Nectin-4 antibodies and antibody-drug conjugates

Abstract

To provide an antibody that binds human nectin-4, an antibody-drug conjugate comprising the antibody, a pharmaceutical composition, and a method of treating cancer.SOLUTION: Provided is an antibody that binds human nectin-4, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 having specific sequences, and the light chain variable region comprising light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 having specific sequences. Also provided is an antibody-drug conjugate comprising the antibody conjugated to a cytotoxic agent.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Reference to sequence listing) This application has been submitted with a sequence listing in ST.26 XML format. It is provided as a file titled "30650_WO" created on May 3, The size of the sequence listing information is 120 kilobytes. It is incorporated herein by reference.

[0002] FIELD OF THEINVENTION The present disclosure relates to the field of medicine. More specifically, the present disclosure relates to a Nectin-4 antibody drug conjugate. The present invention relates to a benzodiazepine and pharmaceutical compositions thereof, and their use in the treatment of cancer. [Background technology]

[0003] Nectin-4 is a Ca2+-independent immunoglobulin-like cell adhesion molecule that mediates the binding of nectin to the cellular cellular membrane. Unlike other members of the nectin family, Nectin-4 expression is predominantly in the placenta or embryo, but also in the urothelium, breast, lung, stomach, colorectum, and pancreas. It is overexpressed in several tumor types, including pancreatic and ovarian cancers. High Nectin-4 expression in certain cancer types has been associated with tumor development.

[0004] Antibody-drug conjugates (ADCs) for use as tumor treatments have the potential to penetrate tumor cells and Tumor-targeting antibodies conjugated to payloads designed to kill cells upon administration The specific Nectin-4 antibody contains the MMAE payload (International Publication No. WO 201247 724) and certain camptothecin analogues (WO 2022112356 and It has been used to generate ADCs having the same molecular weight as the nucleotide sequence (No. 2021151984).

[0005] ADCs used in oncology have the potential to be highly specific, well tolerated, and targeted to tumor targets rather than healthy cells. The toxicity achieved while maintaining the desired activity against bystander tumor cells and intracellular delivery. and labile payloads that maintain good physical and chemical stability while enabling delivery of This compound is very difficult to design because multiple aspects of the molecule must be balanced. It is an object. Summary of the Invention

[0006] There remains a need for Nectin-4 ADCs to treat cancer. Nectin-based drugs with a sufficient therapeutic index based on better tolerability and / or better efficacy 4 ADCs not only effectively kill tumor cells but are also well tolerated by patients. In particular, effector null antibodies are required to support a sufficiently high dose. There remains a need for Nectin-4 ADCs with endothelial and topoisomerase I payloads. In particular, it has been shown to be effective against Nectin-4-low tumors while allowing for lower doses. There remains a need for Nectin-4 ADCs with enhanced bystander activity In particular, it is hoped that the dermatological events seen with certain Nectin-4 ADCs can be avoided or improved. There remains a need for Nectin-4 ADCs that allow for better management of cancer. Signaling optic and / or peripheral neuropathy seen in specific Nectin-4 ADCs There remains a need for Nectin-4 ADCs that avoid these diseases. In particular, Nectin-4 with established in vivo pharmacokinetics and suitable chemical and physical stability There remains a need for ADCs that have the following characteristics: Better antitumor activity as measured in vivo and enhanced biosynthesis against Nectin-4low tumors Standard activity, lower immunogenicity, no measurable antibody effector function, and and / or better physical and chemical stability of Nectin-4 A. There remains a need for DCs. The ADCs provided herein address these needs. Address one or more.

[0007] Provided herein are certain Nectin-4 ADCs and methods for administering Nectin-4 ADCs to patients with cancer. The composition also includes a Nectin-4 ADC or a Nectin-4 ADC for cancer in a subject. Methods of using compositions comprising the -4 ADC are also provided herein.

[0008] In one aspect, there is provided herein an antibody that binds to human Nectin-4, the antibody comprising: The heavy chain variable region (HCVR) and the light chain variable region (LCVR) are included. Complementarity determining region (HCDR) includes HCDR1, HCDR2, and HCDR3, and LCVR is , light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3; a) HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, and HCDR3 comprises SEQ ID NO:6, LCDR1 comprises SEQ ID NO:7, and LCDR2 comprises SEQ ID NO:8. and LCDR3 comprises SEQ ID NO:9. b) HCDR1 comprises SEQ ID NO: 18 and HCDR2 comprises SEQ ID NO: 19; R3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 21, and LCDR2 comprises the sequence and LCDR3 comprises SEQ ID NO: 23; c) HCDR1 comprises SEQ ID NO: 28 and HCDR2 comprises SEQ ID NO: 29; R3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO: 31, and LCDR2 comprises the sequence and LCDR3 comprises SEQ ID NO: 33; d) HCDR1 comprises SEQ ID NO: 38 and HCDR2 comprises SEQ ID NO: 39; R3 comprises SEQ ID NO: 40, LCDR1 comprises SEQ ID NO: 41, and LCDR2 comprises the sequence and LCDR3 comprises SEQ ID NO: 43; e) HCDR1 comprises SEQ ID NO: 48 and HCDR2 comprises SEQ ID NO: 49; R3 comprises SEQ ID NO:50, LCDR1 comprises SEQ ID NO:41, and LCDR2 comprises the sequence SEQ ID NO: 42, and LCDR3 comprises SEQ ID NO: 51, f) HCDR1 comprises SEQ ID NO: 56 and HCDR2 comprises SEQ ID NO: 57; R3 comprises SEQ ID NO:58, LCDR1 comprises SEQ ID NO:59, and LCDR2 comprises the sequence and LCDR3 comprises SEQ ID NO: 61; g) HCDR1 comprises SEQ ID NO: 66 and HCDR2 comprises SEQ ID NO: 67; R3 comprises SEQ ID NO: 68, LCDR1 comprises SEQ ID NO: 69, and LCDR2 comprises the sequence 70, and LCDR3 comprises SEQ ID NO: 71, or h) HCDR1 comprises SEQ ID NO: 76 and HCDR2 comprises SEQ ID NO: 77; R3 comprises SEQ ID NO: 78, LCDR1 comprises SEQ ID NO: 69, and LCDR2 comprises the sequence number 70, and LCDR3 comprises SEQ ID NO:79.

[0009] In a further aspect, there is provided herein an antibody that binds to human Nectin-4, comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), a) the HCVR comprises SEQ ID NO: 10 and the LCVR comprises SEQ ID NO: 11; b) the HCVR comprises SEQ ID NO: 14 and the LCVR comprises SEQ ID NO: 15; c) the HCVR comprises SEQ ID NO: 24 and the LCVR comprises SEQ ID NO: 25; d) the HCVR comprises SEQ ID NO: 34 and the LCVR comprises SEQ ID NO: 35; e) the HCVR comprises SEQ ID NO: 44 and the LCVR comprises SEQ ID NO: 45; f) the HCVR comprises SEQ ID NO: 52 and the LCVR comprises SEQ ID NO: 53; g) the HCVR comprises SEQ ID NO: 62 and the LCVR comprises SEQ ID NO: 63; h) the HCVR comprises SEQ ID NO: 72 and the LCVR comprises SEQ ID NO: 73; or i) the HCVR comprises SEQ ID NO:80 and the LCVR comprises SEQ ID NO:81.

[0010] In another aspect, there is provided herein an antibody that binds to human Nectin-4, the antibody comprising , a heavy chain (HC) and a light chain (LC), a) HC comprises amino acids 2 to 444 of SEQ ID NO: 2, and LC comprises amino acids 2 to 444 of SEQ ID NO: 3. 2-215 inclusive, b) HC comprises amino acids 2 to 444 of SEQ ID NO: 12, and LC comprises amino acids 2 to 444 of SEQ ID NO: 13. Contains acids 2 to 215 c) HC comprises amino acids 2 to 443 of SEQ ID NO: 16, and LC comprises SEQ ID NO: 17. R, d) HC comprises amino acids 2 to 447 of SEQ ID NO: 26, and LC comprises SEQ ID NO: 27. R, e) HC comprises amino acids 2 to 446 of SEQ ID NO: 36, and LC comprises SEQ ID NO: 37. R, f) HC comprises amino acids 2 to 446 of SEQ ID NO: 46, and LC comprises SEQ ID NO: 47. R, g) HC comprises amino acids 2 to 446 of SEQ ID NO: 54, and LC comprises SEQ ID NO: 55. R, h) HC comprises amino acids 2 to 450 of SEQ ID NO: 64, and LC comprises amino acids 2 to 450 of SEQ ID NO: 65. Contains acids 2 to 216, or i) HC comprises amino acids 2 to 450 of SEQ ID NO: 74, and LC comprises amino acids 2 to 450 of SEQ ID NO: 75. Contains 2 to 216 acids.

[0011] In another aspect, provided herein is a compound that is capable of binding to a cytotoxic agent directly or via a linker. and an antibody-drug conjugate comprising the Nectin-4 antibody disclosed herein conjugated to the Nectin-4 antibody. It is an ADC.

[0012] In a further aspect, there is provided an ADC herein, wherein the cytotoxic agent comprises the formula: XY and a camptothecin analog comprising the formula: Y is of the formula:

[0013] [ka] X is * -CH 2 O-, * -(CH 2 ) 2 O-, * -(CH 2 ) 3 O-, * -(CH 2 ) 4 O-, * -CH 2 NH-, * -(CH 2 ) 2 NH-, * -(CH 2 ) 3 NH-, * - (CH 2 ) 4 NH-, * -CH 2 N(CH 3 )-,* -(CH 2 ) 2 N(CH 3 )-、 * -(CH 2 ) 3 N(CH 3 )-、 * -(CH 2 ) 4 N(CH 3 )-、 * -CH 2 N(R 1 )-、 * -(CH 2 ) 2 N(R 1 )-、 * -(CH 2 ) 3 N(R 1 )-、 * -(CH 2 ) 4 N(R 1 )-、 * -CH 2 N(CH 3 )C(=O)CH 2 O- * -CH 2 N(CH 2 R 1 )C(=O)CH 2 O- * -CH 2 NHC(=O)CH 2 O- * -CH 2 NHC (=O)(CH 2 ) 2 O- * -CH 2 NHC(=O)(CH 2 ) 3 O- * -CH 2 N HC(=O)(CH 2 ) 4 O- * -CH 2 NHC(=O)(CH 2 ) 5 O- *-CH 2 NHC(=O)CH 2 -, * -CH 2 NHC(=O)(CH 2 ) 2 -, * -CH 2 NH C(=O)(CH 2 ) 3 -, * -CH 2 NHC(=O)(CH 2 ) 4 -, * -CH 2 NH C(=O)(CH 2 ) 5 -, * -CH 2 SCH 2 -, * -CH 2 S(CH 2 ) 2 -, * - CH 2 S(CH 2 ) 3 -, * -CH 2 S(CH 2 ) 4 -or * -CH 2 S(CH 2 ) 5 - and * is the moiety covalently attached to Y, and R 1 is phenyl.

[0014] In another aspect, provided herein is an antibody-drug conjugate (ADC) of the formula: R,

[0015] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0016] In another embodiment, the Nectin-4 antibody disclosed herein and one or more pharma- ceutically acceptable and a carrier, diluent, or excipient thereof. In another embodiment, a Nectin-4 ADC disclosed herein and one or more pharma- ceutically acceptable and a carrier, diluent, or excipient as provided herein. .

[0017] In another embodiment, a patient in need of treatment for cancer is administered an effective amount of the nephrectomy disclosed herein. Provided herein are methods of treating cancer comprising administering a quinolone-4 ADC to a patient. In a further aspect, a patient in need of treatment for cancer is administered an effective amount of the nephrectomy disclosed herein. Provided herein are methods of treating cancer comprising administering a cutin-4 ADC. The cancers were urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, neck, ovarian, or prostate cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Nectin-4 As used herein, "human nectin-4" refers to poliovirus receptor-related 4, Ig superfamily receptor LNIR, or poliovirus receptor-related protein 4 ( It refers to the human Nectin-4 protein or polypeptide, also known as PVRL4. The amino acid sequence of human Nectin-4 includes the signal peptide provided in SEQ ID NO:1. It can be found in NP_112178.2.

[0019] Nectin-4 antibody (also known as anti-Nectin-4 antibody) As used herein, the term "antibody" refers to an immunoglobulin molecule that binds to an antigen. Antibodies may be of any class (e.g., IgG, IgE, IgM, IgD, IgA). and any subclass (e.g., IgG1, IgG2, IgG3, IgG4). Good too.

[0020] An exemplary antibody of the disclosure comprises four polypeptide chains: The immunoglobulin G ( The amino-terminal portion of each of the four polypeptide chains is responsible for antigen recognition. Contains variable regions of about 100 to 125 amino acids involved in the synthesis of four polypeptide chains The carboxy-terminal portion of each contains a constant region primarily responsible for effector function. Each heavy chain is composed of a heavy chain variable region (VH, also known as HCVR) and a heavy chain constant region. Each light chain comprises a light chain variable region (VL, also known as LCVR) and a light chain constant region (LCVR). The IgG isotype is divided into subclasses (e.g., IgG1, IgG2, I IgG1, IgG2, and IgG3).

[0021] The VH and VL regions are called framework regions (FR), The complementarity determining regions are interspersed with regions that are more conserved. The CDRs are located on the surface of the protein. These regions are exposed to the antigen and are important regions of the antibody for antigen-binding specificity. It consists of three CDRs and four FRs: FR1, CDR1, FR2, and CDR2. , FR3, CDR3, and FR4 are arranged in this order from the amino terminus to the carboxy terminus. In the specification, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3." The three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." CDR The CDRs contain most of the residues that form specific interactions with the antigen. The assignment to is based on the Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest,” Nat ional Institutes of Health,Bethesda,Md.( 1991)), Chothia (Chothia et al., “Canonical structures for the hypervariable region s of immunoglobulins”, Journal of Molecular ar Biology,196,901-917(1987), Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins” ,Journal of Molecular Biology,273,927-94 8 (1997)), North (North et al., “A New Clust ering of Antibody CDR Loop Conformations ”,Journal of Molecular Biology,406,228-2 56(2011)), or IMGT (the international Immun oGeneTics database, available at www.imgt.org, Lef ranc et al., Nucleic Acids Res.1999;27:20 This can be done according to well-known schemes, including those described in (E. G., et al., 1999-212). The CDRs shown are determined by North.

[0022] Certain antibodies described herein are derived from an IgG1 Fc region or human IgG1. Fc regions, e.g., modified IgG1 Fc regions with altered Fc effector functions. IgG1 has the ability to inhibit antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CD Some antibodies disclosed herein have an IgG1 Fc region that is induced by The amino acid substitutions described herein alter the effector function. According to the EU index numbering, the mutations are at positions 234 and 235 of the Fc region. According to some disclosures herein, the mutations are introduced into the Fc region. Introduced at positions 234, 235, and 265 (according to EU index numbering) In some embodiments, the Nectin-4 antibody of the present disclosure has an alpha amino acid sequence identical to that of residues 234 and 235. and a modified human IgG1 Fc region containing serine at position 265 (EU index Also referred to as hIgG1 effector null or hIgG1EN Fc region, based on numbering In further embodiments, some antibodies contain a glutamine at position 297, an alamycin at position 298, and an arylamine at position 299. or glycine, alanine or glutamine at position 322, alanine or glutamine at position 329 containing glycine or alanine at position 331, and / or serine or alanine at position 332 (EU In In some embodiments, the Fc region is a 2-membered Fc-region, and the Fc region is a 1-membered Fc-region. These antibody mutations are: alanine at position 234, glutamic acid at position 235, alanine at position 237, serine at position 330, and serine at position 331 (EU Index In a further embodiment, these antigens introduced into the IgG1 Fc region are The amino acid substitutions reduced or eliminated measurable antibody effector functions.

[0023] In certain aspects of the disclosure, the Nectin-4 antibody comprises one or more engineered cysteine ​​residues. In a further aspect, the antibody has a modified human IgG1 or human IgG4 constant domain comprising: The heavy chain constant domain 1 (CH1), the heavy chain constant domain 2 (CH2), and / or the heavy chain constant domain It contains engineered cysteines at one or more sites within common domain 3 (CH3).

[0024] Mammalian antibody expression typically results in glycosylation. Glycosylation of antibodies is typically Typically, glycosylation is either N-linked or O-linked. N-linked glycosylation is The attachment of a carbohydrate moiety to the side chain of a residue. O-linked glycosylation refers to the attachment of a hydroxyamino The attachment of sugars, such as N-acetylgalactosamine, galactose, or xylose, to the acid Typically, glycosylation is performed at highly conserved N-glycosylation sites (e.g., According to the IMGT or EU index numbering, it is at position 297 in IgG1. Occurs in the Fc region of an antibody. Glycosylation sites are engineered to alter glycosylation. It is possible to block or reduce glycosylation or to modify the amino acid sequence. (e.g., by modifying the ribozyme to generate additional or diverse glycosylation).

[0025] Expression of mammalian antibodies from the IgG subclass involves the C-terminal This can result in clipping of amino acids, e.g., in the case of an IgG1 antibody, one or The two C-terminal amino acids can be removed. In the case of IgG1 antibodies, the C-terminal lysine is present. In this case, the heavy chain may be truncated or cut off during expression. The third glycine may be truncated or deleted from the heavy chain as well.

[0026] Expression of an antibody in a mammal can also result in modification of the N-terminal amino acid. For example, or pyroglutarate, if the most N-terminal amino acid of the light chain is glutamine or glutamic acid For example, the most C-terminal amino acid of a heavy or light chain may be modified to a lysine or glycine. If it is thin, it can be removed.

[0027] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein. , incorporating natural nucleotides, modified nucleotides, and / or nucleotide analogs. Single-stranded and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, A polynucleotide of the present disclosure also refers to a polymer of nucleotides, e.g., DNA. or RNA polymerase or a substrate incorporated therein by a synthetic reaction. good.

[0028] The polynucleotides of the disclosure can be, for example, those sequences in which the polynucleotide is operably linked to an expression control sequence. Once linked, they can be expressed in a host cell. Expression control sequences capable of expressing the gene are well known in the art. The promoter contains one or more signal peptides that facilitate secretion of the polypeptide from the host cell. The signal peptide may include, for example, an immunoglobulin signal peptide. The signal peptide may be a heterologous or heterologous signal peptide. The expression vector containing the polynucleotide encoding the polypeptide can be prepared by a method well known in the art. Additionally, expression vectors can be used to infect host cells containing the desired polynucleotide sequence. To aid in the detection of host cells transformed with, for example, tetracycline, neomycin, The gene may include one or more selectable markers such as gene expression vector, gene for genomic DNA, gene for transcription factor receptor 2 (RGD), gene for transcription factor activator 2 (RIG), gene for transcription factor activator ..., and gene for dihydrofolate reductase.

[0029] The host cells are stably expressing one or more expression vectors that express all or a portion of the antibodies of the present disclosure. This includes cells that have been transfected, transformed, transduced or infected either transiently or intracellularly. According to some embodiments, the host cell is a gene encoding an antibody HC polypeptide of the present disclosure. The expression vector expressing the LC polypeptide is stably or transiently transfected with the expression vector. In some embodiments, the host cell may be transfected, transformed, transduced or infected. The host cells are harboring expression vectors expressing the HC and LC polypeptides of the antibody of the present disclosure, and The cells of the present disclosure may be transfected, transformed, transduced or infected either statically or transiently. Antibodies can be produced using CHO, NS0, HEK293 or COS cells according to techniques well known in the art. The polypeptide may be produced in mammalian cells, such as cells.

[0030] The medium into which the antibodies of the present disclosure are secreted is subjected to ion exchange and hydrophobic interaction chromatography. The medium can be purified by conventional techniques, such as mixed mode methods. For example, the medium can be purified by conventional The method is used to apply and elute from a Protein A or Protein G column. and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be isolated by size exclusion, hydrophobic interactions, ionic interactions, and the like. It can be effectively removed by common techniques including hydroxyapatite chromatography or by The product may be immediately frozen, for example at -70°C, refrigerated, or frozen. Various methods of protein purification may be employed, such as those described herein. Methods in E nzymology 182:83-89(1990), and Scopes, Prote. in Purification:Principles and Practice, 3rd Edition, Springer, NY (1994).

[0031] In one aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain The heavy chain complementarity determining region (HCVR) and the light chain variable region (LCVR) are included. The light chain constant region (HCDR) includes HCDR1, HCDR2, and HCDR3. The LCVR is a light chain constant region (LCVR). Complementarity determining region (LCDR) including LCDR1, LCDR2, and LCDR3; HCDR HCDR1 comprises SEQ ID NO:4, HCDR2 comprises SEQ ID NO:5, and HCDR3 comprises SEQ ID NO:6 wherein LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8, and LCDR 3 comprises SEQ ID NO: 9. In another embodiment, an antibody that binds to human Nectin-4 is described herein. HCDR1 comprises SEQ ID NO: 18, HCDR2 comprises SEQ ID NO: 19, R3 comprises SEQ ID NO:20, LCDR1 comprises SEQ ID NO:21, and LCDR2 comprises SEQ ID NO:2 2 and LCDR3 comprises SEQ ID NO: 23. In another embodiment, Provided herein is an antibody having a nucleotide sequence similar to that of SEQ ID NO: 1, wherein HCDR1 comprises SEQ ID NO: 28 and HCDR2 comprises SEQ ID NO: 29, HCDR3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO: 31, DR2 comprises SEQ ID NO: 32 and LCDR3 comprises SEQ ID NO: 33. Provided herein is an antibody that binds to cutin-4, wherein HCDR1 comprises SEQ ID NO: 38, CDR2 comprises SEQ ID NO: 39, HCDR3 comprises SEQ ID NO: 40, and LCDR1 comprises SEQ ID NO: LCDR2 comprises SEQ ID NO: 42 and LCDR3 comprises SEQ ID NO: 43. In one embodiment, an antibody that binds to human Nectin-4 is provided herein, wherein HCDR1 is selected from the group consisting of the sequence SEQ ID NO: 48, HCDR2 comprises SEQ ID NO: 49, and HCDR3 comprises SEQ ID NO: 50; LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 42, and LCDR3 comprises the sequence In another aspect, provided herein is an antibody that binds to human Nectin-4. HCDR1 comprises SEQ ID NO: 56, HCDR2 comprises SEQ ID NO: 57, and HCDR3 comprises SEQ ID NO: 58. Contains sequence number 58, LCDR1 contains sequence number 59, and LCDR2 contains sequence number 60 and LCDR3 comprises SEQ ID NO: 61. In another embodiment, the antibody that binds to human Nectin-4 Provided herein, HCDR1 comprises SEQ ID NO: 66 and HCDR2 comprises SEQ ID NO: 67. HCDR3 comprises SEQ ID NO: 68, LCDR1 comprises SEQ ID NO: 69, and LCDR2 comprises In another embodiment, the human nectin- Provided herein is an antibody that binds to SEQ ID NO: 4, wherein HCDR1 comprises SEQ ID NO: 76 and HCDR2 comprises SEQ ID NO: 77. comprises SEQ ID NO: 77, HCDR3 comprises SEQ ID NO: 78, and LCDR1 comprises SEQ ID NO: 69 wherein LCDR2 comprises SEQ ID NO:70 and LCDR3 comprises SEQ ID NO:79.

[0032] In one aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain The antibody comprises a HCVR and a light chain variable region (LCVR), the antibody comprising SEQ ID NO: 10. In another embodiment, the human Nectin-4 comprises an HCVR comprising SEQ ID NO: 11 and an LCVR comprising SEQ ID NO: 12. Provided herein is an antibody that binds to the HCVR comprising SEQ ID NO: 14 and SEQ ID NO: In another embodiment, an antibody that binds to human Nectin-4 is described herein. The antibody is provided in accordance with the present invention, which comprises a HCVR comprising SEQ ID NO:24 and a LCVR comprising SEQ ID NO:25. In another aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising the sequence In another embodiment, the human Provided herein is an antibody that binds to Nectin-4, the antibody comprising an HCV polypeptide comprising SEQ ID NO: 44. R and an LCVR comprising SEQ ID NO: 45. In another embodiment, the polypeptide binds to human Nectin-4. Antibodies are provided herein, the antibodies comprising an HCVR comprising SEQ ID NO:52 and an HCVR comprising SEQ ID NO:53. In another aspect, provided herein is an antibody that binds to human Nectin-4. and the antibody comprises a HCVR comprising SEQ ID NO:62 and a LCVR comprising SEQ ID NO:63. In an aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising the sequence represented by SEQ ID NO: 7. 2 and LCVR comprising SEQ ID NO: 73. Provided herein is an antibody that binds to HCVR and sequence comprising SEQ ID NO: 80. Includes LCVR containing column number 81.

[0033] In a further aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising The antibody comprises a human IgG1 or In a further embodiment, the antibody has a human IgG1 isotype. In a further aspect, the antibody has residues 234 and 235 (EU index numbering): In a further embodiment, the antibody comprises an alanine at position 265 (according to EU index no. In another embodiment, the antibody is of the human IgG4 isotype. has.

[0034] In one aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain a light chain (LC) and a heparin chain (HC), the heparin chain comprising amino acids 2 to 444 of SEQ ID NO: 2; C comprises amino acids 2 to 215 of SEQ ID NO: 3. In one embodiment, it binds to human nectin-4. Provided herein is an antibody that binds to a nucleotide sequence that is identical to that of the nucleotide sequence ... LC comprises amino acids 2 to 444 of SEQ ID NO: 12, and LC comprises amino acids 2 to 21 of SEQ ID NO: 13. In one aspect, provided herein is an antibody that binds to human Nectin-4, comprises a heavy chain (HC) and a light chain (LC), the HC being amino acids 2 to 443 of SEQ ID NO: 16. and LC consists of SEQ ID NO: 17. In one embodiment, an antibody that binds to human nectin-4 The antibody is provided herein, the antibody comprising a heavy chain (HC) and a light chain (LC), the HC having the sequence In one embodiment, the LC comprises amino acids 2 to 447 of SEQ ID NO: 27. Provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein HC comprises amino acids 2 to 446 of SEQ ID NO: 36, and LC comprises SEQ ID NO: 37. In one aspect, provided herein is an antibody that binds to human Nectin-4, The antibody comprises a heavy chain (HC) and a light chain (LC), the HC comprising amino acids 2-4 of SEQ ID NO:46. 46, and the LC consists of SEQ ID NO: 47. In one embodiment, Provided herein is an antibody comprising a heavy chain (HC) and a light chain (LC), the HC comprising: In one embodiment, the LC comprises amino acids 2 to 446 of SEQ ID NO: 55. Provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain (HC) and a light chain (LC), the HC comprising amino acids 2-450 of SEQ ID NO: 64, the LC comprising the sequence In one embodiment, the antibody binds to human Nectin-4. Provided herein is an antibody comprising a heavy chain (HC) and a light chain (LC), the HC being represented by SEQ ID NO: 74, and LC comprises amino acids 2-216 of SEQ ID NO: 75. .

[0035] In a further aspect, provided herein is an antibody that binds to human Nectin-4, wherein HC is a sequence 2 and the LC consists of SEQ ID NO: 3. Provided herein is an antibody comprising the sequence of SEQ ID NO: 12, wherein the HC comprises the sequence of SEQ ID NO: 1 In another aspect, provided herein is an antibody that binds to human Nectin-4, comprising H C consists of SEQ ID NO: 16 and LC consists of SEQ ID NO: 17. Provided herein is an antibody that binds to Chin-4, wherein the HC consists of SEQ ID NO: 26 and the LC consists of In another embodiment, an antibody that binds to human Nectin-4 is described herein. wherein the HC consists of SEQ ID NO: 36 and the LC consists of SEQ ID NO: 37. Provided herein is an antibody that binds to human Nectin-4, wherein HC is selected from the group consisting of SEQ ID NO: 46 and SEQ ID NO: 47. and LC consists of SEQ ID NO: 47. In another embodiment, an antibody that binds to human Nectin-4 provided herein, wherein HC consists of SEQ ID NO: 54 and LC consists of SEQ ID NO: 55. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein HC is represented by the sequence In another embodiment, the LC comprises SEQ ID NO: 65. Provided herein is an antibody that binds, wherein the HC consists of SEQ ID NO: 74 and the LC consists of SEQ ID NO: It consists of 75.

[0036] In another embodiment, SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO: 16 and SEQ ID NO:17, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:37 , SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 64 and SEQ ID NO: A polypeptide having the amino acid sequence of SEQ ID NO: 65, or SEQ ID NO: 74 and SEQ ID NO: 75 Different mammalian cells containing DNA molecules that contain the polynucleotide sequences encoding the The cells are provided herein and are capable of expressing the Nectin-4 antibodies disclosed herein.

[0037] In another aspect, a mammalian cell comprising the first DNA molecule and the second DNA molecule is described herein. a first DNA molecule encoding a polypeptide having an amino acid sequence as follows: and the second DNA molecule comprises a polynucleotide sequence encoding The cell comprises a polynucleotide sequence encoding a polypeptide that binds to the polypeptide disclosed herein. It is possible to express Nectin-4 antibodies.

[0038] [Table 1]

[0039] In another aspect, the mammalian cells disclosed herein are cultured under conditions such that the antibodies are expressed. and recovering the expressed antibody. A process for producing is provided herein.

[0040] In another embodiment, SEQ ID NO:2 and SEQ ID NO:3, SEQ ID NO:12 and SEQ ID NO:13, SEQ ID NO: 16 and SEQ ID NO:17, SEQ ID NO:26 and SEQ ID NO:27, SEQ ID NO:36 and SEQ ID NO:37 , SEQ ID NO: 46 and SEQ ID NO: 47, SEQ ID NO: 54 and SEQ ID NO: 55, SEQ ID NO: 64 and SEQ ID NO: A polypeptide having the amino acid sequence of SEQ ID NO: 65, or SEQ ID NO: 74 and SEQ ID NO: 75 A mammalian cell containing a DNA molecule that contains a polynucleotide sequence encoding the antibody is and recovering the expressed antibody. Antibodies that can be used to treat the inflammatory bowel disease are provided herein.

[0041] In another aspect, there is provided a mammalian cell comprising a first DNA molecule and a second DNA molecule, The first DNA molecule is a polynucleotide encoding a polypeptide having the amino acid sequence and the second DNA molecule encodes a polypeptide having the amino acid sequence culturing a cell containing the polynucleotide sequence under conditions such that the antibody is expressed; and recovering the expressed antibody.

[0042] [Table 2]

[0043] As used herein, the term "enfortumab" refers to a compound expressed using standard conditions. and purified, as disclosed in Figures 3A and 3B of WO 2012047724. This refers to a fully human anti-Nectin-4 IgG1 kappa monoclonal antibody having a sequence identical to that of the human Nectin-4 antibody.

[0044] payload The Nectin-4 antibodies of the present disclosure can be administered with various payloads (including pharma- ceutically acceptable salts thereof). to form an antibody drug conjugate (ADC). Suitable moieties for conjugation to the Nectin-4 antibodies disclosed herein include cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes or compounds, toxins, and and other payloads known in the art.

[0045] Exemplary ADCs herein include those that are capable of carrying a camptothecin-based payload (e.g., camptothecin-based Camptothecin analogues have been shown to have anticancer activity. Camptothecin and its derivatives are topoisomerase I (TOPO1) inhibitors. Prevents reannealing of partially cut DNA, the accumulation of which leads to cell death Binds to the TOPO1 / DNA complex. Other topoisomerase I inhibitors can be used as payloads.

[0046] Other payloads for the ADCs described herein include maytansinoids (e.g., DM1 and DM4), pyrrolobenzodiazepines (e.g., PBD dimers), auristatins peptides (e.g., MMAE and MMAF), duocarmycins, calicheamicins , DNA minor groove binders (e.g., enediynes and lexitropsins), and taxanes (e.g., For example, paclitaxel and docetaxel.

[0047] In certain aspects, provided herein are ADCs, wherein the camptothecin analog has the formula : XY, wherein Y is of formula I,

[0048] [ka] During the ceremony, R 1 , F, C.H. 3 , or CF 3 and R 2 H, F, OR 3 , S.R. 3 , S(O)R 4 , -S(O) 2 R 4 , C 1 ~C 6 Al Kill or C 1 ~C 6 fluoroalkyl, or R 1 and R 2 The conclusion is that Together with the carbon atom to which it is attached, methylenedioxy or difluoromethylenedioxy Forming a ring, R 3 is H or C 1 ~C 6 is alkyl, R 4 is C 1 ~C 6 is alkyl, X is the formula of AB, During the ceremony, There is no B, -(C 1 ~C 6 alkylene)-, -(C 1 ~C 6 (alkylene)-X 1 -(C 1 ~C 6 alkylene)-, -X 1 '-(C 1 ~C6 Alkylene)- * , or -(C 1 ~C 6 (alkylene)-X 1 -L 2 - * and * is the site of covalent attachment to A, X 1 -O-, -S-, -S(O)-, -S(O) 2 -, -C(-O)-, -NR 5 -, -NR 5 C(-O)- or -C(-O)NR 5 - and Each R 5 are independently -H, C 1 ~C 6 Alkyl, C 1 ~C 6 Fluoroalkyl, C 3 ~C 6 cycloalkyl, aryl, heteroaryl, or benzyl; X 1 ' is -O-, -S-, -S(O)-, or -S(O) 2 - and L 2 is phenylene, A is -H or -X 2 and X 2 OR 6 , S.R. 6 , S(O)R 6 , S(O) 2 R 6 , SSR 6 , or N(R 6 ) 2 and Each R 6 are independently H, C 1 ~C 6 Alkyl, C 1 ~C 6 Fluoroalkyl, C 3 ~ C 6cycloalkyl, aryl, heteroaryl, or benzyl; B and L 2 are each independently halogen, -CN, -OR 7 , -SR 7 , -N(R 7 ) 2 , C 1 ~C 6 Alkyl, C 1 ~C 6 Fluoroalkyl, C 1 ~C 6 Heteroalkyl, C 3 ~C 6 Cycloalkyl, C 2 ~C 10 Heterocycloalkyl, aryl, or heteroa is optionally substituted with 1 to 4 substituents selected from Each R 7 are independently H, C 1 ~C 6 Alkyl, C 1 ~C 6 Fluoroalkyl, C 3 ~ C 6 cycloalkyl, aryl, heteroaryl, or benzyl; However, R 1 is F, B is -(C1-C6 alkylene)-, (C 1 ~C 6 a Rukiren)-X 1 -(C 1 ~C 6 alkylene)-, -X 1 '-(C 1 ~C 6 Alkylene) - * , or -(C 1 ~C 6 (alkylene)-X 1 -L 2 - * and * is covalently bonded to A A is the site where -X 2 where R1 is F and R 2 Is -OMe In this case, -BA is -NH 2 That's not possible.

[0049] In certain aspects, provided herein are ADCs, wherein the camptothecin analog has the formula : XY, wherein Y is of formula I,

[0050] [ka] During the ceremony, R 1 , F, C.H. 3 , or CF 3 and R 2 H, F, OR 3 , S.R. 3 , S(O)R 4 , -S(O) 2 R 4 , C 1 ~C 6 Al Kill or C 1 ~C 6 fluoroalkyl, or R 1 and R 2 The conclusion is that Together with the carbon atom to which it is attached, methylenedioxy or difluoromethylenedioxy Forming a ring, R 3 is H or C 1 ~C 6 is alkyl, R 4 is C 1 ~C 6 is alkyl, X is * -CH 2 O-, * -(CH 2 ) 2 O-, * -(CH 2 ) 3 O-, *-(CH 2 ) 4 O- * -CH 2 NH- * -(CH 2 ) 2 NH- * -(CH 2 ) 3 NH- * - (CH 2 ) 4 NH- * -CH 2 N(CH 3 )-、 * -(CH 2 ) 2 N(CH 3 )-、 * -(CH 2 ) 3 N(CH 3 )-、 * -(CH 2 ) 4 N(CH 3 )-、 * -CH 2 N(R 5 )-、 * -(CH 2 ) 2 N(R 5 )-、 * -(CH 2 ) 3 N(R 5 )-、 * -(CH 2 ) 4 N(R 5 )-、 * -CH 2 N(CH 3 )C(=O)CH 2 O- * -CH 2 N(CH 2 R 5 )C(=O)CH 2 O- * -CH 2 NHC(=O)CH 2 O- * -CH2 NHC (=O)(CH 2 ) 2 O-、 * -CH 2 NHC(=O)(CH 2 ) 3 O-、 * -CH 2 N HC(=O)(CH 2 ) 4 O-、 * -CH 2 NHC(=O)(CH 2 ) 5 O-、 * -CH 2 NHC(=O)CH 2 -、 * -CH 2 NHC(=O)(CH 2 ) 2 -、 * -CH 2 NH C(=O)(CH 2 ) 3 -、 * -CH 2 NHC(=O)(CH 2 ) 4 -、 * -CH 2 NH C(=O)(CH 2 ) 5 -、 * -CH 2 SCH 2 -、 * -CH 2 S(CH 2 ) 2 -、 * - CH 2 S(CH 2 ) 3 -、 * -CH 2 S(CH 2 ) 4 -、 or * -CH 2 S(CH 2 ) 5 - and *is the moiety covalently attached to Y, and R 5 is phenyl.

[0051] In certain aspects, provided herein are ADCs, wherein the camptothecin analog has the formula : XY, wherein Y is of formula II,

[0052] [ka] X is * -CH 2 O-, * -(CH 2 ) 2 O-, * -(CH 2 ) 3 O-, * -(CH 2 ) 4 O-, * -CH 2 NH-, * -(CH 2 ) 2 NH-, * -(CH 2 ) 3 NH-, * - (CH 2 ) 4 NH-, * -CH 2 N(CH 3 )-, * -(CH 2 ) 2 N(CH 3 )-, * -(CH 2 ) 3 N(CH 3 )-, * -(CH 2 ) 4 N(CH 3 )-, * -CH 2 N(R 1 )-, * -(CH 2 )2 N(R 1 )-、 * -(CH 2 ) 3 N(R 1 )-、 * -(CH 2 ) 4 N(R 1 )-、 * -CH 2 N(CH 3 )C(=O)CH 2 O- * -CH 2 N(R 1 ) C(=O)CH 2 O- * -CH 2 NHC(=O)CH 2 O- * -CH 2 NHC(=O )(CH 2 ) 2 O- * -CH 2 NHC(=O)(CH 2 ) 3 O- * -CH 2 NHC( =O)(CH 2 ) 4 O- * -CH 2 NHC(=O)(CH 2 ) 5 O- * -CH 2 NH C(=O)CH 2 - * -CH 2 NHC(=O)(CH 2 ) 2 - * -CH 2 NHC(= O)(CH 2 ) 3 - * -CH 2 NHC(=O)(CH 2 ) 4 - * -CH 2 NHC(= O)(CH 2 ) 5 -, * -CH 2 SCH 2 -, * -CH 2 S(CH 2 ) 2 -, * -CH 2 S(CH 2 ) 3 -, * -CH 2 S(CH 2 ) 4 -or * -CH 2 S(CH 2 ) 5 -Yes the law of nature, * is the moiety covalently attached to Y, and R 1 is benzyl.

[0053] In another aspect, provided herein is an ADC, wherein the camptothecin analog is Contains one of the expressions.

[0054] [ka]

[0055] In a further aspect, provided herein is an ADC, wherein the camptothecin analog is Contains formula III.

[0056] [ka]

[0057] In a further aspect, provided herein is an ADC, wherein the camptothecin analog is Contains formula IV.

[0058] [ka]

[0059] In a further aspect, provided herein is an ADC, wherein the camptothecin analog is Contains formula V.

[0060] [ka]

[0061] In a further aspect, provided herein is an ADC, wherein the camptothecin analog is Contains formula VI.

[0062] [ka]

[0063] In a further aspect, provided herein is an ADC, wherein the camptothecin analog is Contains formula VII.

[0064] [ka]

[0065] Self-destructive unit The self-immolation, or self-removal, nature of some ADCs is Self-immolation can be designed into the overall structure of the DC. Typically, the self-immolation is achieved by activating a trigger group. and then undergoes chemical and / or biological reactions to form the group itself, i.e. the self-immolative unit. The self-immolative unit induces targeted elimination of the ADC by cellular cleavage. It provides favorable properties to ADCs, such as providing space to reduce steric hindrance of cellular proteases. can be provided.

[0066] In some embodiments of the disclosure, the ADCs described herein contain a self-immolative unit. If present, the self-immolative unit is located on the ADC after the peptide unit of the linker, and Thus, the trigger group is exposed after protease cleavage of the peptide unit of the ADC. In some embodiments, the self-immolative unit is -NH-CH 2 - group, para-aminobenzyloxycarbonyl ortho-aminobenzyl carbonate (PABC), ortho-aminobenzyl carbonate (OABC), or Other self-immolative units are known.

[0067] In some embodiments of the disclosure, the ADCs described herein do not contain a self-immolative unit. In these embodiments, the terminal amine from the camptothecin analogs described herein may be , linked directly to the peptide unit.

[0068] Linker As disclosed herein, the payload can be prepared by methods understood by those of skill in the art. , conjugated to a Nectin-4 antibody, and One example of such a conjugate is the peptide described herein. The antibody comprises a linker connected to the Nectin-4 antibody described herein.

[0069] The linker used in the ADC is chosen to allow time for the ADC to localize to the target cell. The drug is designed for stability in plasma. If the payload is released too quickly, all types of Damage to non-target tissues reduces the therapeutic index of the ADC. Once internalized, the linker acts as a lysing agent to allow the payload to function as designed. It should provide a mechanism for payload release.

[0070] Linkers known to those of skill in the art include, for example, cleavable and non-cleavable portions. Thus, provided herein are compounds that can be used to treat cancer, such as cancers, in which a payload, e.g., a camptothecin analog, is cleaved. It is conjugated to the antibody via a linker having a cleavable moiety or a non-cleavable The ADCs are those in which the antibody is conjugated to an antibody via a linker having a reactive moiety.

[0071] Any suitable linker known in the art can be used in preparing the ADCs of the disclosure. In certain aspects, a linker can be used to link an antibody of the present disclosure with a drug or cytotoxic agent. An example is N-succinimidyl-4-(N- Maleimidomethyl)-cyclohexane-l-carboxylate (SMCC), V-succinimide Nimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), bis-maleimidyl Polyethylene glycol (BMPEO), BM (PEO) 2 , B.M. (P.E.O.) 3 , N- (b-Maleimidopropyloxy)succinimide ester (BMPS), g-Maleimidopropyloxy N-Hydroxy-2-methylbutyric acid (GMBS), N-Hydroxy-2-methylbutyric acid (N-Hydroxy-2-methylbutyric acid) 5-maleimidovaleric acid NHS, HBVS, N -Succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy- (6-Amidocaproate), m-Maleimidobenzoyl-N-hydroxysuccinimide dosester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HC 1 salt (MPBH), N-succinimidyl 3-(bromoacetamido)propionate (SB AP), N-succinimidyl iodoacetate (SIA), k-maleimidoundecanoic acid N-Succinimidyl ester (KMUA), N-Succinimidyl 4-(p-maleimido) Succinimidyl-6-(-maleimidopropionate)-butyrate (SMPB), Succinimidyl-(4-vinylsulfonyl)benzoate (SMPH), Dithiobis-maleimidoethane (DTME), l,4-bis-maleimidoethane (SVSB), Imidobutane (BMB), l,4-bismaleimidyl-2,3-dihydroxybutane (B MDB), Bis-maleimidohexane (BMH), Bis-maleimidoethane (BMOE) , Sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate Silane (Sulfo-SMCC), Sulfosuccinimidyl (4-iodo-acetyl)amino Benzoate (Sulfo-SIAB), m-Maleimidobenzoyl-N-hydroxysulfo Succinimide ester (sulfo-MBS), N-(y-maleimidobutyryloxy)succinimide Sulfosuccinimide ester (sulfo-GMBS or sGMBS), N-(e-maleimide) N-(K-maleoxy)sulfosuccinimide ester (sulfo-EMCS), imidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and and sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB ), Succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), Succinimidyl 4-hydrazide terephthalate hydrochloride (SHTH), Succinimidyl Hydrazinium nicotinate hydrochloride (SHNH), succinimidyl-p-formaldehyde Succinimidyl-p-formylphenoxyacetate (SFB) and succinimidyl-p-formylphenoxyacetate (SFPA), V-succinimidyl-3-(2-pyridyldithio)propionate (SP DP), V-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-Succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and V- Succinimidyl-4-(2-pyridyldithio) 2-sulfobutanoate (Sulfo-SPD B), but are not limited to these.

[0072] In certain aspects of the disclosure, the ADCs comprise a cleavable linker. Different mechanisms for releasing drugs or cytotoxic agents are known in the art. These mechanisms include (1) the activation of cellular proteases (e.g., cathepsin B or β-glucuronidase); (2) utilizing a protease cleavage site in the linker that is cleaved by a protease; By using the lower pH of lysosomes to induce hydrolysis of the acid-labile units in the linker, or (3) utilizing higher intracellular levels of glutathione to disulfide bridges in the linker. This includes reducing the alkyl bridges.

[0073] In some embodiments of the disclosure, the ADCs comprise peptides that provide sites for protease cleavage. In some embodiments, the peptide unit comprises a linker comprising a -Gly-Gly peptide unit. ly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -V al-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -L ys-Lys-, -Ala-Lys-, -Lys-Ala-, -Phe-Cit-, -C it-Phe,-Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Ph e-Ala-, -Ala-Phe-, -Phe-Phe-Lys-, -Lys-Phe- Phe-, -Gly-Phe-Lys-, -Lys-Phe-Gly-, -Leu-Al a-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val- Ala-Val-, -Ala-Leu-Ala-Leu-(SEQ ID NO: 100), -Leu -Ala-Leu-Ala-(SEQ ID NO: 101), -Gly-Phe-Leu-Gly- (SEQ ID NO: 103), -Gly-Leu-Phe-Gly-(SEQ ID NO: 104), -Va l-Arg-, -Arg-Val-, -Arg-Arg-, -Ala-Ala-, -Al a-Met-, -Met-Ala-, -Thr-Thr-, -Thr-Met-, -Me t-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gl n-Val-, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Se r-Ala-, -Ser-Gly-, -Ala-Ser-, -Gly-Ser-, -Le u-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Ty r-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Va l-Thr-, -Thr-Val-, -Met-Tyr-, -Tyr-Met-, -Al a-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Ala-Ala-(配 列番号105), -Gly-Ala-Gly-Gly-(SEQ ID NO: 106), -Gly- Gly-Ala-Gly-(SEQ ID NO: 107), -Gly-Val-Gly-Gly-( 配列番号108), -Gly-Gly-Val-Gly-(SEQ ID NO: 109), -Gly -Phe-Gly-Gly-(SEQ ID NO: 110), or -Gly-Gly-Phe-Gl y- (SEQ ID NO: 102). Citrulline is represented by Cit. In an embodiment of the present disclosure, The peptide units contain all naturally occurring amino acids in the L-amino acid form. The peptide units may contain all D- or L-amino acids, or a combination thereof. It may include.

[0074] In some embodiments, the ADCs described herein further comprise a linker connecting the antibody to the cytotoxic agent. In a further aspect, the linkers provided herein include peptide units. In a further aspect, the peptide units provided herein include Ala-Ala-Ala, Val-Cit, or Gly-Gly-Phe-Gly (SEQ ID NO: 102). In some embodiments, the peptide unit comprises Ala-Ala-Ala. In one embodiment, the peptide unit is Gly-Gly-Phe. -Gly (SEQ ID NO: 102). In a further embodiment, the peptide unit comprises an L-amino acid form It contains all natural amino acids in all forms.

[0075] In some aspects of the disclosure, the ADCs are provided by modifying the cysteines of the antibodies disclosed herein. The spacer moiety herein is connected to the peptide units and / or payloads described herein. The linker includes a spacer unit called the Ser unit A. Some of the chemistries used in the art are maleimides, succinimides, or bromines. Further compounds of the present disclosure may be used in the ADCs of the present disclosure. In an embodiment, maleimidocaproyl (mc) or maleimidomethylcyclohexane-1-caproyl (mc) Maleimide-type spacers such as carboxylates can be used.

[0076] In some embodiments of the disclosure, an ADC having a spacer unit A of formula VIII is provided in the document,

[0077] [ka] In the formula, z is 1 to 5.

[0078] In some embodiments of the disclosure, an ADC having a spacer unit A of formula IX is described herein. Provided in

[0079] [ka] In the formula, z is 1 to 5.

[0080] In certain aspects of the disclosure, an antibody-drug conjugate (ADC) is provided herein. and the ADC has the formula:

[0081] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0082] In another aspect, provided herein is an ADC having the formula X,

[0083] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0084] In another aspect, provided herein is an ADC having formula XI:

[0085] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0086] In another aspect, provided herein is an ADC having formula XII:

[0087] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0088] In another aspect, provided herein is an ADC having formula XIII:

[0089] [ka] wherein Ab is a Nectin-4 antibody disclosed herein and n is from about 1 to about 16. .

[0090] In a further embodiment, n is from about 2 to about 12. In another embodiment, n is from about 2 to about 8. In one embodiment, n is from about 4 to about 8. In another embodiment, n is from about 8 to about 12. In another embodiment, n is about 2. In another embodiment, n is about 4. In another embodiment, n is about 6. In another embodiment, n is about 8. In another embodiment, n is about 10. In another embodiment, n is about 12.

[0091] In certain embodiments disclosed herein, the Ab comprises amino acids 2-444 of SEQ ID NO:2. The present invention comprises an HC and an LC comprising amino acids 2 to 215 of SEQ ID NO: 3, and n is about 8. In one embodiment disclosed herein, the Ab comprises an HC comprising amino acids 2-444 of SEQ ID NO:2; and LC comprising amino acids 2 to 215 of SEQ ID NO: 3, where n is about 4. In a further embodiment, the Ab has an HC consisting of SEQ ID NO:2 and an LC consisting of SEQ ID NO:3. Includes.

[0092] In certain embodiments disclosed herein, the Ab comprises amino acids 2-444 of SEQ ID NO:12. and LC including amino acids 2 to 215 of SEQ ID NO: 13, and n is about 8. In certain embodiments disclosed herein, the Ab comprises an H sequence comprising amino acids 2-444 of SEQ ID NO:12. C and LC comprising amino acids 2 to 215 of SEQ ID NO: 13, and n is about 4. In a further embodiment disclosed herein, the Ab comprises an HC consisting of SEQ ID NO:12 and an HLA consisting of SEQ ID NO:13. and LC consisting of:

[0093] In certain embodiments disclosed herein, the Ab comprises amino acids 2-443 of SEQ ID NO:16. and a LC consisting of SEQ ID NO: 17, where n is about 8. In one embodiment, the Ab has an HC comprising amino acids 2-443 of SEQ ID NO:16 and an HLA-binding domain comprising SEQ ID NO:17. and n is about 4. In further embodiments disclosed herein, Ab comprises , an HC consisting of SEQ ID NO:16, and an LC consisting of SEQ ID NO:17.

[0094] In certain embodiments disclosed herein, the Ab comprises amino acids 2-447 of SEQ ID NO:26. wherein LC consists of SEQ ID NO: 27 and n is about 8. Ab comprises amino acids 2 to 447 of SEQ ID NO:26, and LC comprises SEQ ID NO:27. In a further embodiment disclosed herein, the Ab consists of SEQ ID NO:26. and a LC consisting of SEQ ID NO:27.

[0095] In certain embodiments disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:36. In one embodiment disclosed herein, the LC comprises SEQ ID NO: 37, and n is about 8. Ab comprises amino acids 2 to 446 of SEQ ID NO: 36, and LC comprises SEQ ID NO: 37. In a further embodiment disclosed herein, the Ab consists of SEQ ID NO: 36. and a LC consisting of SEQ ID NO:37.

[0096] In certain embodiments disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:46. wherein LC consists of SEQ ID NO: 47 and n is about 8. Ab comprises amino acids 2 to 446 of SEQ ID NO: 46, and LC comprises SEQ ID NO: 47. In a further embodiment disclosed herein, the Ab consists of SEQ ID NO: 46. and a LC consisting of SEQ ID NO:47.

[0097] In certain embodiments disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:54. In one embodiment disclosed herein, the LC consists of SEQ ID NO: 55, and n is about 8. Ab comprises amino acids 2-446 of SEQ ID NO:54, and LC comprises SEQ ID NO:55. In a further embodiment disclosed herein, the Ab consists of SEQ ID NO:54. and a LC consisting of SEQ ID NO:55.

[0098] In certain embodiments disclosed herein, the Ab comprises amino acids 2-450 of SEQ ID NO:64. LC comprises amino acids 2 to 216 of SEQ ID NO: 65, and n is about 8. In one embodiment, Ab comprises amino acids 2-450 of SEQ ID NO:64, and LC comprises amino acids 2-450 of SEQ ID NO: No. 65, wherein n is about 4. , the Ab comprises a HC consisting of SEQ ID NO:64 and a LC consisting of SEQ ID NO:65.

[0099] In certain embodiments disclosed herein, the Ab comprises amino acids 2-450 of SEQ ID NO:74. LC comprises amino acids 2 to 216 of SEQ ID NO: 75, and n is about 8. In one embodiment, Ab comprises amino acids 2-450 of SEQ ID NO:74, and LC comprises amino acids 2-450 of SEQ ID NO: No. 75, wherein n is about 4. , the Ab comprises a HC consisting of SEQ ID NO:74 and a LC consisting of SEQ ID NO:75.

[0100] Conjugates for anti-nectin-4 antibodies Conjugating the antibodies disclosed herein to the payloads disclosed herein Methods are known in the art. In some methods, the antibody is a first reaction The antibody is then conjugated to a linker in a second reaction. In some methods, the antibody is conjugated to a payload in a single reaction. The compound is conjugated to a payload or payload / linker in

[0101] In some aspects of the present disclosure, the Nectin-4 antibodies described herein are 4. The antibody may be a cysteine ​​residue or a thiol group of one or more cysteine ​​residues located on the antibody. In a further embodiment, the conjugate is covalently attached to a camptothecin analog. The cysteine ​​residues that are interchain disulfide cysteine ​​residues are each interchain disulfide cysteine ​​residues. A method for controlling the reduction of the peptide to allow conjugation to the participating cysteines is desired. In other embodiments, other than those used for the interchain disulfides, The cysteine ​​residues used for conjugation are engineered into the antibody.

[0102] In another aspect of the disclosure, the Nectin-4 antibody described herein is via the amino group of one or more lysine residues located on the campto Covalently attached to the tesin analogue.

[0103] In certain aspects, provided herein are ADCs that are capable of binding to a cytotoxic agent, a cytotoxic agent-autophagy agent, or a combination thereof. The connection of the self-immolative spacer, or cytotoxic agent-self-immolative spacer-linker to the antibody is In a further embodiment, the thiol group on one or more cysteines of Each of the stains is a natural cysteine ​​in the hinge region of the antibody.

[0104] Drug-to-Antibody Ratio (DAR) In the present disclosure, the drug loading in the formula is the number of drug molecules per antibody (drug-to-antibody ratio or D Depending on the context, the subscript n may be used to denote an individual It represents the number of drug molecules bound to each antibody molecule (and is therefore an integer value), or it represents the average drug The average drug loading represents the drug loading in the composition (and therefore may be an integer or non-integer value). represents the average number of drug-linker molecules per antibody.

[0105] A higher DAR can generate more potent ADCs, but a higher DAR still This can lead to destabilization, aggregation, increased off-target toxicity, and reduced drug clearance from the systemic circulation. This may result in enhancement.

[0106] In an embodiment of the present disclosure, the average drug loading, when referring to a composition comprising a population of ADCs, is about 1 In a further embodiment, the DAR is from about 2 to about 8. In a further embodiment, the DAR is 4. In another embodiment, the DAR is about 6 to about 10. In a further embodiment, the DAR is 8. The DAR in the preparation can be determined by mass spectrometry, HIC, ELISA assays, or HPLC, or other techniques known in the art. It can be characterized by the means.

[0107] The present disclosure provides a method of producing an ADC, the method comprising: (a) reducing the Nectin-4 antibody disclosed herein with a reducing agent to obtain a reduced antibody; Producing a Nectin-4 antibody; (b) contacting the reduced Nectin-4 antibody with a compound of the present disclosure to form a conjugate; and producing a compound comprising one or more of formulas I-IX. In a further embodiment, the reducing agent is DTT or TCEP.

[0108] The conjugates of the present disclosure or salts thereof can be readily prepared by a variety of procedures known to those skilled in the art. Several methods, some of which are illustrated in the preparations and examples below, are known in the art. The inventors will now describe a method for preparing the conjugates or salts thereof of the present disclosure using each of the described routes. Certain synthetic steps involved may be combined in different ways or different schemes may be used. The product of each step is extracted, evaporated, precipitated, etc. Conventional methods known in the art, including precipitation, chromatography, filtration, trituration, and crystallization, can be used to separate the nuclei. All substituents, unless otherwise indicated, are as previously defined. The reagents and starting materials are readily available to those skilled in the art. The following preparations, examples, and assays further illustrate the present disclosure but in no way limit the scope of the disclosure. It should not be construed as limiting.

[0109] therapeutic use In another aspect, a patient in need of treatment for cancer is provided with an effective amount of a nephrectomy drug described herein. Methods for treating cancer comprising administering a quinolone-4 ADC or pharmaceutical composition as described herein. In a further aspect, a patient in need of treatment for cancer is provided with an effective amount of the method described herein. A method for treating cancer, comprising administering the Nectin-4 ADC or pharmaceutical composition described in The method of the present invention is provided herein, wherein the cancer is bladder cancer, breast cancer, lung cancer, stomach cancer, colon cancer, or the like. rectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In certain aspects, the ADCs or pharmaceutical compositions described herein are used in perioperative, adjuvant, or is administered in a neoadjuvant setting.

[0110] In a further aspect, a method of treating cancer is provided, wherein the cancer is bladder cancer. In one aspect, a method of treating cancer is provided, wherein the cancer is urothelial cancer. In one embodiment, the urothelial cancer is metastatic urothelial carcinoma (muC). In a further aspect, a method of treating cancer is provided, wherein the cancer is breast cancer. and the cancer is lung cancer. In a further aspect, a method of treating cancer is provided, the cancer being In a further aspect, a method of treating cancer is provided, the cancer being colorectal, gastric, or gastric cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is pancreatic cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is head and neck cancer. In one embodiment, a method of treating cancer is provided, wherein the cancer is ovarian cancer. A method of treating cancer is provided, wherein the cancer is cervical cancer. The method is provided, wherein the cancer is prostate cancer.

[0111] In a further aspect, the patient has relapsed after receiving enfortumab vedotin or Or the patient has become refractory to enfortumab vedotin or standard of care. In aspects, a patient being treated with an ADC or pharmaceutical composition described herein has In a further aspect, the patient is ineligible for treatment with rutumab vedotin. C or the pharmaceutical composition is naïve to treatment with enfortumab vedotin. In a further aspect, a patient treated with an ADC or pharmaceutical composition described herein Participants should be considered for programmatic inclusion in the neoadjuvant / adjuvant, locally advanced or metastatic setting. Death receptor-1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitors, and In a further aspect, the patient has previously received gold-containing chemotherapy. Patients being treated with the pharmaceutical composition are administered a combination of enfortumab and pembrolizumab. or a combination of nivolumab and ipilimumab, or atezolizumab and cisplatin / The patient had become refractory or relapsed to the combination with gemcitabine. In this case, a patient being treated with an ADC or pharmaceutical composition described herein may be Refractory to or relapsed after chemotherapy with cyclosporine or the combination of carboplatin and gemcitabine In a further aspect, a combination of an ADC or pharmaceutical composition described herein with a PD-1 inhibitor is Patients receiving chemotherapy in combination with a cisplatin-containing anti-PD-L1 inhibitor or PD-L1 inhibitor were The patient is ineligible for chemotherapy treatment.

[0112] In a further aspect, an effective amount of an ADC or pharmaceutical composition described herein is administered to one or more The method includes administering the compound in combination with an anti-tumor agent simultaneously, separately or sequentially. In a further aspect, an effective amount of an ADC or pharmaceutical composition described herein is administered to a subject The combination of PD-L1 inhibitors or PD-L1 inhibitors may be administered simultaneously, separately, or sequentially. A method is provided that includes:

[0113] In another aspect, an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein is and administering the FGFR compound simultaneously, separately or sequentially in combination. Methods are provided herein. In a further aspect, the cancer is urothelial cancer. The FGFR compounds are erdafitinib, LOXO-435, futibatinib, and bofatab. Mab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogalati nib, FGF401, or pemigatinib.

[0114] In another aspect, the present invention provides a method for the treatment of a Nectin-4 ADC or a medicament comprising administering to a subject the method of the present invention a method for the treatment of a subject therapies comprising administering to a subject the method of the present invention a Nectin-4 ADC or a medicament for use in treatment of a subject therapies. In a further aspect, the present invention provides a pharmaceutical composition for use in the treatment of cancer. In a further aspect, the cancer is a urinary tract cancer. Skin cancer, breast cancer, lung cancer, stomach cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, In a further aspect, the ADC or therapeutic agent described herein is The pharmaceutical composition is for use in a perioperative, adjuvant, or neoadjuvant setting. be.

[0115] In a further aspect, the present invention provides a method for the treatment of bladder cancer comprising administering to a subject a therapeutically effective amount of Nectin-4 as described herein. ADCs or pharmaceutical compositions are provided herein. In a further aspect, the ADCs or pharmaceutical compositions are used to treat urothelial carcinoma. The Nectin-4 ADC or pharmaceutical composition described herein for use in In a further aspect, the urothelial carcinoma is metastatic urothelial carcinoma (muC). In some embodiments, the ADC or pharmaceutical composition described herein is used to treat breast cancer. In a further aspect, the method of claim 1, further comprising administering to said patient a therapeutically effective amount of the compound described herein for use in the treatment of lung cancer. Provided herein is an ADC or pharmaceutical composition comprising the ADC or pharmaceutical composition for use in the treatment of gastric cancer. Provided herein are ADCs or pharmaceutical compositions as described herein that can be used in In embodiments, an ADC or pharmaceutical composition described herein is used to treat colorectal cancer. In a further aspect, a method for treating pancreatic cancer is provided herein. The described ADC or pharmaceutical composition is provided herein. In a further aspect, the ADC or pharmaceutical composition is used in the treatment of head and neck cancer. Provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of In a further aspect, the ADC or therapeutic agent described herein is used to treat ovarian cancer. A pharmaceutical composition is provided herein. In a further aspect, the present invention is directed to a method for treating cervical cancer. Provided herein is an ADC or pharmaceutical composition as described herein. The ADC or pharmaceutical composition described herein for use in the treatment of prostate cancer is Provided.

[0116] In a further aspect, the present invention provides a method for the treatment of cancer comprising administering to a subject a Nectin-4 AD as described herein. C or a pharmaceutical composition is provided herein, wherein the cancer is treated with enfortumab vedotin. The cancer has recurred or the cancer has become refractory to enfortumab vedotin. In a further aspect, the Nectin-4 ADC described herein for use in the treatment of cancer. or a pharmaceutical composition provided herein, wherein the cancer is treated with enfortumab vedotin. The cancer has recurred or has become refractory to standard treatments. For use in the treatment of cancer where the prior use of enfortumab vedotin was contraindicated Provided herein is an ADC or pharmaceutical composition as described herein. For use in the treatment of cancer without prior use of enfortumab vedotin Provided herein are ADCs or pharmaceutical compositions described herein for use in administering the invention to patients having autism or bronchitis. In certain aspects, the ADC or pharmaceutical composition described herein for use in the treatment of cancer is As provided herein, neoadjuvant / adjuvant, locally advanced or metastatic In settings with prior use of PD-1 or PD-L1 inhibitors and platinum-containing chemotherapy or the combination of enfortumab and pembrolizumab, nivolumab and ipilimumab In combination with mab or in combination with atezolizumab and cisplatin / gemcitabine In a further aspect, the present invention provides a method for the treatment of cancer comprising administering to a subject a therapeutically effective amount of PD The present invention relates to a combination of a PD-1 inhibitor or a PD-L1 inhibitor, either simultaneously, separately or sequentially. and the cancer is a cisplatin-containing compound. It cannot be treated with chemotherapy.

[0117] In a further aspect, a compound for use in the treatment of cancer, in combination with one or more anti-tumor agents, The ADCs or pharmaceutical compositions described herein, either separately or in sequential combination, are In a further embodiment, the anti-tumor agent is a PD-1 inhibitor or a PD-L1 inhibitor. It is.

[0118] In another embodiment, a compound, either simultaneously or separately, for use in the treatment of cancer. or a Nectin-4 ADC or pharmaceutical composition described herein, which are sequentially combined In a further aspect, the cancer is urothelial carcinoma. The FGFR compounds are erdafitinib, LOXO-435, futibatinib, and bofatab. Mab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogalati nib, FGF401, or pemigatinib.

[0119] In another aspect, the use of a nectin molecule as described herein in the manufacture of a medicament for the treatment of cancer. Provided herein are uses of the -4 ADC or pharmaceutical composition for the treatment of cancer. The present invention also relates to the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for treating The cancers provided in the supplement are bladder cancer, breast cancer, lung cancer, stomach cancer, colorectal cancer, and pancreatic cancer. , head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer.

[0120] In a further aspect, the use of the compounds described herein in the manufacture of a medicament for the treatment of cancer. Use of a vasopressin-4 ADC or pharmaceutical composition is provided herein, wherein the cancer is treated with enfortumab vehi The cancer has recurred after treatment with enfortumab vedotin or target In a further embodiment, the prior use of enfortumab vedotin is associated with refractory disease. In the manufacture of a medicament for the treatment of cancer, In a further aspect, the use of enfortumab vedotin or a pharmaceutical composition thereof is provided herein. The use of the compounds described herein in the manufacture of a medicament for the treatment of cancer without prior use of the compounds described herein Use of the ADC or pharmaceutical composition thereof is provided herein. Adjuvant / PD-1 or PD-2 in the locally advanced or metastatic setting In the manufacture of drugs for the treatment of cancer, the prior use of L1 inhibitors and platinum-containing chemotherapy Provided herein is the use of an ADC or pharmaceutical composition described herein in In an embodiment, the ADC or pharmaceutical agent described herein in the manufacture of a medicament for the treatment of cancer. Use of the composition is provided herein, the cancer being treated with cisplatin-containing chemotherapy. and such agents may be administered simultaneously, separately, or in combination with a PD-1 inhibitor or a PD-L1 inhibitor. It is administered continuously.

[0121] In a further aspect, the ADC described herein in the manufacture of a medicament for the treatment of cancer. or the use of a pharmaceutical composition is provided herein, such an agent being co-administered with one or more antitumor agents. In a further aspect, the method comprises administering the method of the present invention to a subject in need of treatment for cancer. Provided herein is the use of an ADC or pharmaceutical composition described herein in The agent may be administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor. can be.

[0122] In another aspect, the use of a nectin molecule as described herein in the manufacture of a medicament for the treatment of cancer. Use of an ADC or pharmaceutical composition is provided herein, such an agent comprising an FGFR compound. In a further aspect, the cancer is urothelial cancer. In a further embodiment, the FGFR compound is erdafitinib, LOXO-435, fuchi Vatinib, bofatamab, bemarituzumab, derazantinib, infigratinib, pemi Gatinib, rogaratinib, FGF401, or pemigatinib.

[0123] In a further aspect, the bladder cancer is urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma. In certain aspects, the bladder cancer is non-invasive, non-muscle invasive, or muscle invasive. In a further aspect, the bladder cancer is post-cystectomy muscle invasive bladder cancer. is of the bladder, renal pelvis, ureter, or urethra. In a further aspect, the breast cancer is HR positive, HER2-negative breast cancer, or triple-negative breast cancer (TNBC). In a further aspect, the breast cancer is ductal or lobular carcinoma. In a further aspect, the lung cancer is non-squamous cell lung cancer (NSCLC) or non-squamous NSCLC. In a further aspect, the prostate cancer is metastatic. In a further aspect, the gastric cancer is esophageal cancer or gastroesophageal junction cancer. In a further aspect, the ovarian cancer is serous or mucinous. The ovarian cancer is fallopian tube cancer or peritoneal cancer.

[0124] In a further embodiment, the anti-tumor agent may be a chemotherapy treatment agent, including a platinum-containing chemotherapy agent. and / or cisplatin, carboplatin, dacarbazine, liposomal doxorubicin docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, Paclitaxel, Paclitaxel Protein-bound Particle Injection Suspension, Ixabepilone, Pecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin) , FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabi topotecan, liposomal irinotecan, pemetrexed, methotrexate, In a further aspect, the chemotherapeutic agent may include blastine, and cetuximab. The combination of cisplatin (or carboplatin) and gemcitabine, including In a further embodiment, the antitumor agent is nivolumab, ipilimumab, pidilizumab, pembro Limulizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat and durvalumab. do.

[0125] Pharmaceutical Compositions and Methods of Administration The antibodies or ADCs described herein can be formulated as pharmaceutical compositions and used in medicine. The pharmaceutical composition may be administered, for example, orally, topically, or subcutaneously, to provide a biologically useful antibody or ADC. The compound may be administered by any route that makes the compound available.

[0126] Also provided herein are methods for combining an antibody or ADC with a physiologically acceptable carrier, diluent, and one or more agents selected from the group consisting of excipients and auxiliary agents. Provided in the specification.

[0127] The antibodies or ADCs of the present disclosure, or pharmaceutical compositions comprising same, can be administered parenterally (e.g., via the skin). The antibodies or ADCs of the disclosure may be administered intravenously or intravenously. It may be administered alone to a patient, in combination with a carrier, diluent or excipient, in either single or multiple doses. The pharmaceutical compositions described herein can be prepared by methods well known in the art (e.g., Rem. ington:The Science and Practice of Pharm acy, 22nd ed. (2012), A. Loyd et al., Pharmac. The antibodies disclosed herein can be prepared by the National Atomic Press. or ADC, and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0128] In one embodiment, the antibodies disclosed herein are combined with one or more pharma- ceutically acceptable carriers, diluent, Disclosed herein is a pharmaceutical composition comprising a diluent, a diluent, or an excipient. and one or more pharma- ceutically acceptable carriers, diluents, or excipients. Disclosed herein is a pharmaceutical composition comprising:

[0129] definition As used herein, in the context of this disclosure (especially in the context of the claims), The terms "a," "an," "the," and similar terms are used herein unless otherwise defined in this specification. Unless otherwise clearly contradicted by context, both the singular and plural forms are used. should be interpreted as a bar.

[0130] As used herein, the term "bond" means a chemical bond or refers to the ability of a protein or molecule to form an attractive interaction with another protein or molecule. It is intended to mean that the ion exchange rate is 100%, as determined by common methods known in the art. This brings the two proteins or molecules into close proximity so that the two proteins or molecules can be linked together.

[0131] As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic result. The effective amount of protein or conjugate refers to the amount (duration and means of administration) required. The disease state, age, sex and weight of the individual, and the protein or The effective amount may vary depending on factors such as the capacity of the conjugate. Any toxic or detrimental effects of the conjugate are outweighed by the therapeutically beneficial effects. .

[0132] As used herein, the terms "treat," "treatment," or "treating" may slow, control, delay, or halt the progression of a disorder or disease disclosed herein; refers to any process that may ameliorate a disorder or disease symptom, but does not necessarily apply to all disorders or disease symptoms. However, it does not necessarily indicate complete disappearance of disease symptoms.

[0133] As used herein, the term "patient" refers to a human patient.

[0134] Certain abbreviations are defined as follows: "ACN" is acetonitrile "DCM" refers to dichloromethane, and "DIPEA" refers to N,N-dichloromethane. isopropylethylamine, and "DBU" is 1,8-diazabicyclo[5.4.0 ]undec-7-ene, and "DMTMM" refers to (4-(4,6-dimethoxy-1,3 ,5-triazin-2-yl)-4-methyl-morpholinium chloride, and "DMA "C" stands for dimethylacetamide, and "DMF" stands for N,N-dimethylformamide. "DTT" refers to dithiothreitol, and "EtOAc" refers to ethyl acetate. "EDTA" refers to ethylenediaminetetraacetic acid, "FA" refers to formic acid, and "HMPA " refers to hexamethylphosphoramide, "h" refers to time, and "HEPES" refers to ( N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) " refers to N-methylmorpholine, and "NMP" refers to (N-methyl-2-pyrrolidone). "Su" stands for succinimide, and "PPTS" stands for p-toluenesulfonic acid picrate. "THF" stands for tetrahydrofuran. n), "TsOH" refers to p-toluenesulfonic acid, and "TCEP" refers to triphenylphosphine oxide. It refers to bis(2-carboxyethyl)phosphine. EXAMPLES

[0135] Example 1: Generation of Nectin-4 antibody The amino acid sequences of the CDRs, variable regions, complete heavy chains and light chains of antibodies 1 to 8, and the coding regions thereof The nucleotide sequences to be loaded are listed below in the section entitled "Amino Acid and Nucleotide Sequences." In addition, the CDRs, light chains, heavy chains, light chain variable regions, and heavy chain variable regions of antibodies 1 to 8 are also included. The sequence numbers for are shown in Tables 1 and 2.

[0136] The anti-Nectin-4 antibodies of the present disclosure, including but not limited to Antibodies 1 to 8, are essentially Antibodies can be expressed and purified as follows: or a single expression system encoding both the HC and LC. HEK293 or GFP-transfected cells were either transiently or stably transfected with the vector system. The expression plasmid contains the LC and H of the antibody. C gene (e.g., as shown in Table 3), Commonly used and widely used vectors for this purpose, such as those based on the viral major immediate-early promoter, It is expressed from a suitable construct.

[0137] The medium into which the antibodies of the present disclosure are secreted is subjected to ion exchange and hydrophobic interaction chromatography. The medium can be purified by conventional techniques, such as mixed mode methods. For example, the medium can be purified by conventional The method is used to apply and elute from a Protein A or Protein G column. and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be isolated by size exclusion, hydrophobic interactions, ionic interactions, and the like. It can be effectively removed by common techniques including hydroxyapatite chromatography or by The product can be immediately frozen, for example at -70°C, refrigerated, or lyophilized. Various methods of protein purification may be employed and such methods are well known in the art. The method is well known in the art and is described, for example, in 30 Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Pro tein Purification:Principles and Practical e, 3rd Edition, Springer, NY (1994). can be frozen immediately at -70°C, stored at 2-8°C for several months, or lyophilized. The mixture may be allowed to stand or stored at 4° C. for immediate use.

[0138] [Table 3]

[0139] [Table 4]

[0140] [Table 5]

[0141] Example 2: Generation of Nectin-4 ADC Synthesis of camptothecin analogues. ACS Med.Chem.Lett.2019,10,1386-1392 and International The present disclosure, such as A1, is essentially as provided in Publication No. 2020219287. Amputothecin analogs can be synthesized as follows.

[0142] [ka]

[0143] Step 1: To a flask containing anhydrous 1,2-dichloroethane (50 mL), add 1 in DCM M BCl 3 (9.95 mL, 9.95 mmol) was added, followed by ice H 2 O bath at 0 °C. 3-Fluoro-4-methylaniline 1 (1.56 g, 12.4 mmol) was added to Add in small portions and then stir at 0° C. for 10 min, after which 5-bromovaleronitrile 2(1 0.72mL, 14.9mmol) was added, followed by addition of AlCl 3 (2.16g, 16.2mm ol) was added. The ice bath was removed and the reaction solution was allowed to warm gradually to room temperature. Stir at room temperature for 10 minutes. After that, the mixture was heated to reflux for 39 h. The solution was cooled to room temperature and diluted with cold H 2 O (25 mL) After slow addition, 5% aqueous HCl was added. After 30 min, the solution was diluted with DCM (50 mL). The organic layer was diluted with H 2 Wash with anhydrous NaCl and brine. 2 SO 4 After drying with The filtrate was concentrated under reduced pressure, and the residue was subjected to reverse phase chromatography using the following conditions: It was purified by Column: C18 (100 g), H 2 Elute with 25% ACN in O for 5 min, then Come H 2 25%–95% ACN in O for 15 min, followed by H 2 Dip in 95% ACN in O for 5 min. Compositional switching gave compound 3 as an off-white solid (1.42 g, 40%).

[0144] Step 2: Compound 3 (3.15 g, 15.64 mmol) in toluene (200 mL), Compound 4 (3.92 g, 14.89 mmol) and PPTS (0.037 g, 0.15 mmol) mol) was added to a 50 mL flask equipped with a reflux condenser containing anhydrous toluene (10 mL). The reaction was heated to reflux for 40 hours with magnetic stirring under an argon atmosphere overnight, The mixture was then allowed to cool to room temperature, and the solid was filtered and washed with toluene (5 mL) to give 5. Obtained (4.74g, 74%).

[0145] Step 3: Compound 5 (0.860) in HMPA (5 mL) and deionized water (0.9 mL) g, 1.67 mmol) was heated at 101 °C for 18 h. After cooling to room temperature, the solution The product was loaded onto a C18 cartridge and purified by reversed-phase chromatography using the following conditions: Column: C18 (30 g), H 2 Elute with 25% ACN in O, then H 2 25% in O ~95% ACN for 15 min, then H 2 Switch to a 5 min gradient of 95% ACN in O to mix. The mixture was diluted with CH 2 Cl 2 A linear gradient of 0 to 20% MeOH in 15 min was used. Further purification by silica gel chromatography afforded compound A1 as an off-white solid. Obtained (0.392 g, 51% yield).

[0146] Synthesis scheme of linker + self-immolative unit + camptothecin analogue ACS Med.Chem.Lett.2019,10,1386-1392 and International The camptothecin derivatives of the present disclosure are essentially as provided in Publication No. 2020219287. The analogues of cyclohexyl ether were conjugated to a linker (using a maleimide compound) and a self-immolative unit as follows: It can be adjudged.

[0147] [ka]

[0148] Step 4: Fmoc-GGFG (1 g, 1.59 mmol, 1.5 eq.) in NMP, A 1 (0.48 mg, 1.06 mmol, 1 equiv.) and 4 Å molecular sieves (3 g, A A mixture of 1 (3×wt. of 1) was added to HCl (4M, 4.5 equiv.) in 1,4-dioxane at 20° C. The reaction was quenched by adding H2O. The solids were removed by filtration. The organic layer was extracted with EtOAc. 2 SO 4 Dry, filter, and evaporate under reduced pressure. The residue was purified by normal phase chromatography (SiO2 , 0-10% MeOH in DCM) Further purification gave Fmoc-GGFG-A1 (0.75 g, yield 69%).

[0149] Step 5: Fmoc-GGFG-A1 (0.75g, 0.73mmol, 1 equivalent) in DMF amount) was treated with DBU (0.11 mL, 0.77 mmol, 1.05 equiv) at 0 °C. The reaction was quenched by adding TsOH (0.25 g, 1.47 mmol, 2 equiv.). The resulting mixture was stirred at 0° C. for 4 h and then carried to the next step without further purification. was used directly.

[0150] Step 6: Crude mixture of GGF-A1 (0.73 mmol, 1 eq.) and TsOH in DMF The mixture was basified to pH 7 with NMM at 0° C., followed by additional NMM (0.16 mL, 1.47 mmol, 2 eq.) and compound 6 (0.283 g, 0.917 mmol, 1.2 The resulting mixture was stirred at 0°C for 4-6 hours, then quenched and The compound was purified by phase chromatography (SiO2, 0-10% MeOH in DCM) to give mc -GGFG-A1 (0.51 g, yield 70%) was obtained.

[0151] Camptothecin analogs of the present disclosure may be prepared by cleaving a linker (bromoacetamide compound) as follows: and self-immolative units.

[0152] [ka]

[0153] Step 1: Fmoc-GGGF-A1 (0.5 g, 0.489 mm) in DMF (5 mL) ol) solution, Et 2NH (0.025 g, 0.34 mmol) was added. The mixture was The mixture was stirred at 15-25°C for 1 hour. The reaction was then quenched by the addition of THF, followed by reducing The residue was purified by reverse phase chromatography using the following conditions: Column, C18, 150 x 30 mm x 5 μm, H 2 Elution was performed with 22–45% ACN in O (0 0.225% FA), and GGFG-A1 was obtained as the FA salt (0.196 g, 47% yield).

[0154] Step 2: To a solution of compound 7 (2.0 g, 12.26 mmol) in 20 mL of THF, Compound 8 (2.9 g, 12.29 mmol) was added at 0° C. The reaction mixture was cooled to 20° C. Stirring was continued at 20° C. for 18 h, after which additional compound 8 (0.5 g, 3.06 m After stirring at 20° C. for an additional 4 h, the reaction was concentrated under reduced pressure. The product was purified by reverse phase chromatography. Column: C18 column, 150×30 mm× 5μm, H 2 Elution with 22–45% ACN in O (0.225% FA) gave compound 9 ( 1.15g, 33% yield).

[0155] Step 3: Compound 9 (430 mg, 1.51 mmol) and D in DMAC (11 mL) A mixture of MTMMT (340 mg, 1.04 mmol) was cooled to 0° C. DMAC (4 GGF-A1 (0.500 g, 0.63 mmol) and DIPEA (0.0 mL) A mixture of 90 g, 0.70 mmol) was added dropwise. Stirring was continued at 0° C. for 0.5 h. DCM (300 mL) was then added. The mixture was diluted with 10% NaBr (3×50 mL The organic layer was washed with Na 2 SO 4 Dry at 40°C, filter, and concentrate under reduced pressure at 0-10°C. The residue was purified by reverse phase chromatography. Column: C18 column, 150 × 3 0mm×5μm, H 2 Elution with 15-45% ACN in O (0.225% FA) and Br-G GFG-A1 was obtained (0.23 g, yield 34.7%).

[0156] Part I. Preparation of ADCs with maleimide linker-payload To prepare antibody-drug conjugates with eight drugs per antibody, IgG1 The antibody is diluted with 6-8 molar equivalents of a reducing agent such as DTT or TCEP at 37°C for 2 hours. The reduced antibody is then diluted with 50 mM HEPES containing 2 mM EDTA. The buffer was exchanged using a PD-10 desalting column containing ES (pH 7.0), and the eluate was purified with HE Adjust the protein concentration to 5-10 mg / mL with PES buffer. Remove excess linker-Pe Loading (e.g., 10 molar equivalents) was added over 1 hour to provide a substantial excess of L-cysteine. The conjugation reaction can be quenched by the addition of (eg, 6 molar equivalents). The resulting mixture of ADCs was equilibrated with 25 mM histidine, 9% sucrose (pH 5.5). The product was purified on a PD-10 desalting column with a 30 kDa MWCO centrifuge unit. Perform three centrifugation cycles using 100% ethanol to remove unreacted linker-payload related species. Finally, the resulting ADC was sterile filtered through a 0.2 μM filter for future use. It can be stored at 4°C or -80°C for use.

[0157] Part II. Preparation of ADCs with bromoacetyl linker-payloads To prepare antibody-drug conjugates with eight drugs per antibody, IgG1 The antibody is diluted with 6-8 molar equivalents of a reducing agent such as DTT or TCEP at 37°C for 2 hours. The reduced antibody is then diluted with 50 mM HEPES containing 2 mM EDTA. The buffer was exchanged using a PD-10 desalting column containing ES (pH 7.4), and the eluate was purified with HE Adjust the protein concentration to 5-10 mg / mL with PES buffer. Remove excess linker PAL. Add 12 molar equivalents of L-cysteine ​​over a period of 2-3 hours to obtain a substantial excess of L-cysteine. The conjugation reaction can be stopped by adding 10 molar equivalents of The resulting ADC mixture was diluted with 25 mM histidine, 9% sucrose (pH 5.5) and diluted with 10% ethanol. Purification was performed using equilibrated PD-10 desalting columns, followed by centrifugation in 30 kDa MWCO centrifuge units. Three centrifugation cycles using 100% ethanol were performed to remove unreacted linker-payload related species. Finally, the resulting ADC was sterile filtered through a 0.2 μM filter for future use. The mixture can be stored at 4°C or -80°C for further use.

[0158] Example 3: Antibody Binding Affinity, Cross-Reactivity, and Selectivity Characterization of human and cross-species binding of nectin-4 ADCs by surface plasmon resonance Using Biacore 8K+ equipment (Cytiva (Marlborough, MA)) Using this method, a Nectin-4 ADC (conjugated as in Formula XI, with a DAR of 8) was Recombinant HIS-tagged human (Acr Biosystems, Catalog No. NE4-H52H3 (Newark, DE) , cynomolgus monkey (Acro Biosystems, Catalog No. NE4-C52H4) , and rats (R&D Systems, Minneapolis, MN, catalog no. The kinetic and affinity parameters of the binding interaction with β-lactamase (No. 9997-N4-050) were determined. Ta.

[0159] The anti-human Fc sensor surface was coated with goat anti-human IgG Fc (Southern Biotec ch, catalog number 2014-01 (Birmingham, AL) Series S CM4 (Cytiva, catalog number BR-100534) sensor The antibody was prepared by amine coupling to the surface at 25°C. Immobilization was performed in 10 mM HEPES. , 150 mM NaCl, 0.05% Tween-20, pH 7.4 running buffer All eight channels of flow cells 1 and 2 were filled with 400 mM 1-ethyl-3 -(3-Dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 nM N -10 μL / mL of a 1:1 (v / v) mixture of hydroxysuccinimide (NHS) The anti-human IgG Fc capture reagent was then added at a flow rate of 10 μL / min for 7 min. The sensor surface ( The antibody was bound to 100 mM EtOH (diluted to 50 μg / mL in 10 mM acetate pH 4.5 buffer). Add ethylenediamine (in 200 mM borate buffer, pH 8.5) to all flow cells and The remaining active groups were blocked by injecting the solution into the column at a flow rate of 10 μL / min for 7 min. The total volume was then analyzed by three successive 1-min injections of 75 mM phosphoric acid at 10 μL / min. All channels and flow cells were preconditioned.

[0160] For kinetic / affinity analysis, running buffer and sample dilution buffer contained 10 mM phosphate. Sodium chloride, 150 mM NaCl, 0.05% Tween-20, pH 7.4, 1 mM g / mL bovine serum albumin (BSA) and the assay temperature was 37°C.

[0161] In each analytical cycle, a different ADC is injected at 5 μg / mL and 10 μL / min. After capture, the same analytes were then added to flow cell 2 of each channel by 30 μl of ethanol. L / min for 2 min into flow cells 1 and 2 of all eight channels and monitor dissociation for 10 min. After dissociation, 75 mM phosphoric acid was injected at 10 μL / min for 1 min, and this was repeated three times. All surfaces were regenerated by repeating the ADC capture and analyte cycles for 0, 2 Analysis of each ADC at concentrations of 0.5, 7.4, 22, 67, 200 and 600 nM Nectin-4 The product bond was obtained.

[0162] Sensorgram data was analyzed using Biacore Insight Evaluation Default 1:1 binding model in Software v3.0.12.15655 A global fit was performed using the following formula: The kinetic and affinity parameters of the ADCs are shown in Table 4. .

[0163] [Table 6] * Although binding is clearly observed, the data show kinetic heterogeneity, which supports a 1:1 binding model. The results are poorly fitted. k a is the association rate constant, and k d is the dissociation rate constant, K D is the equilibrium dissociation constant (K D = k d / k a where n is the number of iterations. For n=1 or 2, , indicate repeated values, n=3, mean ± standard deviation.

[0164] Cell surface binding of Nectin-4 antibodies in cell lines expressing the Nectin-4 receptor A panel of nine Nectin-4 antibodies was assayed for cell expression in two Nectin-4-expressing tumor cell lines. The surface binding of the antibody was tested in SUM190PT tumor cells, which show high endogenous expression, and in SUM190PT tumor cells, which show lower endogenous expression. Using NCI-H1781 tumor cells, which show high endogenous expression, we investigated the expression of β-lactam IgG1 in tumors with high receptor density and low receptor densities. Cell lines were selected to represent somatic densities. SUM190PT and NCI H1781 cells were , and were determined to have antibody binding capacities of 108,000 and 20,000, respectively (MES F quantification kit, Bangs Laboratories). Antibody binding was quantified by flow cytometry. EC of binding curve 50 Both the mean binding MFI and the maximum binding MFI for each antibody were recorded.

[0165] The cells were dissociated using non-enzymatic dissociation buffer for 5 min at 37°C. The cells were counted and 10 in a polypropylene 96-well plate 5 Cells were dispensed at 1800r / well. The mixture was centrifuged for 5 min at 37 °C and the supernatant was discarded. Assay buffer (1% BSA and 0.09% Starting at 300 nM, dilute 1:4 in 1x PBS containing sodium azide. An 11-point antibody dilution series was prepared. The dilution series was added to the cells at 100 μL / well. Several untreated control wells / cell lines were assayed. The cells and antibodies were incubated for 1 hour at 4°C on an orbital shaker. After incubation, the assay plate was centrifuged and 300 μL / well was added. The cell pellet was then washed twice with 100% Al assay buffer. A 1:500 dilution of exa647-conjugated mouse anti-human IgG secondary antibody (100 μL) The assay plate was incubated in the dark for 1 hour at 4°C with shaking until stained with 100 µL of ink. After incubation, the plates were centrifuged and the cells were diluted with 300 μL / well. The plate was washed twice with 1× PBS assay buffer. Dispense 100 μL / well of a 1:5000 solution of Car (BioLegend) onto the cells. The plates were incubated at 4°C in the dark with shaking for 10 min. The cell pellet was then Wash the plates once with assay buffer and then add 200 μL / well of 4% paraformaldehyde in 1× PBS. The cells were fixed with aldehyde for 15 min at room temperature in the dark. The cells were centrifuged, washed once, and then incubated at 65 °C for 15 min at room temperature. Resuspend in 1 μL of assay buffer and incubate with Sartorious iQue HTFC Cyt Obtained using an ometer.

[0166] Cells were acquired using a Sartorious iQue HTFC Cytometer and F Using oreCyt standard edition (v.6.2.6652) The FCS file was generated. The FCS file was then imported into FlowJo (v10.8.1). Debris was analyzed by forward scatter (FSC) versus side scatter (SSC) gating. Single cells were excluded from the analysis and analyzed using a forward scatter area (FSC-A) versus height (FSC-H) gate. Finally, we selected the dead mice that were positive for Zombie Green staining. The cells were excluded and the MFI of Alexa647-positive live cells was quantified. Fluorescence was subtracted from all samples. Data were graphed and analyzed using GraphPad Primer. Analyzed using sm (v9.5.1). EC 50 agonist vs. response - variable slope (4 parameters) The % maximum binding of Enfortumab was determined via curve fitting for each cell line. The mean of the maximum enfortumab MFI was calculated as 100%.

[0167] Nine Nectin-4 antibodies did not bind to the Nectin-4 negative T24 parent line. As shown, the antibody showed good binding to both high and low Nectin-4 expressing tumor cells.

[0168] [Table 7] MFI: Mean Fluorescence Intensity

[0169] Flow cytometry of normal human epidermal keratinocytes using Nectin-4 antibody and AD C bond characteristics To characterize the binding of the disclosed Nectin-4 antibodies and ADCs to normal human epidermal keratinocytes, To culture the HEKα cells, 150 × 25 mm tissue culture dishes (Corning Inc.) were Complete culture medium (Dermal Cell Basal Medium + Kerat Inocyte Growth Kit (ATCC), 1 million cells were plated per well. The cells were then plated and reached confluence within 24 hours. The differentiation process was continued for another 5–10 min. After induction of differentiation, the cells were incubated with 2 mg / mL Clostridium histolyticum Collagenase from cumin, Type XI (Sigma-Aldrich) was used for 37 The cells were dissociated at 4°C. The cells were counted and plated in V-bottom polypropylene 96-well plates (Thermo Scientific Nunc) 5 Cells / well were dispensed. Antibodies and ADCs were , 300 nM in assay buffer (1×DPBS containing 2% FBS, Gibco). The cells were added in 1:4 serial dilutions starting at 0.05%. The cells were then incubated on a microplate shaker The cells were incubated at 4°C for 1 hour in the dark. Wash twice with 100 µL / well of assay buffer and add 1:1,000 diluted Alexa F Luor 647 Conjugated AffiniPure F(ab')2 Fragment Goat Anti-Human Staining was performed with IgG(H+L) (Jackson ImmunoResearch). 4℃ After 1 hour incubation at 4°C, cells were washed twice with 200 μL / well of assay buffer. Then, Zombie Green fixable viabili diluted 1:2000 The cells were stained with ty dye (BioLegend). The cells were washed with assay buffer and incubated at 4° C. for 20 min. Cells were fixed with BD Cytofix fixation buffer for 20 minutes at 4°C. CytPix flow cytometer (Thermo Fisher Scientific The cells are resuspended in assay buffer for acquisition at 17,000 μl. The antibody-binding capacity was determined (MESF quantification kit, Bangs Laborat ories).

[0170] As shown in Table 6, the tested Nectin-4 antibodies and ADCs (consisting of Formula XI) The DAR is 8) is 100% with enfortumab and enfortumab ADC (formula XI, the DAR is 8. EC similar to or higher than that of 50 Ab1 binds to normal human epidermal keratinocytes. , has a lower affinity for HEKα cells than enfortumab.

[0171] [Table 8] EC 50 = half maximum effective concentration

[0172] Example 4: ADC Binding and Internalization Fluorescence imaging of human nectin-4 positive cells using nectin-4 antibody and A Characterization of DC internalization capacity The T24 cell line was engineered to express human nectin-4-eGFP and analyzed for high expression. The T24-human nectin-4-eGFP clone 3 was 379,000 The antibody binding capacity of the antibody was determined to be 0 (MESF quantification kit, Bangs Laboratories). T24 human nectin-4-eGFP clone 3 cells were cultured in complete medium. Nutrient medium (modified McCoy's 5A+10%FBS+Glutamax+400μg / m L G418+Pen / Strep) Medium, black transparent bottom CellCarrier Ultr a 384-well microplate (Perkin Elmer), 12 per well The plates were sealed with AeraSeal™ Sealing Film. Cover with ms and incubate at 37°C, 5% CO 2 The next day, the antibodies and ADCs were incubated at The plates were sealed with AeraSeal ( Cover with PerkinElmer Opera Phenix S for 24 hours. The data was then placed in an incubator for imaging with the cleaning system. The data was processed and analyzed in Harmony and Microsoft Excel, and GraphP Graphed using ad Prism.

[0173] As shown in Table 7, Nectin-4 antibodies and Nectin-4 ADCs (as in Formula XI) and the DAR is 8. Induces degradation of the nectin-4-eGFP signal. The Cutin-4 ADC has been shown to be more efficient at internalizing than the enfortumab antibody, which is in the same ADC format as antibodies 1 to 8. He had the ability.

[0174] [Table 9] % activity = maximum GFP signal loss compared to enfortumab mAb (100%)

[0175] Example 5: ADC cytotoxicity and bystander activity Characterization of the cytotoxicity of nectin-4 ADCs in low and high nectin-4 expressing cell lines A low-expressing cell line, NCI-H1781 cells, were cultured on white clear-bottom 96-well tissue culture plates. Culture medium (RPMI 1640 + 1x GlutaMAX + 10% heat-inactivated fetal bovine serum) The cells were incubated at 37°C, 5% CO 2 Overnight The next day, ADC was diluted in culture medium from a final working concentration of 100 nM to 1 Serial dilutions of 1:3 were added to the plate. The plate was sealed with Breathe-Easy® sealing membrane. Cover with 5% CO at 37°C. 2 After 5 days of treatment, the cells were incubated with CellTite Using the r-Glo Luminescent Cell Viability Assay Plates were read using 100 μL / well of CellTiter-Glo Reagent. The plate was incubated at room temperature for 10 minutes. Luminescence was measured using a SpectraMax M5e RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. The percentage of cell death was calculated relative to 0% untreated. The data was graphed and shown in Graph Analysis was performed using Pad Prism version 9.5.1. 50 is log(rejection The inhibitor (agonist) versus response was determined by variable slope (4 parameter) curve fitting.

[0176] The T24 cell line was engineered to express human nectin-4 and clonally selected. The current clonal cell line, T24-human nectin-4 clone 108, was cultured in culture medium (McCo y's 5A + 1 × GlutaMAX + 10% heat-inactivated fetal bovine serum + 400 μg / m The ADCs were seeded into 100 μg / ml 100-well plates (G418) at 100 μg / ml in culture medium from a final working concentration of 200 nM. :4 serial dilutions were added for 5 days.

[0177] T24-human nectin-4 clone 108 and NCI H1781 cells were 5 The antibody binding capacities were determined to be 6,000 and 20,000 (MESF quantification kit). , using Bangs Laboratories).

[0178] As shown in Table 8, selected exemplary Nectin-4 ADCs (as shown in Formula XI) and the DAR is 8) , in Nectin-4 cell lines with differential expression levels, in the same ADC format as antibodies 1 to 8 The enfortumab antibody (having an effector null mutation and conjugated to formula XI) The IC showed a similar potent maximum cell killing rate to that of enfortumab, which has a DAR of 8. 50 value Similar results across ADCs tested in T24-human nectin-4 clone 108 cells Potency and variable potency in ADCs tested in NCI H1781 cells No nonspecific cytotoxicity was observed in the T24 nectin-4 negative cells tested. I did.

[0179] [Table 10]

[0180] Characterization of cytotoxicity of Nectin-4 ADC in MMAE-resistant cell lines In T24 nectin-4 cells that are resistant to MMAE, the two Nectin-4 ADC and Enfortumab Vedotin were tested for activity. Nectin-4 clone 14 cells were cultured in white clear-bottom 96-well tissue culture plates at 1 The cells were seeded at 500 cells per well in 100 μL of culture medium and incubated at 37°C, 5% CO 2 Overnight inn The next day, each ADC was serially diluted 1:4 in culture medium at a starting concentration of 400 nM. 100 μL of each ADC dilution was added per well and the plate was cooled to 37°C. Cover with e-Easy® sealing membrane and incubate at 37°C, 5% CO 2 The mixture was incubated at 4°C for 1 h. After 5 days of incubation, remove the plate from the incubator and place it at room temperature for 15 minutes. 100 μL of CellTiter-Gl was added per well. o Reagent was added and the plate was incubated at room temperature for 10 minutes. RLU (Relative Luminescence Units) ) by SpectraMax M5e using SoftMax Pro 5.4 The percentage of cell death was compared to DMSO alone (free payload) or no treatment (ADC). The data were graphed and analyzed using Graphpad Prism version 9.5.1. IC50 was calculated as log(inhibitor) vs. response-variable slope (4-parameter The parameters were determined by curve fitting.

[0181] ADC made with Ab1 and Ab2 (conjugated with formula XI, DAR is 8) retained activity against MMAE-resistant cells, whereas enfortumab vedotin has lost its effectiveness.

[0182] Comparison of enfortumab vedotin with exemplary linkers / payloads Bystander activity of rutuzumab We engineered the UMUC3 cell line to express human nectin-4 and have distinct expression levels Two clonal populations were selected. 3-h Nectin4 clone F7 / UMUC3-Luc-GFP cells were mixed and cultured in assay medium. Medium (MEM + 1x GlutaMAX + 10% heat-inactivated fetal bovine serum + 1 mM pyruvate A total of 1500 cells / well / 100 μL were seeded at a 4:1 ratio in 100 μL of UM UC3-hNectin4 clone E3 / UMUC3-Luc-GFP cells were mixed and assayed. A total of 2100 cells / well / 100 μL were seeded in a 6:1 ratio in U-medium. -Luc-GFP cells were negative for Nectin-4 expression. The plates were incubated at 37 °C for 1 h. , 5% CO 2 The next day, ADC was added at 100 nM to a final working concentration of 100 nM. The antibodies were then added in a 1:3 serial dilution in assay medium. To assess sensitivity, free payload was titrated from a final working concentration of 200 nM to 100 nM in the assay. Serial dilutions of 1:3 in medium were added. The plates were then washed with Breathe-Easy®. Cover with sealing membrane and incubate at 37°C, 5% CO 2 After 5 days of treatment, ONE Plates were read using the -Glo™ Luciferase Assay System. Add 100 μL / well of ONE-Glo™ Assay Reagent to the plate for 10 minutes at room temperature. The luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) was obtained using .xPro 5.4. UMUC3-Luc-GF The percentage of P mortality was calculated relative to 0% untreated. Data were graphed and analyzed using Graphpa. d Analyzed using Prism version 9.5.1. IC 50 Bystander Activity was measured using log(inhibitor) vs. response (3-parameter) curve fitting, free Load cytotoxicity measured by log(inhibitor) vs. response (4-parameter) curve fitting It was decided by.

[0183] UMUC3 nectin-4 clone F7 and UMUC3 nectin-4 clone E3 cells , and were determined to have antibody binding capacities of 113,000 and 567,000, respectively (M (ESF quantification kit, Bangs Laboratories)

[0184] As shown in Table 9, the enols conjugated as in Formula XI and having a DAR of 8 are Rutumab showed significantly higher IL-18 activity against UMUC3-Luc-GFP cells compared to enfortumab vedotin. In particular, Table 10 shows that UMUC3-Luc -GFP cell lines were similarly sensitive to free payload XI and MMAE. Higher expression on positive cell lines indicates higher binding with enfortumab vedotin. The potency of the compound is dependent on the conjugated endonucleases of formula XI. remains lower than holtumab.

[0185] [Table 11]

[0186] [Table 12]

[0187] In experiments performed essentially as described herein, UMUC3-hNectin The 4-clonal F7 / UMUC3-luciferase-GFP cell pair was used to Bystander efficacy of the four described nectin-4 ADCs and enfortumab vedotin The effect of ADCs on cell proliferation was evaluated. Cells were plated at a 4:1 ratio and treated with serial dilutions of each ADC for 5 days. To determine the bystander effect of the ADC, ONE-Glo™ Luciferase was used. Luciferase luminescence was monitored using the enzyme assay system. Antibody 1, ADCs made with 1a, 2, and 7 (conjugated with formula XI, DAR 8) showed a strong bystander effect, whereas enfortumab vedotin showed showed a much weaker bystander effect.

[0188] Bystander effect of nectin-4 ADC in the cell line T24 nectin-4 negative cells Fruit characteristics We engineered the T24 cell line to express mScarlet and tested it in a bystander assay. The clones were selected for the study. T24-mScarlet clone 5 and T24-human nec Mix 100, 100 and 108 clones and incubate in culture medium (McCoy's s 5A + 1×GlutaMAX + 10% heat-inactivated fetal bovine serum + 400 μg / mL G418) at a ratio of 9:1, with a total of 1000 cells / well / 100 μL. 4 mScarlet clone 5 cells were negative for nectin-4 expression. The cells were incubated at 37°C and 5% CO 2 The next day, the ADC was added to the final working concentration The plates were then added in 1:4 serial dilutions starting at 50 nM in culture medium. Cover with e-Easy® sealing film, place in BioSpa, and perform 5 min. Scans were performed daily for 24 hours.

[0189] Images of T24-mScarlet cells were acquired using Gen5 Image Prime 3.11 and analyzed by Cytation 5. T24-mScarlet cells The percentage of death was calculated by the reduced integrated intensity (area × average intensity) at time 0 and in the control group. Data were graphed and analyzed using GraphPad Prism version 9.5 The analysis was performed using IC 50 is log(inhibitor) vs. response-variable slope (four parameter t) Determined by curve fitting.

[0190] As shown in Table 11, certain Nectin-4 ADCs of the present disclosure (conjugates as in Formula XI) and a DAR of 8), each of which is exemplified herein by antibodies 1-8, T24 mScarlet clone 5, same as antibodies 1-8 in nectin-4 negative cells Enfortumab in ADC form (enfortumab conjugated with formula XI, DAR is 8) The study showed a bystander effect of similar efficacy and strength to rituximab.

[0191] [Table 13]

[0192] Other payloads and other linker compounds in low and high Nectin-4 expressing cell lines Characterization of the activity of Nectin-4 ADCs NCI-H1781 cells were cultured in culture medium (RP) in white clear bottom 96-well tissue culture plates. MI 1640 + 1xGlutaMax + 10% heat inactivated fetal bovine serum + 1mM Pyrubicin The cells were incubated at 37°C and 5% CO 2 Incubated overnight at The next day, ADCs were serially diluted 1:4 in culture medium starting from a final working concentration of 100 nM. The plate was covered with Breathe-Easy® sealing membrane and incubated at 37°C for 5 %CO 2 After 5 days of treatment, the cells were incubated with CellTiter-Glo Lum Read the plate using the inescent Cell Viability Assay. Add 100 μL / well of CellTiter-Glo reagent to the plate at room temperature. Incubated for 10 minutes. Luminescence was read on a SpectraMax M5e. RLU (relative light units) were obtained using ftMax Pro 5.4. The percentage of cell death was calculated using the , calculated relative to 0% untreated. Data were graphed and analyzed using GraphPad Prism Analysis was performed using version 9.5.1. 50 is log(inhibitor) vs. response-variable The slope was determined by four-parameter curve fitting.

[0193] The UMUC3 cell line was engineered to express human nectin-4 and cloned for high expression. The UMUC3-human nectin4 clone F7 modified cell line was cultured in culture medium (ME M+1×GlutaMAX+10% heat-inactivated fetal bovine serum+1mM sodium pyruvate The ADC was added to a final working concentration of 100 nM. were serially diluted 1:4 in culture medium and added for 6 days.

[0194] NCI H1781 and UMUC3-hNectin4 clone F7 and cells were used, respectively. The antibody binding capacities of the two strains were determined to be 20,000 and 113,000, respectively (MESF quantitative kit). (Bangs Laboratories, Inc.).

[0195] As shown in Table 12, Ab conjugated as in Formula XI and having a DAR of 8 2 ADC and PEG8-VA-exatecan, Ab2 ADC in DAR8 format Both were against NCI-H1781 and UMUC3-human nectin-4 clone F7 cell lines. It exhibits potent cytotoxic effects.

[0196] [Table 14]

[0197] Example 6: ADCC, ADCP, and / or CDC Assays In vitro antibody-dependent cell-mediated cytotoxicity (ADCC) assay of Nectin-4 antibody The T24 cell line was engineered to express human nectin-4 and clones were selected for high expression. T24-human nectin 4 clone 147 had an antibody binding capacity of 411,000. (Using the MESF quantification kit, Bangs Laboratories) The target cells, T24-human nectin4 clone 147, were cultured in test medium (IMDM+1xG lutaMax+10% heat-inactivated fetal bovine serum+Pen-Strep 100U / mL ~100μg / mL) in a clear tissue culture 96-well plate and incubated at 37℃, 5% O 2 The next day, 40 μL / well of antibody was added at a final working concentration of 20 μl / well. Antibodies were added in 1:4 serial dilutions in test medium starting from 0 nM. Antibodies were incubated at 37°C and 5% CO 2 The cells were then incubated with 100 mM NaCl for 1 hour. Add NFAT-CD16 at 200k / 80μL / well and incubate at 37℃, 5% CO 2 By After 23 hours, 20 μL of the supernatant and 50 μL of the previously prepared QUAN TI-Luc / well was mixed in a white opaque plate. Luminescence was measured using SpectraMax The data was read on an M5e. RLU (relative luminescence units) were calculated using SoftMax Pro 5.4. ) and plotted the compound on the X-axis using GraphPad Prism version 9.5.1. Plotted on the Y-axis against concentration.

[0198] Unlike the enfortumab antibody, which has a wild-type IgG1 Fc, antibodies 1-8 have It is a null antibody and did not induce antibody-dependent cellular cytotoxicity.

[0199] In vitro antibody-dependent cellular phagocytosis (ADCP) assay of Nectin-4 antibody The T24 cell line was engineered to express high levels of human nectin4-eGFP and cloned. The target cells, T24-human nectin4 eGFP clone 3, were cultured in the test medium ( IMDM+1xGlutaMax+10% heat-inactivated fetal bovine serum+Pen-Strep 100U / mL to 100μg / mL) into a transparent tissue culture 96-well plate. , 37°C, 5% CO 2 The next day, 40 μL / well of mAb was added to Add serial dilutions of 1:4 in test medium starting from a final working concentration of 200 nM and incubate at 37 °C for 5 min. %CO 2 The effector cells, Jurkat-Lu Add cia NFAT-CD32 at 200k / 80μL / well and incubate at 37℃, 5% CO 2 After 23 hours, 20 μL of the supernatant and 50 μL of the previously prepared Q UANTI-Luc / well was mixed in a white opaque plate. Luminescence was measured by Spectra The data were read using a Max M5e. RLU (relative light units) were calculated using SoftMax Pro 5.4. The x-axis (light units) was calculated using GraphPad Prism version 9.5.1. Plotted on the Y-axis against compound concentration.

[0200] Unlike the enfortumab antibody, which has a wild-type IgG1 Fc, antibodies 1-8 have It is a null antibody and did not induce antibody-dependent cellular phagocytosis.

[0201] In vitro Complement-Dependent Cytotoxicity (CDC) Assay of Nectin-4 Lead Antibodies The T24 cell line was engineered and clonally selected to express high levels of human nectin-4. T24-human nectin 4 clone 147 cells were cultured in assay medium (McCoy's 5 A + 1xGlutaMax + 10% heat inactivated fetal bovine serum) in white clear bottom 96 well Add the cells to tissue culture plates and incubate at 37°C, 5% CO 2 Incubate overnight in an incubator. The next day, 50 μL / well of antibody was added at a final working concentration of 200 nM to the assay. Add serial dilutions of 1:3 in medium and incubate at 37 °C in 5% CO 2 Incubated for 1 hour at Diluted human serum complement (1:3) was then added at 50 μL / well in assay medium. , 37°C, 5% CO 2 The plate was incubated for 3 hours with CellTiter-Glo Lu Plates were analyzed using the minescent Cell Viability Assay. Read. Add 100 μL / well of CellTiter-Glo reagent to the plate at room temperature. The plates were incubated at 4°C for 10 min. Luminescence was read on a SpectraMax M5e. RLU (relative light units) were obtained using oftMax Pro 5.4. Percentage of cell death was calculated relative to untreated. Data were graphed and analyzed using GraphPad Prism Analysis was performed using version 9.5.1.

[0202] As a positive control, Jeko-1 cells were cultured in assay medium (RPMI1640 + 1xGlu Treated as above with anti-CD20 antibody in taMAX + 10% heat-inactivated fetal bovine serum Ta.

[0203] Similar to enfortumab, antibodies 1–8 inhibited CD4+ expression in the nectin-4-expressing T24 cell line. The anti-CD20 control antibody did not result in CDC activity and did not inhibit CD20-expressing Jeko-1 cells were used as a positive control.

[0204] Example 7: Efficacy of Nectin-4 ADC in tumor xenograft models The efficacy of Nectin-4 ADCs of the present disclosure having effector null mutations in the Fc region The efficacy of the antibody was tested and it has a wild-type Fc and the same payload background as that disclosed herein. To compare the enfortumab antibody in ADCs with high or moderate nectin levels, Two tumor xenograft models with T-4 expression were tested as described. Immunodeficient female mice (nu / nu) weighing 18–20 g were cultured in the right flank area, and high nectin- UM-UC-3 Nectin 4 clone F7 cells expressing Nectin 4 were transplanted into one side of the body. The tumors grew approximately 1 50~250mm 3 When tumor volume reached 100 μg / kg, animals were divided into treatment or control groups according to tumor volume and dosed. Drug administration was started (day 0, n=8 per group). The test substance was formulated in 5% dextrose. The tumors were measured biweekly for up to 40 days.

[0205] In another study, 5- to 6-week-old female NSG mice were treated with 100-mg IFN-γ-α (100-mg / kg bw) of 100% glycerol (100-mg / kg bw) with moderate Nectin-4 expression. MDA-MB-468 cells were injected subcutaneously. 3 reached At that time, the animals were divided into treatment or control groups according to tumor volume, and dosing was started (day 0, group-specific The test substance was formulated using 5% dextrose and administered as a single dose (2 mg / kg). Tumors were measured biweekly until day 71.

[0206] In both models, Nectin-4 ADC (conjugated as in Formula XI, The DAR is 8, as exemplified herein for each of antibodies 1-8) or a benchmark Enfortumab ADC (formula XI) conjugated with enfortumab having a DAR of 8 A single dose of 2mg / kg of either UMUC3 or nectin-4 was used. on day 40 for Lone F7 and on day 71 for MDAMB468 xenografts. Tumor growth was measured using effector null antibodies 1 to 8 as shown in Table 13. Treatment with Nectin-4 ADCs inhibited tumor growth and inhibited tumor growth in mice with wild-type Fc regions. Equal to or, for certain ADCs, better than the benchmark enfortumab ADC The efficacy was good.

[0207] [Table 15] SEM = standard error of the mean

[0208] Xenograft experiments were performed as described using UM-UC-3 nectin4 clone F7 cells. In the study, the Nectin-4 ADC described herein was administered at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg in combination with cisplatin and gemcitabine. Combination of SOC agents with Nectin-4 ADC at low doses of 0.5 and 1 mg / kg demonstrated antitumor activity Increased tumor activity was observed.

[0209] Amino acid and nucleotide sequences SEQ ID NO:1 (human nectin-4) MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDV VTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQEL ALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQ ADEGEYECRVSTFPAGSFQARLRLRVLVPPLPSLNPGPAL EEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHS RSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHI LHVSFLAEASVRGLEDQNLWHIGREGAMLKCLSEGQPPPS YNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSN EFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAAL LFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIR RLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEE PEGRSYSTLTTVREIETQTELLSPGSGRAEEEEDQDEGIK QAMNHFVQENGTLRAKPTGNGIYINGRGHLV SEQ ID NO:2 (HC of Ab1) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTA YMELSRLRSDDTAVYYCAREDWDFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO:3 (LC of Ab1) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQ KPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQYGSSPITFGQGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQ ID NO: 4 (HCDR1 of Ab1 and 1a) KASGYTFTGYYIH SEQ ID NO:5 (HCDR2 of Ab1 and 1a) WINPNSGGTN SEQ ID NO:6 (HCDR3 of Ab1 and 1a) AREDWDFDY SEQ ID NO: 7 (LCDR1 of Ab1 and 1a) RTSQSVSSSYLA SEQ ID NO:8 (LCDR2 of Ab1 and 1a) YGASNRAT SEQ ID NO: 9 (LCDR3 of Ab1 and 1a) QQYGSSPIT SEQ ID NO: 10 (HCVR of Ab1) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTA YMELSRLRSDDTAVYYCAREDWDFDYWGQGTLVTVSS SEQ ID NO: 11 (LCVR of Ab1) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQ KPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRL EPEDFAVYYCQQYGSSPITFGQGTKVEIK SEQ ID NO: 12 (HC of Ab1a) QVQLVQSGAQVKKPGASVKVSCKASGYTFTGYYIHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTA YMELSRLRSDDTAVYFCAREDWDFDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSV FLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: 13 (LC of Ab1a) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQ KPGQAPRLLIYGASNRATDIPDRFSGSGSGTDFTLTINRL EPEDFAVYYCQQYGSSPITFGQGTRLEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC SEQ ID NO: 14 (HCVR of Ab1a) QVQLVQSGAQVKKPGASVKVSCKASGYTFTGYYIHWVRQ APGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTA YMELSRLRSDDTAVYFCAREDWDFDYWGQGTLVTVSS SEQ ID NO: 15 (LCVR of Ab1a) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQ KPGQAPRLLIYGASNRATDIPDRFSGSGSGTDFTLTINRL EPEDFAVYYCQQYGSSPITFGQGTRLEIK SEQ ID NO: 16 (HC of Ab2) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQ ATGKGLEWVSAIGTVGDTYYPGSVKGRFTISRENAKNSLY LQMNSLRAGDTAVYYCAREWNGMDVWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICN VNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVF LFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALAPIEKTISKAKGQPREPQVYTLPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK SEQ ID NO: 17 (LC of Ab2) DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTL TISSLQAEDVAVYYCQQFYTTPYSFGQGTKVEIKRTVAAP SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNA LQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 18 (HCDR1 of Ab2) AASGFTFSSYDMH SEQ ID NO: 19 (HCDR2 of Ab2) AIGTVGDTY SEQ ID NO:20 (HCDR3 of Ab2) AREWNGMDV SEQ ID NO:21 (LCDR1 of Ab2) KSSQSVLYNSNNKNYLA SEQ ID NO:22 (LCDR2 of Ab2) YWASTRES SEQ ID NO:23 (LCDR3 of Ab2) QQFYTTPYS SEQ ID NO:24 (HCVR of Ab2) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQ ATGKGLEWVSAIGTVGDTYYPGSVKGRFTISRENAKNSLY LQMNSLRAGDTAVYYCAREWNGMDVWGQGTTVTVSS SEQ ID NO:25 (LCVR of Ab2) DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYL AWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTL TISSLQAEDVAVYYCQQFYTTPYSFGQGTKVEIK SEQ ID NO:26 (HC of Ab3) EVQLVESGGGLVKPGGSLRLSCAASGFNLNHYNMNWVRQ APGKGLEWVSSVSSGGGFRYYADSVRGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARGAVFHDAFDIWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQT YICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGG PSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK SEQ ID NO:27 (LC of Ab3) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLAWYQQK PGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQINSYPFTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC SEQ ID NO:28 (HCDR1 of Ab3) AASGFNLNHYNMN SEQ ID NO:29 (HCDR2 of Ab3) SVSSGGGFRY SEQ ID NO: 30 (HCDR3 of Ab3) ARGAVFHDAFDI SEQ ID NO:31 (LCDR1 of Ab3) RASQDISSYLA SEQ ID NO:32 (LCDR2 of Ab3) YVASTLQS SEQ ID NO: 33 (LCDR3 of Ab3) QQINSYPFT SEQ ID NO: 34 (HCVR of Ab3) EVQLVESGGGLVKPGGSLRLSCAASGFNLNHYNMNWVRQ APGKGLEWVSSVSSGGGFRYYADSVRGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARGAVFHDAFDIWGQGTLVTVSS SEQ ID NO: 35 (LCVR of Ab3) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLAWYQQK PGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQINSYPFTFGQGTKVEIK SEQ ID NO: 36 (HC of Ab4) QVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQ PPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARLGIFFDAFDIWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 37 (LC of Ab4) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQFNSYPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC SEQ ID NO: 38 (HCDR1 of Ab4) TVSGGSISGYYWS SEQ ID NO:39 (HCDR2 of Ab4) YIYYSGSTN SEQ ID NO: 40 (HCDR3 of Ab4) ARLGIFFDAFDI SEQ ID NO: 41 (LCDR1 of Ab4 and Ab5) RASQGISSYLA SEQ ID NO: 42 (LCDR2 of Ab4 and Ab5) YAASTLQS SEQ ID NO: 43 (LCDR3 of Ab4) QQFNSYPWT SEQ ID NO: 44 (HCVR of Ab4) QVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQ PPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFS LKLSSVTAADTAVYYCARLGIFFDAFDIWGQGTLVTVSS SEQ ID NO: 45 (LCVR of Ab4) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQFNSYPWTFGQGTKVEIK SEQ ID NO: 46 (HC of Ab5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYVHWVRQ APGQGLEWMGIINPSIISTSYAQKFQARVTMTRDTTSTSTV YMELSSLRSEDTAVYYCARGLNFDAFDIWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO: 47 (LC of Ab5) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQLNNYPFTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC SEQ ID NO: 48 (HCDR1 of Ab5) KASGYTFTSYYVH SEQ ID NO: 49 (HCDR2 of Ab5) IINPSIISTS SEQ ID NO:50 (HCDR3 of Ab5) ARGLNFDAFDI SEQ ID NO:51 (LCDR3 of Ab5) QQLNNYPFT SEQ ID NO:52 (HCVR of Ab5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYVHWVRQ APGQGLEWMGIINPSIISTSYAQKFQARVTMTRDTTSTSTV YMELSSLRSEDTAVYYCARGLNFDAFDIWGQGTLVTVSS SEQ ID NO:53 (LCVR of Ab5) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQK PGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQLNNYPFTFGQGTKVEIK SEQ ID NO:54 (HC of Ab6) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARGRLGTYFDYWGQGTLVTVSSA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTY ICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGP SVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK SEQ ID NO:55 (LC of Ab6) SYVLTQPPSVSVVAPGQTARITCGGNNIGSKSVHWYQQKP GQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEA GDEADYYCQVWDRSSDQVVFGGGTKLTVLGQPKAAPSVTL FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKA GVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECS SEQ ID NO:56 (HCDR1 of Ab6) KASGYTFTGYYMH SEQ ID NO:57 (HCDR2 of Ab6) IINPSGGSTT SEQ ID NO:58 (HCDR3 of Ab6) ARGRLGTYFDY SEQ ID NO:59 (LCDR1 of Ab6) GGNNIGSKSVH SEQ ID NO: 60 (LCDR2 of Ab6) YDDSDRPS SEQ ID NO:61 (LCDR3 of Ab6) QVWDRSSDQVV SEQ ID NO:62 (HCVR of Ab6) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQ APGQGLEWMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCARGRLGTYFDYWGQGTLVTVSS SEQ ID NO: 63 (LCVR of Ab6) SYVLTQPPSVSVVAPGQTARITCGGNNIGSKSVHWYQQKP GQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEA GDEADYYCQVWDRSSDQVVFGGGTKLTVL SEQ ID NO: 64 (HC of Ab7) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGQGLEWMGIINPSSGSASYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCAREGVGRDILQAFDIWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 65 (LC of Ab7) QSVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVNWYQQ LPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGL QSEDEADYYCAAWDDSLNGHVFGGGTKLTVLGQPKAAPSV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS SEQ ID NO: 66 (HCDR1 of Ab7) KASGYTFTNYYMH SEQ ID NO: 67 (HCDR2 of Ab7) IINPSSGSAS SEQ ID NO: 68 (HCDR3 of Ab7) AREGVGRDILQAFDI SEQ ID NO: 69 (LCDR1 of Ab7 and Ab8) SGSSSNIGNNAVN SEQ ID NO: 70 (LCDR2 of Ab7 and Ab8) YYDDLLPS SEQ ID NO: 71 (LCDR3 of Ab7) AAWDDSLNGHV SEQ ID NO: 72 (HCVR of Ab7) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQ APGQGLEWMGIINPSSGSASYAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCAREGVGRDILQAFDIWGQGTLVT VSS SEQ ID NO: 73 (LCVR of Ab7) QSVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVNWYQQ LPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGL QSEDEADYYCAAWDDSLNGHVFGGGTKLTVL SEQ ID NO: 74 (HC of Ab8) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQ APGQGLEWMGIINPISGRTSSAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCAKEGVGGELLRAFDIWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEA AGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVK FNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK SEQ ID NO: 75 (LC of Ab8) QSVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVNWYQQ LPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGL QSEDEADYYCAAWDDSLNGFVFGGGTKLTVLGQPKAAPSV TLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECS SEQ ID NO: 76 (HCDR1 of Ab8) KASGYTFTSYYIH SEQ ID NO: 77 (HCDR2 of Ab8) IINPISGRTS SEQ ID NO:78 (HCDR3 of Ab8) AKEGVGGELLRAFDI SEQ ID NO:79 (LCDR3 of Ab8) AAWDDSLNGFV SEQ ID NO: 80 (HCVR of Ab8) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQ APGQGLEWMGIINPISGRTSSAQKFQGRVTMTRDTSTSTV YMELSSLRSEDTAVYYCAKEGVGGELLRAFDIWGQGTLVT VSS SEQ ID NO: 81 (LCVR of Ab8) QSVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVNWYQQ LPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGL QSEDEADYYCAAWDDSLNGFVFGGGTKLTVL SEQ ID NO: 82 (DNA of HC for Ab1) CAAGTTCAGCTGGTGCAGTCTGGAGCCGAAGTGAAGAAG CCCGGAGCCTCAGTGAAAGTGTCCTGTAAGGCGTCCGGCT ATACCTTCACTGGTTACTATATCCACTGGGTACGCCAGGC TCCAGGACAGGGCTTGGAGTGGATGGGTTGGATCAATCCT AACTCTGGGGGCACCAACTACGCACAGAAATTCCAAGGGA GAGTCACCATGACGCGGGATACTAGCATTAGCACAGCTTA TATGGAGCTCTCGCGTCTGAGGAGTGACGATACTGCCGTC TACTACTGCGCACGAGAAGACTGGGACTTTGATTATTGGG GGCAGGGCACACTTGTGACAGTTTCAAGTgcctccaccaa gggcccatcggtcttccccctggcaccctcctccaagagc acctctgggggcacagcggccctgggctgcctggtcaagg actacttccccgaaccggtgacggtgtcgtggaactcagg cGCGctgaccagcggcgtgcacaccttcccggctgtccta cagtcctcaggactctactccctcagcagcgtggtgaccg tgccctccagcagcttgggcacccagacctacatctgcaa cgtgaatcacaagcccagcaacaccaaggtggacaagaga gttgagcccaaatcttgtgacaaaactcacacatgcccac cgtgcccagcacctgaagccgccgggggaccgtcagtctt cctcttccccccaaaacccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtggtggtgtccgtgagcc acgaagaccctgaggtcaagttcaactggtatgtggacgg cgtggaggtgcataatgccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtggtcagcgtcctcaccg tcctgcaccaagactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacagg tgtacaccctgcccccatcccgggaggagatgaccaagaa ccaagtcagcctgacctgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggagagcaatgggcagccgg agaacaactacaagaccacgcctcccgtgctggactccga cggctccttcttcctctattccaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtga tgcatgaggctctgcacaaccactacacgcagaagagcct ctccctgtctccgggcaaa SEQ ID NO: 83 (DNA of LC against Ab1) GAGATCGTGCTGACCCAGAGTCCAGGAACACTCAGCCTG TCCCCCGGAGAACGGGCTACTTTGTCATGCCGTACGAGCC AGAGCGTGTCCTCTTCTTATCTGGCTTGGTACCAGCAAAA GCCTGGACAAGCGCCTCGATTACTTATCTATGGTGCCTCT AACCGCGCCACAGGCATTCCAGACAGATTCTCAGGGTCTG GCAGTGGCACCGACTTTACACTAACCATTTCCAGGCTCGA ACCCGAGGATTTCGCAGTCTACTACTGTCAGCAGTATGGG TCGAGCCCGATTACTTTTGGCCAGGGTACCAAGGTTGAGA TCAAAagaactgtggcggcgccatctgtcttcatcttccc gccatctgatgagcagttgaaatccggaactgcctctgtt gtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgccctccaatcgggtaactc ccaggagagtgtcacagagcaggacagcaaggacagcacc tacagcctcagcagcaccctgacgctgagcaaagcagact acgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgc Sequence No. 84 (DNA of HC for Ab1a) CAGGTTCAACTTGTGCAAAGTGGAGCACAGGTTAAGAAG CCTGGCGCATCCGTGAAAGTCTCTTGCAAAGCATCCGGGT ACACATTCACCGGCTACTATATCCATTGGGTACGGCAGGC CCCAGGTCAGGGGCTTGAATGGATGGGATGGATAAATCCC AATAGCGGTGGTACCAATTACGCCCAGAAGTTTCAGGGAC GAGTCACAATGACTAGAGATACAAGTATCTCAACCGCATA CATGGAACTCTCAAGACTGAGATCAGATGACACCGCAGTC TATTTCTGTGCTCGCGAGGATTGGGACTTCGATTACTGGG GCCAAGGCACCCTGGTTACCGTCAGCAGCgcctccaccaa gggcccatcggtcttccccctggcaccctcctccaagagc acctctgggggcacagcggccctgggctgcctggtcaagg actacttccccgaaccggtgacggtgtcgtggaactcagg cGCGctgaccagcggcgtgcacaccttcccggctgtccta cagtcctcaggactctactccctcagcagcgtggtgaccg tgccctccagcagcttgggcacccagacctacatctgcaa cgtgaatcacaagcccagcaacaccaaggtggacaagaga gttgagcccaaatcttgtgacaaaactcacacatgcccac cgtgcccagcacctgaagccgccgggggaccgtcagtctt cctcttccccccaaaacccaaggacaccctcatgatctcc cggacccctgaggtcacatgcgtggtggtgtccgtgagcc acgaagaccctgaggtcaagttcaactggtatgtggacgg cgtggaggtgcataatgccaagacaaagccgcgggaggag cagtacaacagcacgtaccgtgtggtcagcgtcctcaccg tcctgcaccaagactggctgaatggcaaggagtacaagtg caaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacagg tgtacaccctgcccccatcccgggaggagatgaccaagaa ccaagtcagcctgacctgcctggtcaaaggcttctatccc agcgacatcgccgtggagtgggagagcaatgggcagccgg agaacaactacaagaccacgcctcccgtgctggactccga cggctccttcttcctctattccaagctcaccgtggacaag agcaggtggcagcaggggaacgtcttctcatgctccgtga tgcatgaggctctgcacaaccactacacgcagaagagcct ctccctgtctccgggcaaa SEQ ID NO: 85 (DNA of LC for Ab1a) GAAATCGTGCTTACCCAATCCCCTGGTACTCTCTCCCTC AGCCCCGGCGAACGGGCCACCCTCTCCTGCCGAACAAGCC AAAGTGTGTCTAGCTCCTACCTCGCCTGGTATCAACAGAA ACCCGGCCAAGCACCACGACTTCTTATCTATGGTGCAAGC AACAGGGCTACTGACATTCCAGATCGCTTCAGTGGCTCTG GCTCAGGAACAGACTTCACTCTGACTATCAACAGGTTGGA ACCTGAGGATTTTGCCGTATATTATTGCCAACAATACGGC TCCTCTCCTATAACCTTTGGTCAAGGTACACGCCTGGAAA TAAAGagaactgtggcggcgccatctgtcttcatcttccc gccatctgatgagcagttgaaatccggaactgcctctgtt gtgtgcctgctgaataacttctatcccagagaggccaaag tacagtggaaggtggataacgccctccaatcgggtaactc ccaggagagtgtcacagagcaggacagcaaggacagcacc tacagcctcagcagcaccctgacgctgagcaaagcagact acgagaaacacaaagtctacgcctgcgaagtcacccatca gggcctgagctcgcccgtcacaaagagcttcaacagggga gagtgc Sequence number 86 (DNA of HC for Ab2) GAAGTCCAACTAGTGGAGAGCGGCGGGGGCCTGGTCCAG CCCGGGGGTTCTCTCCGTCTCTCTTGCGCTGCATCCGGAT TCACATTCTCATCCTATGATATGCACTGGGTGCGACAGGC AACGGGGAAGGGCCTGGAGTGGGTTAGCGCCATTGGTACT GTGGGCGACACTTACTATCCTGGAAGCGTGAAGGGACGGT TTACAATCTCAAGGGAGAATGCCAAAAACAGTCTGTACCT TCAGATGAACAGTTTGCGCGCTGGCGACACCGCCGTTTAC TATTGTGCGAGAGAATGGAATGGAATGGATGTGTGGGGCC AGGGTACCACCGTCACAGTATCCTCTgcctccaccaaggg cccatcggtcttccccctggcaccctcctccaagagcacc tctgggggcacagcggccctgggctgcctggtcaaggact acttccccgaaccggtgacggtgtcgtggaactcaggcGC Gctgaccagcggcgtgcacaccttcccggctgtcctacag tcctcaggactctactccctcagcagcgtggtgaccgtgc cctccagcagcttgggcacccagacctacatctgcaacgt gaatcacaagcccagcaacaccaaggtggacaagagagtt gagcccaaatcttgtgacaaaactcacacatgcccaccgt gcccagcacctgaagccgccgggggaccgtcagtcttcct cttccccccaaaacccaaggacaccctcatgatctcccgg acccctgaggtcacatgcgtggtggtgtccgtgagccacg aagaccctgaggtcaagttcaactggtatgtggacggcgt ggaggtgcataatgccaagacaaagccgcgggaggagcag tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcc tgcaccaagactggctgaatggcaaggagtacaagtgcaa ggtctccaacaaagccctcccagcccccatcgagaaaacc atctccaaagccaaagggcagccccgagaaccacaggtgt acaccctgcccccatcccgggaggagatgaccaagaacca agtcagcctgacctgcctggtcaaaggcttctatcccagc gacatcgccgtggagtgggagagcaatgggcagccggaga acaactacaagaccacgcctcccgtgctggactccgacgg ctccttcttcctctattccaagctcaccgtggacaagagc aggtggcagcaggggaacgtcttctcatgctccgtgatgc atgaggctctgcacaaccactacacgcagaagagcctctc cctgtctccgggcaaa Sequence number 87 (DNA of LC for Ab2) GACATTGTCATGACCCAGTCACCTGACTCCTTGGCGGTG AGCCTGGGAGAAAGAGCAACAATTAACTGCAAATCCAGTC AATCGGTCCTCTACAACTCCAATAACAAGAATTACCTGGC TTGGTATCAGCAGAAGCCCGGCCAGCCACCTAAGCTCCTG ATCTATTGGGCTAGTACTCGGGAGTCCGGAGTTCCCGATA GGTTCTCTGGTTCAGGGTCTGGCACCGACTTTACCCTTAC AATAAGCAGCCTACAGGCCGAAGATGTAGCCGTGTATTAC TGTCAACAGTTCTATACAACGCCATACTCTTTTGGCCAGG GGACTAAAGTGGAGATCAAGagaactgtggcggcgccatc tgtcttcatcttcccgccatctgatgagcagttgaaatcc ggaactgcctctgttgtgtgcctgctgaataacttctatc ccagagaggccaaagtacagtggaaggtggataacgccct ccaatcgggtaactcccaggagagtgtcacagagcaggac agcaaggacagcacctacagcctcagcagcaccctgacgc tgagcaaagcagactacgagaaacacaaagtctacgcctg cgaagtcacccatcagggcctgagctcgcccgtcacaaag agcttcaacaggggagagtgc Sequence number 88 (DNA of HC for Ab3) GAGGTGCAGCTGGTTGAGAGCGGCGGCGGGTTAGTCAAG CCTGGTGGCTCCCTTCGGCTGTCCTGTGCAGCCTCGGGGT TCAATCTGAACCACTACAACATGAACTGGGTGCGTCAAGC CCCCGGCAAGGGACTCGAATGGGTCTCTAGTGTTTCCTCA GGGGGTGGATTTCGCTACTATGCTGACAGCGTAAGGGGAC GATTTACCATTTCTCGGGATAATGCCAAAAACAGCTTGTA CCTGCAGATGAATAGTCTAAGAGCAGAAGATACCGCTGTG TATTATTGCGCGAGAGGTGCCGTCTTCCATGACGCTTTCG ACATCTGGGGACAGGGCACTCTCGTGACAGTGTCATCTgc ctccaccaagggcccatcggtcttccccctggcaccctcc tccaagagcacctctgggggcacagcggccctgggctgcc tggtcaaggactacttccccgaaccggtgacggtgtcgtg gaactcaggcGCGctgaccagcggcgtgcacaccttcccg gctgtcctacagtcctcaggactctactccctcagcagcg tggtgaccgtgccctccagcagcttgggcacccagaccta catctgcaacgtgaatcacaagcccagcaacaccaaggtg gacaagagagttgagcccaaatcttgtgacaaaactcaca catgcccaccgtgcccagcacctgaagccgccgggggacc gtcagtcttcctcttccccccaaaacccaaggacaccctc atgatctcccggacccctgaggtcacatgcgtggtggtgt ccgtgagccacgaagaccctgaggtcaagttcaactggta tgtggacggcgtggaggtgcataatgccaagacaaagccg cgggaggagcagtacaacagcacgtaccgtgtggtcagcg tcctcaccgtcctgcaccaagactggctgaatggcaagga gtacaagtgcaaggtctccaacaaagccctcccagccccc atcgagaaaaccatctccaaagccaaagggcagccccgag aaccacaggtgtacaccctgcccccatcccgggaggagat gaccaagaaccaagtcagcctgacctgcctggtcaaaggc ttctatcccagcgacatcgccgtggagtgggagagcaatg ggcagccggagaacaactacaagaccacgcctcccgtgct ggactccgacggctccttcttcctctattccaagctcacc gtggacaagagcaggtggcagcaggggaacgtcttctcat gctccgtgatgcatgaggctctgcacaaccactacacgca gaagagcctctccctgtctccgggcaaa Sequence No. 89 (DNA of LC for Ab3) GATATCCAACTGACCCAGTCTCCAAGTTTCCTGTCTGCT TCAGTGGGCGATAGGGTCACAATCACCTGTCGGGCCTCCC AAGACATCTCCTCGTACCTGGCGTGGTACCAGCAGAAGCC TGGCAAGGCTCCCAAATTGCTCATATACGTTGCAAGTACC CTTCAGAGCGGAGTGCCATCCAGATTCTCAGGGTCCGGAA GCGGGACTGAATTTACACTAACGATCAGCAGCCTCCAGCC CGAGGACTTCGCCACCTATTATTGCCAGCAGATTAACTCT TATCCTTTTACATTTGGCCAGGGTACTAAAGTAGAGATTA AGagaactgtggcggcgccatctgtcttcatcttcccgcc atctgatgagcagttgaaatccggaactgcctctgttgtg tgcctgctgaataacttctatcccagagaggccaaagtac agtggaaggtggataacgccctccaatcgggtaactccca ggagagtgtcacagagcaggacagcaaggacagcacctac agcctcagcagcaccctgacgctgagcaaagcagactacg agaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagag tgc SEQ ID NO: 90 (DNA of HC for Ab4) CAAGTGCAGCTGCAGGAGAGTGGACCAGGTCTCGTGAAG CCTAGTGAGACACTTTCACTCACTTGTACAGTAAGTGGCG GTTCTATCAGCGGGTATTACTGGTCCTGGATTCGGCAGCC TCCCGGGAAGGGATTAGAATGGATCGGATATATCTATTAT TCCGGCAGCACCAATTACAACCCCTCTCTAAAATCCAGGG TTACGATAAGCGTGGATACCTCTAAAAACCAGTTTTCGTT GAAGCTGTCTAGCGTCACCGCAGCTGACACCGCCGTGTAC TACTGCGCTAGACTGGGCATCTTCTTTGACGCCTTCGATA TTTGGGGGCAGGGCACTCTGGTCACAGTTTCCTCAgcctc caccaagggcccatcggtcttccccctggcaccctcctcc aagagcacctctgggggcacagcggccctgggctgcctgg tcaaggactacttccccgaaccggtgacggtgtcgtggaa ctcaggcGCGctgaccagcggcgtgcacaccttcccggct gtcctacagtcctcaggactctactccctcagcagcgtgg tgaccgtgccctccagcagcttgggcacccagacctacat ctgcaacgtgaatcacaagcccagcaacaccaaggtggac aagagagttgagcccaaatcttgtgacaaaactcacacat gcccaccgtgcccagcacctgaagccgccgggggaccgtc agtcttcctcttccccccaaaacccaaggacaccctcatg atctcccggacccctgaggtcacatgcgtggtggtgtccg tgagccacgaagaccctgaggtcaagttcaactggtatgt ggacggcgtggaggtgcataatgccaagacaaagccgcgg gaggagcagtacaacagcacgtaccgtgtggtcagcgtcc tcaccgtcctgcaccaagactggctgaatggcaaggagta caagtgcaaggtctccaacaaagccctcccagcccccatc gagaaaaccatctccaaagccaaagggcagccccgagaac cacaggtgtacaccctgcccccatcccgggaggagatgac caagaaccaagtcagcctgacctgcctggtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggc agccggagaacaactacaagaccacgcctcccgtgctgga ctccgacggctccttcttcctctattccaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgct ccgtgatgcatgaggctctgcacaaccactacacgcagaa gagcctctccctgtctccgggcaaa Sequence number 91 (DNA of LC for Ab4) GATATACAACTAACACAGTCTCCTTCCTTCTTGTCTGCT AGTGTGGGCGACAGGGTCACAATCACTTGCCGGGCAAGCC AGGGAATTTCCTCCTACCTGGCATGGTATCAGCAGAAGCC CGGGAAAGCTCCCAAGCTCCTGATCTATGCCGCCAGCACG CTTCAGAGCGGGGTGCCATCCAGATTTTCGGGTAGCGGCT CTGGCACTGAGTTTACCCTCACAATTAGTTCACTGCAGCC TGAAGACTTTGCCACCTACTACTGTCAACAGTTCAACTCA TATCCATGGACCTTCGGTCAGGGAACCAAAGTTGAGATCA AGagaactgtggcggcgccatctgtcttcatcttcccgcc atctgatgagcagttgaaatccggaactgcctctgttgtg tgcctgctgaataacttctatcccagagaggccaaagtac agtggaaggtggataacgccctccaatcgggtaactccca ggagagtgtcacagagcaggacagcaaggacagcacctac agcctcagcagcaccctgacgctgagcaaagcagactacg agaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagag tgc Sequence number 92 (DNA of HC for Ab5) CAGGTCCAGCTGGTTCAGTCCGGAGCAGAGGTAAAAAAA CCAGGAGCTAGTGTCAAGGTGTCCTGTAAGGCGTCTGGGT ACACTTTTACTTCATACTATGTTCACTGGGTGCGACAGGC CCCTGGGCAGGGCCTCGAATGGATGGGCATCATAAACCCC AGTATCATTTCCACCAGCTACGCTCAAAAGTTTCAAGCCC GGGTGACCATGACGAGGGATACCTCGACATCAACTGTCTA TATGGAGCTATCCTCTTTGAGATCTGAAGATACAGCCGTG TATTACTGCGCACGCGGCCTTTAATTTCGACGCTTCGACA TTTGGGGTCAGGGTACACTGGTGACCGTGAGCAGCgcctc caccaagggcccatcggtcttccccctggcaccctctcc aagagcacctctggggcacagcggccctgggctgcctgg tcaaggactacttccccgaaccggtgacggtgtcgtggaa ctcaggcGCGctgaccagcggcgtgcacaccttccggct gtcctacagtcctcaggactctactccctcagcagcgtgg tgaccgtgccctccagcagcttgggcacccagacctacat ctgcaacgtgaatcacaagcccagcaacaccaaggtggac aagagagttgagcccaaatcttgtgacaaaactcacacat gcccaccgtgcccagcacctgaagccgccgggggaccgtc agtcttcctctccccccaaaacccaaggacaccctcatg atctccccggacccctgaggtcacatgcgtggtggtgtccg tgagccacgaagaccctgaggtcaagttcaactggtatgt ggacggcgtggaggtgcataatgccaagaaagccgcgg gaggagcacacagcacgtaccgtgtggtcagcgtcc tcaccgtcctgcaccaagaactggctgaatggcaagggagat caagtgcaaggtctccaacaaagccctcccagcccccatc gagaaaaccatctccaaagccaaagggcagccccgagaac cacaggtgtacaccctgcccccatcccgggaggagatgac caagaaccaagtcagcctgacctgcctggtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggc agccggagaacaactacaagaccacgcctcccgtgctgga ctccgacggctccttcttcctctattccaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgct ccgtgatgcatgaggctctgcacaaccactacacgcagaa gagcctctccctgtctccgggcaaa SEQ ID NO: 93 (DNA of LC for Ab5) GACATCCAGTTAACTCAGTCTCCTAGCTTCCTGAGCGCT TCTGTTGGAGATAGAGTCACAATTACATGTAGGGCCTCCC AGGGGATTTCATCATATCTGGCTTGGTATCAACAGAAGCC TGGCAAAGCACCAAAGCTCCTGATCTATGCCGCATCTACC CTACAATCGGGTGTGCCCTCCCGGTTCAGTGGCTCCGGGA GTGGAACGGAATTTACCCTTACCATCTCCAGCTTGCAGCC CGAGGACTTCGCCACATACTACTGCCAGCAGCTCAATAAC TACCCATTTACTTTTGGTCAGGGCACCAAAGTGGAGATAA AGagaactgtggcggcgccatctgtcttcatcttcccgcc atctgatgagcagttgaaatccggaactgcctctgttgtg tgcctgctgaataacttctatcccagagaggccaaagtac agtggaaggtggataacgccctccaatcgggtaactccca ggagagtgtcacagagcaggacagcaaggacagcacctac agcctcagcagcaccctgacgctgagcaaagcagactacg agaaacacaaagtctacgcctgcgaagtcacccatcaggg cctgagctcgcccgtcacaaagagcttcaacaggggagag tgc Sequence No. 94 (DNA of HC against Ab6) CAAGTTCAGCTGGTGCAATCCGGAGCTGAGGTCAAGAAG CCTGGTGCAAGCGTGAAAGTGAGTTGTAAAGCCTCAGGCT ATACCTTCACTGGGTATTACATGCACTGGGTTCGGCAGGC TCCAGGACAGGGATTGGAATGGATGGGCATCATTAACCCC AGCGGGGGTTCCACAACGTACGCCCAGAAGTTTCAGGGCC GTGTGACCATGACACGCGATACTTCTACATCTACCGTATA CATGGAACTCAGTTCCCTGCGATCTGAGGACACTGCAGTC TATTACTGCGCCAGGGGCAGACTTGGCACCTATTTCGACT ACTGGGGTCAGGGGACCCTAGTCACAGTGAGCTCAgcctc caccaagggcccatcggtcttccccctggcaccctcctcc aagagcacctctgggggcacagcggccctgggctgcctgg tcaaggactacttccccgaaccggtgacggtgtcgtggaa ctcaggcGCGctgaccagcggcgtgcacaccttccggct gtcctacagtcctcaggactctactccctcagcagcgtgg tgaccgtgccctccagcagcttgggcacccagacctacat ctgcaacgtgaatcacaagcccagcaacaccaaggtggac aagagagttgagcccaaatcttgtgacaaaactcacacat gcccaccgtgcccagcacctgaagccgccgggggaccgtc agtcttcctctccccccaaaacccaaggacaccctcatg atctccccggacccctgaggtcacatgcgtggtggtgtccg tgagccacgaagaccctgaggtcaagttcaactggtatgt ggacggcgtggaggtgcataatgccaagaaagccgcgg gaggagcacacagcacgtaccgtgtggtcagcgtcc tcaccgtcctgcaccaagaactggctgaatggcaagggagat caagtgcaaggtctccaacaaagccctcccagcccccatc gagaaaaccatctccaaagccaaagggcagccccgagaac cacaggtgtacaccctgcccccatcccgggaggagatgac caagaaccaagtcagcctgacctgcctggtcaaaggcttc tatcccagcgacatcgccgtggagtgggagagcaatgggc agccggagaacaactacaagaccacgcctcccgtgctgga ctccgacggctccttcttctctttccaagctcaccgtg gacaagagcaggtggcagcaggggaacgtcttctcatgct ccgtgatgcatgaggctctgcacaaccactacacgcagaa gagcctctccctgtctccgggcaaa SEQ ID NO: 95 (DNA of LC against Ab6) AGCTACGTCCTTACTCAGCCACCTAGTGTGTCAGTAGCG CCTGGGCAAACCGCTCGCATAACTTGCGGGGGAAACAACA TCGGCTCTAAATCCGTCCACTGGTACCAGCAGAAGCCCGG CCAGGCACCCGTATTGGTTGTTTACGACGATAGCGACAGA CCGTCCGGTATCCCAGAGCGGTTTTCCGGTAGTAATTCTG GAAATACAGCTACCCTGACAATTTCTAGGGTGGAAGCCGG GGATGAGGCCGACTATTATTGTCAAGTGTGGGATCGATCA AGCGACCAGGTGGTCTTCGGCGGAGGCACGAAGCTGACCG TGCTCggccagcccaaggctgccccctcggtcactctgtt cccgccctcctctgaggagcttcaagccaacaaggccaca ctggtgtgtctcataagtgacttctacccgggagccgtga cagtggcctggaaggcagatagcagccccgtcaaggcggg agtggagacaaccacaccctccaaacaaagcaacaacaag tacgcggccagcagctatctgagcctgacgcctgagcagt ggaagtcccacagaagctacagctgccaggtcacgcatga agggagcaccgtggagaagacagtggcccctacagaatgt tca SEQ ID NO: 96 (DNA of HC for Ab7) CAGGTGCAACTGGTGCAGAGTGGAGCCGAGGTGAAGAAG CCCGGCGCTTCGGTCAAGGTTTCATGCAAAGCATCCGGGT ATACCTTCACTAACTACTATATGCACTGGGTTCGCCAGGC CCCAGGTCAAGGCTTGGAATGGATGGGAATCATAAATCCT TCTAGCGGTTCTGCATCCTACGCTCAGAAATTTCAGGGAC GTGTCACCATGACAAGAGATACATCTACTTCAACCGTCTA CATGGAGCTAAGTTCCCTGAGGTCCGAAGACACCGCCGTA TATTACTGTGCGCGGGAGGGCGTGGGCCGAGACATTCTCC AGGCTTTCGATATCTGGGGGCAGGGGACGCTTGTGACAGT GAGCAGCgcctccaccaagggcccatcggtcttccccctg gcaccctcctccaagagcacctctgggggcacagcggccc tgggctgcctggtcaaggactacttccccgaaccggtgac ggtgtcgtggaactcaggcGCGctgaccagcggcgtgcac accttcccggctgtcctacagtcctcaggactctactccc tcagcagcgtggtgaccgtgccctccagcagcttgggcac ccagacctacatctgcaacgtgaatcacaagcccagcaac accaaggtggacaagagagttgagcccaaatcttgtgaca aaactcacacatgcccaccgtgcccagcacctgaagccgc cgggggaccgtcagtcttcctcttccccccaaaacccaag gacaccctcatgatctcccggacccctgaggtcacatgcg tggtggtgtccgtgagccacgaagaccctgaggtcaagtt caactggtatgtggacggcgtggaggtgcataatgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtg tggtcagcgtcctcaccgtcctgcaccaagactggctgaa tggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggc agccccgagaaccacaggtgtacaccctgcccccatcccg ggaggagatgaccaagaaccaagtcagcctgacctgcctg gtcaaaggcttctatcccagcgacatcgccgtggagtggg agagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctattcc aagctcaccgtggacaagagcaggtggcagcaggggaacg tcttctcatgctccgtgatgcatgaggctctgcacaacca ctacacgcagaagagcctctccctgtctccgggcaaa Sequence number 97 (DNA of LC against Ab7) CAGTCTGTTCTCACACAACCACCCTCAGTAAGCGAGGCT CCCCGGCAGAGAGTCACAATCTCCTGTAGCGGAAGTAGTT CAAACATAGGGAATAATGCGGTGAACTGGTATCAGCAGTT GCCCGGTAAAGCCCCTAAACTGCTCATTTACTACGACGAC TTACTACCTTCCGGCGTGAGCGACAGGTTTTCAGGAAGCA AGTCTGGCACTTCGGCCTCCCTGGCAATCTCTGGACTTCA GAGTGAGGATGAAGCTGACTATTACTGCGCAGCCTGGGAT GATTCCTTGAACGGCCACGTCTTCGGTGGCGGGACCAAGT TGACCGTGCTGggccagcccaaggctgccccctcggtcac tctgttcccgccctcctctgaggagcttcaagccaacaag gccacactggtgtgtctcataagtgacttctacccgggag ccgtgacagtggcctggaaggcagatagcagccccgtcaa ggcgggagtggagacaaccacaccctccaaacaaagcaac aacaagtacgcggccagcagctatctgagcctgacgcctg agcagtggaagtcccacagaagctacagctgccaggtcac gcatgaagggagcaccgtggagaagacagtggcccctaca gaatgttca Sequence number 98 (DNA of HC for Ab8) CAGGTGCAGCTCGTCCAGAGTGGGGCCGAGGTGAAAAAG CCTGGCGCTTCGGTCAAGGTTTCATGCAAAGCCTCCGGTT ACACCTTCACATCCTATTATATCCACTGGGTGAGACAAGC TCCAGGACAGGGCCTGGAATGGATGGGAATTATAAACCCC ATTAGCGGTCGAACCTCTTCTGCCCAGAAGTTCCAGGGAC GTGTGACTATGACTCGGGATACATCCACAAGCACCGTATA CATGGAGCTATCTTCACTTAGGAGTGAGGACACTGCAGTT TACTATTGTGCGAAGGAAGGTGTCGGGGGCGAGCTGTTGC GCGCATTTGACATCTGGGGGCAAGGCACCCTGGTGACGGT GTCCAGCgcctccaccaagggcccatcggtcttccccctg gcaccctcctccaagagcacctctgggggcacagcggccc tgggctgcctggtcaaggactacttccccgaaccggtgac ggtgtcgtggaactcaggcGCGctgaccagcggcgtgcac accttcccggctgtcctacagtcctcaggactctactccc tcagcagcgtggtgaccgtgccctccagcagcttgggcac ccagacctacatctgcaacgtgaatcacaagcccagcaac accaaggtggacaagagagttgagcccaaatcttgtgaca aaactcacacatgcccaccgtgcccagcacctgaagccgc cgggggaccgtcagtcttcctcttccccccaaaacccaag gacaccctcatgatctcccggacccctgaggtcacatgcg tggtggtgtccgtgagccacgaagaccctgaggtcaagtt caactggtatgtggacggcgtggaggtgcataatgccaag acaaagccgcgggaggagcagtacaacagcacgtaccgtg tggtcagcgtcctcaccgtcctgcaccaagactggctgaa tggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaaaccatctccaaagccaaagggc agccccgagaaccacaggtgtacaccctgcccccatcccg ggaggagatgaccaagaaccaagtcagcctgacctgcctg gtcaaaggcttctatcccagcgacatcgccgtggagtggg agagcaatgggcagccggagaacaactacaagaccacgcc tcccgtgctggactccgacggctccttcttcctctattcc aagctcaccgtggacaagagcaggtggcagcaggggaacg tcttctcatgctccgtgatgcatgaggctctgcacaacca ctacacgcagaagagcctctccctgtctccgggcaaa Sequence number 99 (DNA of LC for Ab8) CAGAGTGTGCTGACCCAGCCCCCAAGCGTCTCCGAGGCA CCCAGACAGAGGGTGACAATCTCCTGCTCCGGAAGCAGTT CAAACATAGGCAACAACGCCGTAAATTGGTACCAGCAGCT GCCAGGTAAGGCCCCTAAACTTCTGATCTATTACGATGAT TTGCTCCCTTCAGGTGTCTCTGACCGCTTTTCCGGAAGCA AATCTGGGACCAGCGCTTCTTTGGCAATTTCTGGCCTGCA ATCGGAAGACGAGGCTGATTACTATTGTGCCGCTTGGGAC GACAGTTTAAATGGGTTCGTTTTCGGCGGCGGAACAAAGC TAACTGTGCTCggccagcccaaggctgccccctcggtcac tctgttcccgccctcctctgaggagcttcaagccaacaag gccacactggtgtgtctcataagtgacttctacccggggag ccgtgacagtggcctggaaggcagatagcagccccgtcaa ggcgggagtggagacaaccacaccctccaaacaaagcaac aacaagtacgcggccagcagctatctgagcctgacgcctg agcagtggaagtcccacagaagctacagctgccaggtcac gcatgaagggagcaccgtggagaagacagtggcccctaca gaatgttca SEQ ID NO: 100-Ala-Leu-Ala-Leu-(also known as ALAL in single letter notation) Known) SEQ ID NO:101-Leu-Ala-Leu-Ala-(also known as LALA in single letter notation) Known) SEQ ID NO: 102 - Gly-Gly-Phe-Gly- (also known as GGFG in single letter notation) Known) Sequence number 103 - Gly-Phe-Leu-Gly- (also known as GFLG in single letter notation) Known) Sequence number 104 - Gly-Leu-Phe-Gly- (also known as GLFG in single letter notation) Known) SEQ ID NO:105 - Ala-Ala-Ala-Ala- (also known as AAAA in single letter notation) Known) SEQ ID NO: 106 - Gly-Ala-Gly-Gly-(can also be expressed as GAGG in single letter notation) Known) SEQ ID NO: 107 - Gly-Gly-Ala-Gly- (also known as GGAG in single letter notation) Known) Sequence number 108 - Gly-Val-Gly-Gly-(also known as GVGG in single letter notation) Known) SEQ ID NO: 109 - Gly-Gly-Val-Gly- (also known as GGVG in single letter notation) Known) SEQ ID NO: 110-Gly-Phe-Gly-Gly-(also known as GFGG in single letter notation) Known)

Claims

1. An antibody that binds to human Nectin-4, the antibody comprising a heavy chain variable region (HCVR) and and a light chain variable region (LCVR), and the HCVR comprises a heavy chain complementarity determining region (HCDR) The LCVR comprises a light chain complementarity determining region (HCDR1, HCDR2, and HCDR3). LCDR) comprising LCDR1, LCDR2 and LCDR3; a) said HCDR1 comprises SEQ ID NO: 4 and said HCDR2 comprises SEQ ID NO: 5; the HCDR3 comprises SEQ ID NO: 6, the LCDR1 comprises SEQ ID NO: 7, R2 comprises SEQ ID NO: 8 and said LCDR3 comprises SEQ ID NO: 9; b) said HCDR1 comprises SEQ ID NO: 18 and said HCDR2 comprises SEQ ID NO: 19 wherein the HCDR3 comprises SEQ ID NO: 20 and the LCDR1 comprises SEQ ID NO: 21; the LCDR2 comprises SEQ ID NO: 22 and the LCDR3 comprises SEQ ID NO: 23; c) said HCDR1 comprises SEQ ID NO:28 and said HCDR2 comprises SEQ ID NO:29 wherein the HCDR3 comprises SEQ ID NO: 30 and the LCDR1 comprises SEQ ID NO: 31; the LCDR2 comprises SEQ ID NO: 32 and the LCDR3 comprises SEQ ID NO: 33; d) said HCDR1 comprises SEQ ID NO: 38 and said HCDR2 comprises SEQ ID NO: 39 wherein the HCDR3 comprises SEQ ID NO: 40 and the LCDR1 comprises SEQ ID NO: 41; the LCDR2 comprises SEQ ID NO: 42 and the LCDR3 comprises SEQ ID NO: 43; e) said HCDR1 comprises SEQ ID NO: 48 and said HCDR2 comprises SEQ ID NO: 49 wherein the HCDR3 comprises SEQ ID NO: 50 and the LCDR1 comprises SEQ ID NO: 41; the LCDR2 comprises SEQ ID NO: 42 and the LCDR3 comprises SEQ ID NO: 51; f) said HCDR1 comprises SEQ ID NO:56 and said HCDR2 comprises SEQ ID NO:57 wherein the HCDR3 comprises SEQ ID NO:58 and the LCDR1 comprises SEQ ID NO:59; the LCDR2 comprises SEQ ID NO: 60, and the LCDR3 comprises SEQ ID NO: 61; g) said HCDR1 comprises SEQ ID NO: 66 and said HCDR2 comprises SEQ ID NO: 67 wherein the HCDR3 comprises SEQ ID NO: 68 and the LCDR1 comprises SEQ ID NO: 69; the LCDR2 comprises SEQ ID NO: 70 and the LCDR3 comprises SEQ ID NO: 71; or h) said HCDR1 comprises SEQ ID NO: 76 and said HCDR2 comprises SEQ ID NO: 77 wherein the HCDR3 comprises SEQ ID NO: 78 and the LCDR1 comprises SEQ ID NO: 69; An antibody, wherein the LCDR2 comprises SEQ ID NO: 70 and the LCDR3 comprises SEQ ID NO:

79.

2. The HCDR1 comprises SEQ ID NO: 4, the HCDR2 comprises SEQ ID NO: 5, R3 comprises SEQ ID NO:6, said LCDR1 comprises SEQ ID NO:7, and said LCDR2 comprises SEQ ID NO: 8, and the LCDR3 comprises SEQ ID NO:

9.

3. said HCDR1 comprising SEQ ID NO: 18, said HCDR2 comprising SEQ ID NO: 19, CDR3 comprises SEQ ID NO: 20, said LCDR1 comprises SEQ ID NO: 21, said LCDR2 comprises SEQ ID NO: 22, The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 22 and said LCDR3 comprises SEQ ID NO:

23.

4. said HCDR1 comprising SEQ ID NO:28, said HCDR2 comprising SEQ ID NO:29, CDR3 comprises SEQ ID NO: 30, said LCDR1 comprises SEQ ID NO: 31, said LCDR2 comprises SEQ ID NO: 32, The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 32 and said LCDR3 comprises SEQ ID NO:

33.

5. said HCDR1 comprising SEQ ID NO: 38, said HCDR2 comprising SEQ ID NO: 39, CDR3 comprises SEQ ID NO: 40, said LCDR1 comprises SEQ ID NO: 41, said LCDR2 comprises SEQ ID NO: 42, The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 42 and said LCDR3 comprises SEQ ID NO:

43.

6. said HCDR1 comprising SEQ ID NO: 48, said HCDR2 comprising SEQ ID NO: 49, CDR3 comprises SEQ ID NO:50, said LCDR1 comprises SEQ ID NO:41, said LCDR2 comprises SEQ ID NO:52, The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 42 and said LCDR3 comprises SEQ ID NO:

51.

7. said HCDR1 comprising SEQ ID NO:56, said HCDR2 comprising SEQ ID NO:57, CDR3 comprises SEQ ID NO:58, said LCDR1 comprises SEQ ID NO:59, said LCDR2 comprises SEQ ID NO:59, The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 60 and said LCDR2 comprises SEQ ID NO:

61.

8. said HCDR1 comprising SEQ ID NO: 66, said HCDR2 comprising SEQ ID NO: 67, CDR3 comprises SEQ ID NO:68, said LCDR1 comprises SEQ ID NO:69, and said LCDR2 comprises SEQ ID NO: The antibody of claim 1, wherein said LCDR1 comprises SEQ ID NO: 70 and said LCDR2 comprises SEQ ID NO:

71.

9. said HCDR1 comprising SEQ ID NO: 76, said HCDR2 comprising SEQ ID NO: 77, CDR3 comprises SEQ ID NO:78, said LCDR1 comprises SEQ ID NO:69, and said LCDR2 comprises SEQ ID NO: The antibody of claim 1, wherein said LCDR2 comprises SEQ ID NO: 79 and said LCDR3 comprises SEQ ID NO:

70.

10. a) said HCVR comprises SEQ ID NO: 10 and said LCVR comprises SEQ ID NO: 11; b) the HCVR comprises SEQ ID NO: 14 and the LCVR comprises SEQ ID NO: 15; c) the HCVR comprises SEQ ID NO:24 and the LCVR comprises SEQ ID NO:25; d) the HCVR comprises SEQ ID NO: 34 and the LCVR comprises SEQ ID NO: 35; e) the HCVR comprises SEQ ID NO: 44 and the LCVR comprises SEQ ID NO: 45; f) the HCVR comprises SEQ ID NO:52 and the LCVR comprises SEQ ID NO:53; g) the HCVR comprises SEQ ID NO:62 and the LCVR comprises SEQ ID NO:63; h) the HCVR comprises SEQ ID NO: 72 and the LCVR comprises SEQ ID NO: 73; or i) the HCVR comprises SEQ ID NO: 80 and the LCVR comprises SEQ ID NO: 81, 2. The antibody described in 1.

11. The antibody comprises an HCVR comprising SEQ ID NO: 10 and an LCVR comprising SEQ ID NO:

11. The antibody described in claim 10.

12. The antibody comprises an HCVR comprising SEQ ID NO: 14 and an LCVR comprising SEQ ID NO:

15. The antibody described in claim 10.

13. The antibody comprises an HCVR comprising SEQ ID NO:24 and an LCVR comprising SEQ ID NO:

25. The antibody described in claim 10.

14. The antibody comprises an HCVR comprising SEQ ID NO: 34 and an LCVR comprising SEQ ID NO:

35. The antibody described in claim 10.

15. The antibody comprises an HCVR comprising SEQ ID NO: 44 and an LCVR comprising SEQ ID NO:

45. The antibody described in claim 10.

16. The antibody comprises an HCVR comprising SEQ ID NO:52 and an LCVR comprising SEQ ID NO:

53. The antibody described in claim 10.

17. The antibody comprises an HCVR comprising SEQ ID NO:62 and an LCVR comprising SEQ ID NO:

63. The antibody described in claim 10.

18. The antibody comprises an HCVR comprising SEQ ID NO: 72 and an LCVR comprising SEQ ID NO:

73. The antibody described in claim 10.

19. The antibody comprises an HCVR comprising SEQ ID NO: 80 and an LCVR comprising SEQ ID NO:

81. The antibody described in claim 10.

20. 20. The method of claim 1, wherein the antibody has a human IgG1 or IgG4 isotype. The antibody described in any one of claims 1 to 4.

21. 21. The antibody of claim 20, wherein the antibody has a human IgG1 isotype.

22. containing alanine at residues 234 and 235 (according to EU index numbering) 22. The antibody described in 21.

23. 23. The method of claim 22, further comprising a serine at position 265 (according to the EU index numbering). The antibodies listed are:

24. the antibody comprises a heavy chain (HC) and a light chain (LC); a) the HC comprises amino acids 2-444 of SEQ ID NO:2 and the LC comprises amino acids 2-444 of SEQ ID NO:3 comprising amino acids 2-215; b) the HC comprises amino acids 2 to 444 of SEQ ID NO: 12 and the LC comprises amino acids 2 to 444 of SEQ ID NO: 1 3, comprising amino acids 2 to 215 of c) the HC comprises amino acids 2 to 443 of SEQ ID NO: 16 and the LC comprises amino acids 2 to 443 of SEQ ID NO: 1 Consists of 7, d) the HC comprises amino acids 2 to 447 of SEQ ID NO:26 and the LC comprises amino acids 2 to 447 of SEQ ID NO:2 Consists of 7, e) the HC comprises amino acids 2 to 446 of SEQ ID NO: 36 and the LC comprises amino acids 2 to 446 of SEQ ID NO: 3 Consists of 7, f) the HC comprises amino acids 2 to 446 of SEQ ID NO: 46 and the LC comprises amino acids 2 to 446 of SEQ ID NO: 4 Consists of 7, g) the HC comprises amino acids 2 to 446 of SEQ ID NO:54 and the LC comprises amino acids 2 to 446 of SEQ ID NO:5 Consists of 5, h) the HC comprises amino acids 2 to 450 of SEQ ID NO: 64 and the LC comprises amino acids 2 to 450 of SEQ ID NO: 6 5, or i) the HC comprises amino acids 2-450 of SEQ ID NO:74 and the LC comprises amino acids 2-450 of SEQ ID NO:7 The antibody of claim 1, comprising amino acids 2 to 216 of 5.

25. a) the HC consists of SEQ ID NO:2 and the LC consists of SEQ ID NO:3; b) the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO: 13; c) the HC consists of SEQ ID NO: 16 and the LC consists of SEQ ID NO: 17; d) the HC consists of SEQ ID NO:26 and the LC consists of SEQ ID NO:27; e) the HC consists of SEQ ID NO: 36 and the LC consists of SEQ ID NO: 37; f) the HC consists of SEQ ID NO: 46 and the LC consists of SEQ ID NO: 47; g) the HC consists of SEQ ID NO: 54 and the LC consists of SEQ ID NO: 55; h) the HC consists of SEQ ID NO:64 and the LC consists of SEQ ID NO:65; or i) the HC consists of SEQ ID NO: 74 and the LC consists of SEQ ID NO: 75, claim 24 The antibody described in

26. 24 or 25, wherein the HC consists of SEQ ID NO:2 and the LC consists of SEQ ID NO:3 The antibody described in

27. 24. The method according to claim 24, wherein the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO:

13.

26. The antibody described in 25.

28. 24. The method according to claim 24, wherein the HC consists of SEQ ID NO: 16 and the LC consists of SEQ ID NO:

17.

26. The antibody described in 25.

29. 24, wherein the HC consists of SEQ ID NO:26 and the LC consists of SEQ ID NO:27; or 26. The antibody described in 25.

30. 24. The method of claim 24, wherein the HC consists of SEQ ID NO: 36 and the LC consists of SEQ ID NO:

37.

26. The antibody described in 25.

31. 24. The method of claim 24, wherein the HC consists of SEQ ID NO: 46 and the LC consists of SEQ ID NO:

47.

26. The antibody described in 25.

32. 24. The method of claim 24, wherein the HC consists of SEQ ID NO: 54 and the LC consists of SEQ ID NO:

55.

26. The antibody described in 25.

33. 24. The method of claim 23, wherein the HC consists of SEQ ID NO: 64 and the LC consists of SEQ ID NO:

65.

26. The antibody described in 25.

34. 24. The method of claim 23, wherein the HC consists of SEQ ID NO: 74 and the LC consists of SEQ ID NO:

75.

26. The antibody described in 25.

35. The antibody of any one of claims 1 to 34 conjugated to a cytotoxic agent. , antibody-drug conjugates (ADCs).

36. The cytotoxic agent is a microtubule inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNA 36. The A of claim 35, selected from the group consisting of alkylating agents and DNA minor groove binding agents. D.C.

37. 37. The method of claim 35 or 36, wherein the cytotoxic agent is a topoisomerase I inhibitor. C.

38. 38. The compound according to claim 37, wherein the topoisomerase I inhibitor is a camptothecin analogue. D.C.

39. The camptothecin analog comprises the formula: X-Y, During the ceremony, Y is of the formula: 【Chemistry 1】 Xは、 ＊ -CH ２ O-、 ＊ -(CH ２ ) ２ O-、 ＊ -(CH ２ ) ３ O-、 ＊ -(CH ２ ) ４ O-、 ＊ -CH ２ H ＊ -(CH ２ ) ２ H ＊ -(CH ２ ) ３ H ＊ - (CH ２ ) ４ H ＊ -CH ２ N(CH ３ )-、 ＊ -(CH ２ ) ２ N(CH ３ )-、 ＊ -(CH ２ ) ３ N(CH ３ )-、 ＊ -(CH ２ ) ４ N(CH ３ )-、 ＊ -CH ２ N(R １ )-、 ＊ -(CH ２ ) ２ N(R １ )-、 ＊ -(CH ２ ) ３ N(R １ )-、 ＊ -(CH ２ ) ４ N(R １ )-、 ＊ -CH ２ N(CH ３ )C(=O)CH ２ O-、 ＊ -CH ２ N(R １ ) C(=O)CH ２ O-、 ＊ -CH ２ I'm sorry ２ O-、 ＊ -CH ２ I'm sorry ) ２ ) ２ O-、 ＊ -CH ２ I'm sorry ２ ) ３ O-、 ＊ -CH ２ H =O)(CH ２ ) ４ O-、 ＊ -CH ２ I'm sorry ２ ) ５ O-、 ＊ -CH ２ NH C(=O)CH ２ -、 ＊ -CH ２ I'm sorry ２ ) ２ -、 ＊ -CH ２ H O)(CH ２ ) ３ -、 ＊ -CH ２ I'm sorry ２ ) ４ -、 ＊ -CH ２ H O)(CH ２ ) ５ -、 ＊ -CH ２ SCH ２ -、 ＊ -CH ２ S(CH ２ ) ２ -、 ＊ -CH ２ S (CH ２ ) ３ --, ＊ -CH ２ S (CH ２ ) ４ - or ＊ -CH ２ S (CH ２ ) ５ -Yes the law of nature, ＊ is the moiety covalently attached to Y, and R １ is benzyl. ADC.

40. 40. The camptothecin analog of claim 38 or 39, wherein the camptothecin analog comprises any one of the following formulae: The ADCs described herein. 【Chemistry 2】

41. 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: 【Chemistry 3】

42. 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: 【Chemistry 4】

43. 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: 【Chemistry 5】

44. 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: 【Chemistry 6】

45. 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: 【Chemistry 7】

46. The ADC of any one of claims 35 to 45, further comprising a self-immolative spacer. ADC.

47. The self-immolative spacer is -NH-CH ２ - or absent, The ADCs described herein.

48. 35. The ADC further comprises a linker connecting the antibody to the cytotoxic agent.

48. The ADC of any one of claims 1 to 47.

49. 49. The ADC of claim 48, wherein the linker comprises a peptide unit.

50. The peptide units are Ala-Ala-Ala, Val-Cit, or Gly-Gly. The ADC of claim 49, comprising -Phe-Gly (SEQ ID NO: 102).

51. 51. The ADC of claim 50, wherein the peptide unit comprises Ala-Ala-Ala.

52. 51. The ADC of claim 50, wherein the peptide unit comprises Val-Cit.

53. The peptide unit comprises Gly-Gly-Phe-Gly (SEQ ID NO: 102).

51. The ADC according to item 50.

54. the linker further comprises a spacer unit A between the antibody and the peptide unit; The ADC according to any one of claims 48 to 53.

55. Spacer unit A is of the formula: 【Chemistry 8】 55. The ADC of claim 54, wherein z is 1 to 5.

56. Spacer unit A is of the formula: 【Chemistry 9】 55. The ADC of claim 54, wherein z is 1 to 5.

57. An antibody-drug conjugate (ADC), said ADC having one of the following formulas: 【Chemistry 10】 and During the ceremony, Ab is an antibody according to any one of claims 1 to 34, An ADC, wherein n is about 1 to 16.

58. 58. The ADC of claim 57, wherein n is about 2 to 12.

59. 58. The ADC of claim 57, wherein n is about 2 to 8.

60. 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: 【Chemistry 11】

61. 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: 【Chemistry 12】

62. 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: 【Chemistry 13】

63. 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: 【Chemistry 14】

64. The ADC of any one of claims 57 to 63, wherein n is about 2.

65. The ADC of any one of claims 57 to 63, wherein n is about 4.

66. The ADC of any one of claims 57 to 63, wherein n is about 6.

67. The ADC of any one of claims 57 to 63, wherein n is about 8.

68. the attachment to the antibody occurs via a thiol group of one or more cysteines of the antibody; The ADC of any one of claims 57 to 67.

69. each of the one or more cysteines is a native cysteine ​​in the hinge region of the antibody; The ADC of claim 68.

70. The Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:2 and a HC comprising amino acids 2-444 of SEQ ID NO:

3. and L C 215 or more, wherein n is about 8. The ADC described in any one of claims 1 to 4.

71. The Ab comprises a HC consisting of SEQ ID NO:2 and a LC consisting of SEQ ID NO:

3.

70. An ADC as described in claim 70.

72. The Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:12 and a HC comprising amino acids 2-444 of SEQ ID NO:

13. and LC comprising acids 2 to 215, wherein n is about 8. The ADC according to any one of claims 1 to 5.

73. The Ab comprises a HC consisting of SEQ ID NO: 12 and a LC consisting of SEQ ID NO:

13. The ADC of claim 72.

74. The Ab consists of a HC comprising amino acids 2-443 of SEQ ID NO:16 and a sequence of SEQ ID NO:

17. LC, and n is about 8. ADC included.

75. The Ab comprises a HC consisting of SEQ ID NO: 16 and a LC consisting of SEQ ID NO:

17. The ADC described in claim 74.

76. The Ab comprises amino acids 2 to 447 of SEQ ID NO:26 and a LC consisting of SEQ ID NO:

27. and n is about 8. 。

77. The Ab comprises a HC consisting of SEQ ID NO:26 and a LC consisting of SEQ ID NO:

27. The ADC described in claim 76.

78. The Ab comprises amino acids 2 to 446 of SEQ ID NO: 36 and a LC consisting of SEQ ID NO:

37. and n is about 8. 。

79. The Ab comprises a HC consisting of SEQ ID NO: 36 and a LC consisting of SEQ ID NO:

37. The ADC described in claim 78.

80. The Ab comprises amino acids 2 to 446 of SEQ ID NO: 46 and a LC consisting of SEQ ID NO:

47. and n is about 8. 。

81. The Ab comprises a HC consisting of SEQ ID NO: 46 and a LC consisting of SEQ ID NO:

47. The ADC described in claim 80.

82. The Ab comprises amino acids 2 to 446 of SEQ ID NO:54 and a LC consisting of SEQ ID NO:

55. and n is about 8. 。

83. The Ab comprises a HC consisting of SEQ ID NO:54 and a LC consisting of SEQ ID NO:

55. The ADC described in claim 82.

84. The Ab comprises amino acids 2-450 of SEQ ID NO:64 and amino acids 2-21 of SEQ ID NO:

65. and L C , wherein n is about 8. The ADC described in one aspect.

85. The Ab comprises a HC consisting of SEQ ID NO:64 and a LC consisting of SEQ ID NO:

65. The ADC described in claim 84.

86. The Ab comprises amino acids 2-450 of SEQ ID NO:74 and amino acids 2-21 of SEQ ID NO:

75. and L C , wherein n is about 8. The ADC described in one aspect.

87. The Ab comprises a HC consisting of SEQ ID NO: 74 and a LC consisting of SEQ ID NO:

75. The ADC described in claim 86.

88. An antibody according to any one of claims 1 to 34, and one or more pharma- ceutically acceptable carriers. A diluent or excipient.

89. The ADC according to any one of claims 35 to 87, and one or more pharma- ceutically acceptable carriers. A pharmaceutical composition comprising:

90. A patient in need of cancer treatment is administered an effective amount of any one of claims 35 to 87. A method of treating cancer comprising administering the ADC described herein.

91. The cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, 91. The method of claim 90, wherein the cancer is cervical cancer, ovarian cancer, or prostate cancer.

92. 92. The method of claim 90 or 91, wherein the cancer is urothelial cancer.

93. Whether the treated patient has relapsed after receiving enfortumab vedotin or any one of claims 90 to 92, which has become refractory to enfortumab vedotin.

3. The method according to claim 1 .

94. The method according to any one of claims 90 to 92, wherein the patient is ineligible for treatment with enfortumab vedotin.

2. The method according to any one of claims 1 to 11.

95. Simultaneous, separate, or sequential administration of a PD-1 inhibitor or a PD-L1 inhibitor. The method of any one of claims 90 to 94, further comprising:

96. The ADC of any one of claims 35 to 87 for use in therapy.

97. The ADC of any one of claims 35 to 87, for use in the treatment of cancer.

98. The cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, 98. The ADC for use according to claim 97, wherein the cancer is a gastrointestinal cancer, an ovarian cancer, or a prostate cancer.

99. 99. The ADC for use according to claim 98, wherein the cancer is urothelial cancer.

100. the cancer has recurred after treatment with enfortumab vedotin or the cancer 99. Any one of claims 97 to 99, which is refractory to enfortumab vedotin.

4. An ADC for use as described in item 1.

101. Any one of claims 97 to 99, wherein the prior use of enfortumab vedotin was contraindicated.

2. An ADC for use as described in claim 1.

102. The ADC is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor. AD for use according to any one of claims 97 to 101, administered in combination. C.

103. A method for treating cancer comprising administering to said patient an effective amount of an ADC according to any one of claims 35 to 87. A pharmaceutical composition comprising the compound of formula (I).

104. The cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, 104. The composition for use according to claim 103, wherein the cancer is a cervical cancer, an ovarian cancer, or a prostate cancer.

105. PD-1 inhibitors or PD-L1 inhibitors may be administered simultaneously, separately, or sequentially in combination.

105. The composition of claim 103 or 104,

106. The AD of any one of claims 35 to 87 for the manufacture of a medicament for the treatment of cancer. Use of C.

107. The cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, The use of claim 106, wherein the cancer is cervical cancer, ovarian cancer, or prostate cancer.

108. The drug further comprises a PD-1 inhibitor or a PD-L1 inhibitor.

7. Use according to claim 7.

109. A method for producing an ADC, comprising: reacting an antibody according to any one of claims 1 to 34 with a compound of the formula: The method comprises contacting the compound of formula (I) with a compound of formula (I). 【Chemistry 15】

110. Prior to the contacting, the antibody is reduced with a reducing agent to obtain a reduced Nectin-4 antibody.

110. The method of claim 109, further comprising producing:

111. 111. The method of claim 110, wherein the reducing agent is DTT or TCEP.

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