1, wherein the antibody or antigen-binding unit comprises a VL CDR3 set forth in SEQ ID NO:7, and optionally further comprises one or more of a VH CDR1 set forth in SEQ ID NO:2, a VH CDR2 set forth in SEQ ID NO:3, a VH CDR3 set forth in SEQ ID NO:4, a VL CDR1 set forth in SEQ ID NO:5, and a VL CDR2 set forth in SEQ ID NO:

6.

3. The antibody or antigen-binding unit according to claim 1 or 2, characterized in that the antibody or antigen-binding unit comprises a VL CDR1 set forth in SEQ ID NO: 5, a VL CDR2 set forth in SEQ ID NO: 6, and a VL CDR3 set forth in SEQ ID NO:

7.

4. The antibody or antigen-binding unit according to any one of claims 1 to 3, characterized in that the antibody or antigen-binding unit comprises a VH CDR1 set forth in SEQ ID NO: 2, a VH CDR2 set forth in SEQ ID NO: 3, a VH CDR3 set forth in SEQ ID NO: 4, a VL CDR1 set forth in SEQ ID NO: 5, a VL CDR2 set forth in SEQ ID NO: 6, and a VL CDR3 set forth in SEQ ID NO:

7.

5. the antibody or antigen-binding unit comprises a heavy chain variable region and / or a light chain variable region; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO:8; and / or the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 10; The antibody or antigen-binding unit according to any one of claims 1 to 4.

6. 1. An antibody or antigen-binding unit, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:

10.

7. 7. The antibody or antigen-binding unit according to claim 1, further comprising a heavy chain constant region and / or a light chain constant region, or wherein the heavy chain constant region is selected from the IgG1, IgG2, IgG3, or IgG4 type, and the light chain constant region is a λ or κ chain constant region.

8. the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO:9; and / or the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having one or more conservative amino acid substitutions to the amino acid sequence set forth in SEQ ID NO: 11; The antibody or antigen-binding unit of claim 7.

9. An antibody, characterized in that the heavy chain of the antibody comprises the amino acid sequence shown in SEQ ID NO: 12 and the light chain of the antibody comprises the amino acid sequence shown in SEQ ID NO:

14.

10. A biomaterial comprising: (1) A nucleic acid encoding the antibody or antigen-binding unit according to any one of claims 1 to 9. (2) A vector, a host cell, or a microorganism containing (1), (3) the expression product, suspension, or supernatant of (2) above; Selected from Biomaterials.

11. 10. A method for producing the antibody or antigen-binding unit of any one of claims 1 to 9, comprising culturing the host cell of claim 10 so as to express the antibody or antigen-binding unit of any one of claims 1 to 9, and isolating the antibody or antigen-binding unit from the host cell.

12. 10. An antibody-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising the antibody or antigen-binding unit according to any one of claims 1 to 9 coupled to a drug via a linker.

13. An antibody drug conjugate having the structure of Formula IA, Formula IB, Formula IC, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, 【Chemical 1】 Abu is the antibody or antigen-binding unit of any one of claims 1 to 9, D is a drug, M is 【Chemistry 2】 and R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3-C8 carbocyclyl group, -O-(CH ２ ) ｒ -, arylene group, -(CH ２ ) ｒ -arylene-, -arylene-(CH ２ ) ｒ -, -(CH ２ ) ｒ -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH ２ ) ｒ -, C3-C8 heterocyclyl group, -(CH ２ ) ｒ -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH ２ ) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ ) ｒ -, -(CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ - and - (CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ ) ｒ -, among which each R ｍ are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in Formula I-A, * is linked to Abu and ** is linked to B; in Formula I-B, * is linked to Abu and ** is linked to L; in Formula IC, * is linked to Abu and ** is linked to V; B is 【Chemistry 3】 where * is connected to M, ** is connected to L, and *** is connected to G; L is -(AA) ｉ - (FF) ｆ -, wherein AA is an amino acid or polypeptide, i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, and each FF is independently 【Chemistry 4】 Of these, each R Ｆ are independently a C1 to C6 alkyl group, a C1 to C6 alkoxy group, -NO ２ or halogen, wherein * is linked to AA, ** is linked to D, z is 0, 1, 2, 3 or 4, and f is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; G is 【Chemistry 5】 where n is 1 to 24; V is 【Chemistry 6】 or V is 【Chemistry 7】 wherein * is connected to M and ** is -NH-CH ２ - is connected to p is 1 to 10; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

14. The above B is 【Chemistry 8】 wherein * is linked to M, ** is linked to L, and *** is linked to G, or a pharmaceutically acceptable salt or solvate thereof.

15. 15. The antibody-drug conjugate of claim 13 or 14, or a pharmaceutically acceptable salt or solvate thereof, wherein each AA is independently selected from the amino acid or peptide sequences Val-Cit, Val-Lys, Phe-Lys, Lys-Lys, Ala-Lys, Phe-Cit, Leu-Cit, Ile-Cit, Trp, Cit, Phe-Ala, Phe-Phe-Lys, D-Phe-Phe-Lys, Gly-Phe-Lys, Leu-Ala-Leu, Ile-Ala-Leu, Val-Ala-Val, Ala-Leu-Ala-Leu, β-Ala-Leu-Ala-Leu, and Gly-Phe-Leu-Gly.

16. 16. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to claim 15, wherein AA is Val-Cit.

17. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 16, wherein i is 1.

18. Each FF is independently 【Chemistry 9】 wherein * is linked to AA and ** is linked to D, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 17.

19. FF is 【Chemistry 10】 19. The antibody-drug conjugate of claim 18, or a pharmaceutically acceptable salt or solvate thereof, wherein * is linked to AA and ** is linked to D.

20. 20. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 19, wherein f is 1.

21. L is 【Chemistry 11】 wherein in formula IA, * is linked to B and ** is linked to D; and in formula IB, * is linked to M and ** is linked to D. The antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 20,

22. An antibody-drug conjugate having a structure of formula IA-1 or IA-2 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein said formula IA-1 or IA-2 is as follows: 【Chemistry 12】 Eventually Abu is the antibody or antigen-binding unit of any one of claims 1 to 9, R is -(CH ２ ) ｒ -, -(CHR ｍ ) ｒ -, C3-C8 carbocyclyl group, -O-(CH ２ ) ｒ -, arylene group, -(CH ２ ) ｒ -arylene-, -arylene-(CH ２ ) r-, -(CH ２ ) ｒ -(C3-C8 carbocyclyl)-, -(C3-C8 carbocyclyl)-(CH ２ ) ｒ -, C3-C8 heterocyclyl group, -(CH ２ ) ｒ -(C3-C8 heterocyclyl)-, -(C3-C8 heterocyclyl)-(CH ２ ) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ ) ｒ -, -(CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ ) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -, -(CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ CH ２ O) ｒ -CH ２ - and - (CH ２ CH ２ O) ｒ C(O)NR ｍ (CH ２ ) ｒ -, among which each R ｍ is independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each r is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; D is a drug, n is an integer from 1 to 24, p is 1 to 10; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

23. The R is —(CH ２ ) ｒ - and r is 1 or 5, or a pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 22.

24. An antibody-drug conjugate having a structure of formula IA-3 or IA-4 or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein said formulas IA-3 and IA-4 are as follows: 【Chemistry 13】 Eventually Abu is the antibody or antigen-binding unit of any one of claims 1 to 9, D is a drug, n is an integer from 1 to 24, p is 1 to 10; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

25. 25. The antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 24, wherein n is 4 to 12, or n is 4 to 8, or n is 4 or 8.

26. An antibody-drug conjugate having a structure of formula I-A-5, I-A-6, I-A-7, I-A-8, I-A-9, I-A-10, or I-A-11, or a stereoisomer thereof, or a pharmaceutically acceptable salt or solvate thereof, wherein said formulas I-A-5, I-A-6, I-A-7, I-A-8, I-A-9, I-A-10, and I-A-11 are as follows: 【Chemistry 14】 【Chemistry 15】 【Chemistry 16】 Eventually Abu is the antibody or antigen-binding unit of any one of claims 1 to 9, D is a drug, p is 1 to 10; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

27. The drug is an anti-cancer drug, a cytotoxic drug, a cell differentiation factor, a stem cell trophic factor, a steroid drug, a drug for treating an autoimmune disease, an anti-inflammatory drug, or a drug for treating an infectious disease; or the drug is an anti-cancer drug; or the drug is an anti-cancer drug; or the drug is a tubulin inhibitor, a DNA damaging agent, or a DNA topoisomerase inhibitor; or the tubulin inhibitor is dolastatin, auristatin, maytansine, Alternatively, the drug is an auristatin and is selected from MMAE, MMAF, or AF; alternatively, the drug is a DNA damaging agent and is selected from calicheamicins, duocarmycins, and anthramycin derivatives PBD (pyrrolobenzodiazepine); alternatively, the drug is a DNA topoisomerase inhibitor or a salt thereof, and is selected from irino camptothecin, irinotecan hydrochloride, camptothecin, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, 9-chloro-10-hydroxycamptothecin, camptothecin derivative SN-38, 22-hydroxyacuminatine, topotecan, raltotecan, belotecan, exatecan, homosilatecan, 6,8-dibromo-2-methyl-3-[2-(D-xylopyranosylamino)phenyl]-4(3H)-quinazolidinyl Non, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(phenylmethyl)-(2E)-2-acrylamide, 2-cyano-3-(3,4-dihydroxyphenyl)-N-(3-hydroxyphenylpropyl)-(E)-2-acrylamide, 12-β-D-glucopyranosyl-12,13-dihydro-2,10-dihydroxy-6-[[2-hydroxy-1-(hydroxymethyl)ethyl]amino]-5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,The antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 12 to 26, characterized in that the DNA topoisomerase inhibitor is selected from the group consisting of camptothecin, 10-hydroxycamptothecin, topotecan, belotecan, irinotecan, 22-hydroxyacuminatine, and exatecan.

28. The drug 【Chemistry 17】 and among them X １ and X ２ are each independently H. hydroxy groups, C1 to C6 alkyl groups, a C1-C6 alkyl group substituted by one or more hydroxy groups, halogens, nitro groups or cyano groups; a C2 to C6 alkenyl group, a C2 to C6 alkynyl group, a C1 to C6 alkoxy group, C1 to C6 aminoalkoxy group, halogen, nitro group, cyano group, mercapto group, alkylthio groups, an amino group, an amino group substituted by an amino group-protecting group, a C1-C6 aminoalkyl group optionally substituted at the amino group moiety by an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 aminoalkylamino group optionally substituted at the amino group moiety with an amino group-protecting group or a C1-C6 alkyl group; a C1-C6 alkyl group linked to a heterocycle, said heterocyclyl being optionally substituted with one or more C1-C6 alkyl groups, C1-C6 alkoxy groups, amino groups, halogens, nitro groups, or cyano groups; a C1-C6 alkylamino group linked to a heterocycle, wherein the heterocyclyl is optionally substituted with a C1-C6 alkyl group or a C1-C6 alkoxy group, and the amino group is optionally substituted with an amino group-protecting group, a halogen, a nitro group, a cyano group, or a protecting group; a heterocyclyl group substituted by an amino group, the nitrogen atom or the amino group moiety of which is optionally substituted by a protecting group or one or more C1-C6 alkyl groups; heterocyclylamino groups, the nitrogen atom or amino moiety of which is optionally substituted with a protecting group or a C1-C6 alkyl group; a carbamoyl group optionally substituted with a carbamoyl protecting group or a C1-C6 alkyl group; morpholin-1-yl, or piperidin-1-yl, X ３ is a C1 to C6 alkyl group, X ４ is H, -(CH ２ ) ｑ -CH ３ , -(CHR ｎ ) ｑ -CH ３ , a C3-C8 carbocyclyl group, —O—(CH ２ ) ｑ -CH ３ , arylene-CH ３ , -(CH ２ ) ｑ -arylene-CH ３ , -arylene-(CH ２ ) ｑ -CH ３ , -(CH ２ ) ｑ -(C3-C8 carbocyclyl)-CH ３ , -(C3-C8 carbocyclyl)-(CH ２ ) ｑ -CH ３ , C3-C8 heterocyclyl group, —(CH ２ ) ｑ -(C3-C8 heterocyclyl)-CH ３ , -(C3-C8 heterocyclyl)-(CH ２ ) ｑ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ ) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ -CH ２ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ ) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ２ -CH ３ , -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ３ , -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ CH ２ O) ｑ -CH ２ -CH ３ , or -(CH ２ CH ２ O) ｑ C(O)NR ｎ (CH ２ ) ｑ -CH ３ Of these, each R ｎ are independently H, a C1-C6 alkyl group, a C3-C8 carbocyclyl group, a phenyl group, or a benzyl group, and each q is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; or X ４ is H or a C1-C6 alkyl group, ** is a connection point, y is 0, 1 or 2; Y is O, S or CR １Ｄ R ２Ｄ Among them, R １Ｄ and R ２Ｄ are each independently H or a C1-C6 alkyl group; s and t are each independently 0, 1 or 2, but are not both 0; Alternatively, the drug 【Chemistry 18】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or the halogen is F and ** is a connection point; Alternatively, the drug 【Chemistry 19】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or the halogen is F and ** is a connection point; Alternatively, the drug 【Chemistry 20】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or said halogen is F and ** is a connection point, or said drug is 【Chemical 21】 Among them, X １ and X ２ are each independently a C1 to C6 alkyl group, a halogen, or —OH, or the C1 to C6 alkyl group is —CH ３ or the halogen is F and ** is a connection point; Alternatively, the drug 【Chemical 22】 where ** is a connection point, R ２ is H or a C1-C8 alkyl group, R ３ is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), R ４ is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), R ５ is H or a methyl group, Or, R ４ and R ５ are linked to form a carbocyclyl group and have the formula -(CR ａ R ｂ ) ｊ -, in which R ａ and R ｂ are each independently H, a C1-C8 alkyl group, or a C3-C8 carbocyclyl group; j is 2, 3, 4, 5, and 6; R ６ is H or a C1-C8 alkyl group, R ７ is H, a C1-C8 alkyl group, a C3-C8 carbocyclyl group, an aryl group, a C1-C8 alkyl-aryl group, a C1-C8 alkyl-(C3-C8 carbocyclyl), a C3-C8 heterocyclyl group or a C1-C8 alkyl-(C3-C8 heterocyclyl), Each R ８ are H, OH, and C, respectively. １ ~C ８ alkyl group, a C3-C8 carbocyclyl group, or O—(C1-C8 alkyl); R ９ is H or a C1-C8 alkyl group, and R １０ is -C(R ８ ) ２ -C(R ８ ) ２ -aryl, -C(R ８ ) ２ -C(R ８ ) ２ -(C3-C8 heterocyclyl) or -C(R ８ ) ２ -C(R ８ ２ -(C3-C8 carbocyclyl), Alternatively, the drug 【Chemical 23】 wherein ** is a connection point.

27. The antibody-drug conjugate according to any one of claims 12 to 26, or a pharmaceutically acceptable salt or solvate thereof.

29. An antibody-drug conjugate having a structure of Formula I-A-12, I-A-13, I-A-14, I-A-15, I-A-16, I-A-17, I-A-18, I-A-19, I-A-20, I-A-21, I-A-22, I-A-23, I-A-24, I-A-25, Formula I-B-1 or Formula I-C-1 or a stereoisomer thereof, or a pharmaceutical wherein the compounds of formulae I-A-12, I-A-13, I-A-14, I-A-15, I-A-16, I-A-17, I-A-18, I-A-19, I-A-20, I-A-21, I-A-22, I-A-23, I-A-24, and I-A-25, formula I-B-1, and formula I-C-1 are as follows: 【Chemistry 24】 【Chemistry 25】 【Chemical 26】 【Chemical 27】 【Chemical Formula 28】 【Chemical Formula 29】 Eventually Abu is the antibody or antigen-binding unit of any one of claims 1 to 9, p is 1 to 10; An antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof.

30. 30. The antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof according to any one of claims 13 to 29, characterized in that p is 2 to 8, or p is 4 to 8, or p is 6 to 8, or p is 7 to 8.

31. A pharmaceutical composition comprising the antibody or antigen-binding unit according to any one of claims 1 to 9, or the biomaterial according to claim 10, or the antibody-drug conjugate according to any one of claims 12 to 30, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier, excipient, and / or additive, or optionally other anticancer drugs.

32. Use of the antibody or antigen-binding unit according to any one of claims 1 to 9, or the biomaterial according to claim 10, or the antibody-drug conjugate according to any one of claims 12 to 30 or a pharmaceutically acceptable salt or solvate thereof, or the pharmaceutical composition according to claim 31 in the treatment of a disease or in the manufacture of a medicament for treating a disease, wherein the use further comprises combination use with another anti-cancer drug, or the disease is a disease associated with abnormal expression of Nectin-4, or the disease is a disease associated with abnormal expression of Nectin-4. The disease is a tumor that expresses or overexpresses Nectin-4, or the disease is a cancer that expresses or overexpresses Nectin-4, or the disease is a solid tumor or a blood cancer, or the disease is selected from breast cancer, pancreatic cancer, bladder cancer, urothelial cancer, melanoma, lung cancer, head and neck cancer, cervical cancer, ovarian cancer, choriocarcinoma, skin cancer, esophageal cancer, gastric cancer, uterine cancer, gallbladder cancer, liver cancer, hepatocellular carcinoma, urethral cancer, renal pelvis cancer, ureter cancer, colorectal cancer, colon cancer, and prostate cancer.

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