Nectin-4 antibodies and antibody-drug conjugates

Abstract

To provide nectin-4 antibody-drug conjugates and pharmaceutical compositions thereof.SOLUTION: Provided herein are an antibody that binds to human nectin-4, an antibody-drug conjugate (ADC) comprising the antibody conjugated to a cytotoxic agent, pharmaceutical compositions containing the antibody, and the pharmaceutical compositions for treating urothelial carcinoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer or prostate cancer.SELECTED DRAWING: None

Description

[Technical field]

[0001] (Reference to sequence listing) This application has been submitted with a Sequence Listing in ST.26XML format. The Sequence Listing is provided as a file entitled "30650_WO", created on May 3, 2024, and is 120 kilobytes in size. The Sequence Listing information in ST.26XML format is incorporated herein by reference in its entirety.

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

[0003] Nectin-4 is a member of the nectin family, which is a Ca2+-independent immunoglobulin-like cell adhesion molecule. Unlike other members of the nectin family, nectin-4 expression in healthy tissues is mainly placental or embryonic, but is overexpressed in some tumor types, including urothelial, breast, lung, gastric, colorectal, pancreatic, and ovarian cancer. Studies have linked high nectin-4 expression in some cancer types to tumor development.

[0004] Antibody drug conjugates (ADCs) for use as tumor therapy contain a tumor-targeting antibody conjugated to a payload designed to kill the tumor cell upon entry into the cell. Certain Nectin-4 antibodies have been used to generate ADCs with MMAE payloads (WO201247724) and certain camptothecin analogues (WO2022112356 and WO2021151984).

[0005] ADCs for use in oncology are very challenging compounds to design as they must balance multiple aspects of the molecule, such as sufficient specificity for tumor targets over healthy cells, maintaining the desired activity against bystander tumor cells while at the same time having acceptable toxicity, and a labile payload that maintains good physical and chemical stability while allowing intracellular delivery. Summary of the Invention

[0006] There is still a need for Nectin-4 ADCs for treating cancer. In particular, there is still a need for Nectin-4 ADCs with sufficient therapeutic index based on better tolerability and / or better efficacy to support high enough doses that not only effectively kill tumor cells but are also well tolerated by patients. In particular, there is still a need for Nectin-4 ADCs with effector null antibodies and topoisomerase I payloads. In particular, there is still a need for Nectin-4 ADCs with enhanced bystander activity against Nectin-4-low tumors while allowing lower doses. In particular, there is still a need for Nectin-4 ADCs that allow avoidance or better management of dermatological events seen in certain Nectin-4 ADCs. In particular, there is still a need for Nectin-4 ADCs that avoid optic neuropathy and / or peripheral neuropathy signals seen in certain Nectin-4 ADCs. In particular, there is still a need for Nectin-4 ADCs with low immunogenicity, stable in vivo pharmacokinetics, and suitable chemical and physical stability. Furthermore, there remains a need for Nectin-4 ADCs that have one or more of the following characteristics: better anti-tumor activity as measured in specific tumor models, enhanced bystander activity against Nectin-4low tumors, lower immunogenicity, no measurable antibody effector function, and / or better physical and chemical stability. The ADCs provided herein address one or more of these needs.

[0007] Provided herein are certain Nectin-4 ADCs and compositions comprising Nectin-4 ADCs. Also provided herein are methods of using Nectin-4 ADCs or compositions comprising Nectin-4 ADCs for cancer in a subject.

[0008] In one aspect, there is provided herein an antibody that binds to human Nectin-4, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2 and HCDR3, and the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2 and LCDR3, a) HCDR1 comprises SEQ ID NO: 4, HCDR2 comprises SEQ ID NO: 5, HCDR3 comprises SEQ ID NO: 6, LCDR1 comprises SEQ ID NO: 7, LCDR2 comprises SEQ ID NO: 8 and LCDR3 comprises SEQ ID NO: 9; b) HCDR1 comprises SEQ ID NO: 18, HCDR2 comprises SEQ ID NO: 19, HCDR3 comprises SEQ ID NO: 20, LCDR1 comprises SEQ ID NO: 21, LCDR2 comprises SEQ ID NO: 22 and LCDR3 comprises SEQ ID NO: 23; c) HCDR1 comprises SEQ ID NO: 28, HCDR2 comprises SEQ ID NO: 29, HCDR3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO: 31, LCDR2 comprises SEQ ID NO: 32 and LCDR3 comprises SEQ ID NO: 33; d) HCDR1 comprises SEQ ID NO: 38, HCDR2 comprises SEQ ID NO: 39, HCDR3 comprises SEQ ID NO: 40, LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 42 and LCDR3 comprises SEQ ID NO: 43; e) HCDR1 comprises SEQ ID NO: 48, HCDR2 comprises SEQ ID NO: 49, HCDR3 comprises SEQ ID NO: 50, LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 42 and LCDR3 comprises SEQ ID NO: 51; f) HCDR1 comprises SEQ ID NO: 56, HCDR2 comprises SEQ ID NO: 57, HCDR3 comprises SEQ ID NO: 58, LCDR1 comprises SEQ ID NO: 59, LCDR2 comprises SEQ ID NO: 60 and LCDR3 comprises SEQ ID NO: 61; g) HCDR1 comprises SEQ ID NO: 66, HCDR2 comprises SEQ ID NO: 67, HCDR3 comprises SEQ ID NO: 68, LCDR1 comprises SEQ ID NO: 69, LCDR2 comprises SEQ ID NO: 70 and LCDR3 comprises SEQ ID NO: 71, or h) HCDR1 comprises SEQ ID NO: 76, HCDR2 comprises SEQ ID NO: 77, HCDR3 comprises SEQ ID NO: 78, LCDR1 comprises SEQ ID NO: 69, LCDR2 comprises SEQ ID NO: 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, the antibody comprising 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 215 of SEQ ID NO: 3; b) HC comprises amino acids 2 to 444 of SEQ ID NO: 12, and LC comprises amino acids 2 to 215 of SEQ ID NO: 13; c) HC comprises amino acids 2 to 443 of SEQ ID NO: 16, and LC consists of SEQ ID NO: 17; d) HC comprises amino acids 2 to 447 of SEQ ID NO: 26, and LC consists of SEQ ID NO: 27; e) HC comprises amino acids 2 to 446 of SEQ ID NO: 36, and LC consists of SEQ ID NO: 37; f) HC comprises amino acids 2 to 446 of SEQ ID NO: 46, and LC consists of SEQ ID NO: 47; g) HC comprises amino acids 2 to 446 of SEQ ID NO: 54, and LC consists of SEQ ID NO: 55; h) HC comprises amino acids 2 to 450 of SEQ ID NO: 64 and LC comprises amino acids 2 to 216 of SEQ ID NO: 65; or i) HC comprises amino acids 2 to 450 of SEQ ID NO: 74, and LC comprises amino acids 2 to 216 of SEQ ID NO: 75.

[0011] In another aspect, provided herein is an antibody-drug conjugate (ADC) comprising a Nectin-4 antibody disclosed herein conjugated directly or via a linker to a cytotoxic agent.

[0012] In a further aspect, there is provided an ADC herein, wherein the cytotoxic agent is a camptothecin analog having the formula: XY, wherein: 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- * -CH2 NHC(=O)(CH 2 ) 3 O-, * -CH 2 NHC(=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 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 - and * is the moiety covalently attached to Y, and R 1 is phenyl.

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

[0015] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0016] In another aspect, provided herein is a pharmaceutical composition comprising a Nectin-4 antibody disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. In another aspect, provided herein is a pharmaceutical composition comprising a Nectin-4 ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0017] In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need of cancer treatment an effective amount of a Nectin-4 ADC disclosed herein. In a further aspect, provided herein is a method of treating cancer comprising administering to a patient in need of cancer treatment an effective amount of a Nectin-4 ADC disclosed herein, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[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. The antibody may be of any class (e.g., IgG, IgE, IgM, IgD, IgA) and any subclass (e.g., IgG1, IgG2, IgG3, IgG4).

[0020] An exemplary antibody of the present disclosure is an immunoglobulin G (IgG) type antibody composed of four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds. The amino terminal portion of each of the four polypeptide chains contains a variable region of about 100-125 or more amino acids primarily responsible for antigen recognition. The carboxy terminal portion of each of the four polypeptide chains 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 is composed of a light chain variable region (VL, also known as LCVR) and a light chain constant region. The IgG isotype can be further divided into subclasses (e.g., IgG1, IgG2, IgG3, and IgG4).

[0021] The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs are exposed on the surface of the protein and are the regions of the antibody that are important for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3" and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3". The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be performed using the methods described in Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987), Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international ImMunoGeneTics database available at www.imgt.org; Lefranc et al., Nucleic Acids Res. 1999;27:209-212). The CDRs of the present disclosure are determined according to North.

[0022] Certain antibodies described herein contain an IgG1 Fc region or an Fc region derived from human IgG1, for example, a modified IgG1 Fc region with altered Fc effector functions. IgG1 is known to induce antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Some antibodies of the present disclosure have amino acid substitutions introduced into the IgG1 Fc region to alter effector functions. According to some disclosed herein, mutations are introduced at positions 234 and 235 (according to EU index numbering) of the Fc region. According to some disclosed herein, mutations are introduced at positions 234, 235, and 265 (according to EU index numbering) of the Fc region. In some aspects, the Nectin-4 antibody of the present disclosure comprises a modified human IgG1 Fc region (also referred to as hIgG1 effector null or hIgG1EN Fc region, according to EU index numbering) that includes alanine at residues 234 and 235, and serine at position 265. In further aspects, some antibodies have additional mutations in the Fc region including a glutamine, alanine, or glycine at position 297, an alanine or glutamine at position 322, an alanine or glycine at position 329, and / or an alanine or serine at position 331 (according to EU index numbering). In some aspects, these antibody mutations are an alanine at position 234, a glutamic acid at position 235, an alanine at position 237, a serine at position 330, and a serine at position 331 (according to EU index numbering). In further aspects, these amino acid substitutions introduced into the IgG1 Fc region reduced or eliminated measurable antibody effector function.

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

[0024] Mammalian antibody expression typically results in glycosylation. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, such as N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid. Typically, glycosylation occurs in the Fc region of an antibody at a highly conserved N-glycosylation site (e.g., position 297 in IgG1 according to the IMGT or EU index numbering). Glycosylation sites can be modified to alter glycosylation (e.g., to block or reduce glycosylation, or to change the amino acid sequence to generate additional or diverse glycosylation).

[0025] Expression of antibodies in mammals from the IgG subclass can result in clipping of the C-terminal amino acids from one or both heavy chains, for example, in the case of IgG1 antibodies, one or two C-terminal amino acids may be removed. In the case of IgG1 antibodies, the C-terminal lysine, if present, may be truncated or clipped from the heavy chain during expression. Additionally, the penultimate glycine may be truncated or clipped from the heavy chain as well.

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

[0027] The terms "nucleic acid" or "polynucleotide," as used interchangeably herein, refer to a polymer of nucleotides, including single- and / or double-stranded nucleotide-containing molecules, such as DNA, cDNA, and RNA molecules, that incorporate naturally occurring nucleotides, modified nucleotides, and / or nucleotide analogs. A polynucleotide of the present disclosure can also include substrates incorporated therein, for example, by a DNA or RNA polymerase or a synthetic reaction.

[0028] The polynucleotides of the present disclosure can be expressed in a host cell, for example, after the polynucleotide is operably linked to an expression control sequence. Expression control sequences capable of expressing an operably linked polynucleotide are well known in the art. For example, the expression vector can include a sequence encoding one or more signal peptides that facilitate secretion of the polypeptide from the host cell. The signal peptide can be, for example, an immunoglobulin signal peptide or a heterologous signal peptide. The expression vector containing the polynucleotide of interest (e.g., a polynucleotide encoding an antibody polypeptide) can be transferred into the host cell by well-known methods. Additionally, the expression vector can include one or more selection markers, such as, for example, tetracycline, neomycin, and dihydrofolate reductase, to aid in detection of host cells transformed with the desired polynucleotide sequence.

[0029] Host cells include cells stably or transiently transfected, transformed, transduced or infected with one or more expression vectors expressing all or a portion of the antibody of the present disclosure. According to some embodiments, host cells can be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC polypeptide and an expression vector expressing the LC polypeptide of the antibody of the present disclosure. In some embodiments, host cells can be stably or transiently transfected, transformed, transduced or infected with an expression vector expressing the HC and LC polypeptides of the antibody of the present disclosure. The antibodies of the present disclosure can be produced in mammalian cells, such as CHO, NS0, HEK293 or COS cells, according to techniques well known in the art.

[0030] The medium into which the antibody of the present disclosure is secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods, and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, refrigerated, or lyophilized, for example at -70°C. Various methods of protein purification can be employed, such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein 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 variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3, HCDR1 comprising SEQ ID NO: 4, HCDR2 comprising SEQ ID NO: 5, HCDR3 comprising SEQ ID NO: 6, LCDR1 comprising SEQ ID NO: 7, LCDR2 comprising SEQ ID NO: 8, and LCDR3 comprising SEQ ID NO: 9. In another aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and LCDR3, the HCDR1 comprising SEQ ID NO: 4, the HCDR2 comprising SEQ ID NO: 5, the HCDR3 comprising SEQ ID NO: 6, the LCDR1 comprising SEQ ID NO: 7, the LCDR2 comprising SEQ ID NO: 8, and the LCDR3 comprising SEQ ID NO: 9. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein HCDR1 comprises SEQ ID NO: 28, HCDR2 comprises SEQ ID NO: 29, HCDR3 comprises SEQ ID NO: 30, LCDR1 comprises SEQ ID NO: 31, LCDR2 comprises SEQ ID NO: 32, and LCDR3 comprises SEQ ID NO: 33. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein HCDR1 comprises SEQ ID NO: 38, HCDR2 comprises SEQ ID NO: 39, HCDR3 comprises SEQ ID NO: 40, LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 42, and LCDR3 comprises SEQ ID NO: 43. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein HCDR1 comprises SEQ ID NO: 48, HCDR2 comprises SEQ ID NO: 49, HCDR3 comprises SEQ ID NO: 50, LCDR1 comprises SEQ ID NO: 41, LCDR2 comprises SEQ ID NO: 42, and LCDR3 comprises SEQ ID NO: 51. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein HCDR1 comprises SEQ ID NO: 56, HCDR2 comprises SEQ ID NO: 57, HCDR3 comprises SEQ ID NO: 58, LCDR1 comprises SEQ ID NO: 59, LCDR2 comprises SEQ ID NO: 60, and LCDR3 comprises SEQ ID NO: 61.In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein HCDR1 comprises SEQ ID NO: 66, HCDR2 comprises SEQ ID NO: 67, HCDR3 comprises SEQ ID NO: 68, LCDR1 comprises SEQ ID NO: 69, LCDR2 comprises SEQ ID NO: 70, and LCDR3 comprises SEQ ID NO: 71. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein HCDR1 comprises SEQ ID NO: 76, HCDR2 comprises SEQ ID NO: 77, HCDR3 comprises SEQ ID NO: 78, LCDR1 comprises SEQ ID NO: 69, 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 variable region (HCVR) and a light chain variable region (LCVR), the antibody comprising a HCVR comprising SEQ ID NO: 10 and a LCVR comprising SEQ ID NO: 11. In another aspect, provided herein is an antibody that binds to human nectin-4, the antibody comprising a HCVR comprising SEQ ID NO: 14 and a LCVR comprising SEQ ID NO: 15. In another aspect, provided herein is an antibody that binds to human nectin-4, the antibody comprising 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 a HCVR comprising SEQ ID NO: 34 and a LCVR comprising SEQ ID NO: 35. In another aspect, provided herein is an antibody that binds to human nectin-4, the antibody comprising a HCVR comprising SEQ ID NO: 44 and a LCVR comprising SEQ ID NO: 45. In another aspect, provided herein is an antibody that binds to human nectin-4, the antibody comprising a HCVR comprising SEQ ID NO: 52 and a LCVR comprising SEQ ID NO: 53. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein the antibody comprises a HCVR comprising SEQ ID NO: 62 and a LCVR comprising SEQ ID NO: 63. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein the antibody comprises a HCVR comprising SEQ ID NO: 72 and a LCVR comprising SEQ ID NO: 73. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein the antibody comprises a HCVR comprising SEQ ID NO: 80 and a LCVR comprising SEQ ID NO: 81.

[0033] In a further aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), and the antibody has a human IgG1 or IgG4 isotype. In a further aspect, the antibody has a human IgG1 isotype. In a further aspect, the antibody comprises an alanine at residues 234 and 235 (according to EU index numbering). In a further aspect, the antibody further comprises a serine at position 265 (according to EU index numbering). In another aspect, the antibody has a human IgG4 isotype.

[0034] In one aspect, 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 to 444 of SEQ ID NO: 2, and the LC comprising amino acids 2 to 215 of SEQ ID NO: 3. In one aspect, 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 to 444 of SEQ ID NO: 12, and the LC comprising amino acids 2 to 215 of SEQ ID NO: 13. In one aspect, 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 to 443 of SEQ ID NO: 16, and the LC consisting of SEQ ID NO: 17. In one aspect, 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 to 447 of SEQ ID NO: 26, and the LC consisting of SEQ ID NO: 27. In one aspect, 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 to 446 of SEQ ID NO: 36, and the LC consisting of SEQ ID NO: 37. In one aspect, 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 to 446 of SEQ ID NO: 46, and the LC consisting of SEQ ID NO: 47. In one aspect, 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 to 446 of SEQ ID NO: 54, and the LC consisting of SEQ ID NO: 55. In one aspect, 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 to 450 of SEQ ID NO: 64, and the LC consisting of amino acids 2 to 216 of SEQ ID NO: 65. In one aspect, provided herein is an antibody that binds to human Nectin-4, the antibody comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises amino acids 2-450 of SEQ ID NO: 74, and the 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 the HC consists of SEQ ID NO:2 and the LC consists of SEQ ID NO:3. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:12 and the LC consists of SEQ ID NO:13. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:16 and the LC consists of SEQ ID NO:17. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:26 and the LC consists of SEQ ID NO:27. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:36 and the LC consists of SEQ ID NO:37. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:46 and the LC consists of SEQ ID NO:47. In another aspect, provided herein is an antibody that binds to human nectin-4, wherein the HC consists of SEQ ID NO:54 and the LC consists of SEQ ID NO:55. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein the HC consists of SEQ ID NO: 64 and the LC consists of SEQ ID NO: 65. In another aspect, provided herein is an antibody that binds to human Nectin-4, wherein the HC consists of SEQ ID NO: 74 and the LC consists of SEQ ID NO: 75.

[0036] In another aspect, provided herein are different mammalian cells comprising a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence of 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:65, or SEQ ID NO:74 and SEQ ID NO:75, wherein the cell is capable of expressing a Nectin-4 antibody disclosed herein.

[0037] In another aspect, provided herein is a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having an amino acid sequence as follows:

[0038] [Table 1]

[0039] In another aspect, provided herein is a process for producing a Nectin-4 antibody, comprising culturing one of the mammalian cells disclosed herein under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0040] In another aspect, provided herein is an antibody produced by culturing a mammalian cell containing a DNA molecule comprising a polynucleotide sequence encoding a polypeptide having the amino acid sequence of 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:65, or SEQ ID NO:74 and SEQ ID NO:75, under conditions such that the antibody is expressed, and recovering the expressed antibody.

[0041] In another aspect, provided herein is an antibody produced by culturing a mammalian cell comprising a first DNA molecule and a second DNA molecule, wherein the first DNA molecule comprises a polynucleotide sequence encoding a polypeptide having the amino acid sequence:

[0042] [Table 2]

[0043] The term "enfortumab" as used herein refers to a fully human anti-Nectin-4 IgG1 kappa monoclonal antibody having the sequence disclosed in Figures 3A and 3B of WO2012047724, expressed and purified using standard conditions.

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

[0045] Exemplary ADCs herein utilize camptothecin-based payloads (e.g., camptothecin analogs). Camptothecin is a topoisomerase I (TOPO1) inhibitor that has been shown to have anticancer activity. Camptothecin and its derivatives bind to the TOPO1 / DNA complex, preventing reannealing of partially cleaved DNA, the accumulation of which leads to cell death. Other topoisomerase I inhibitors known in the art, such as SN-38 and DXd, can be used as payloads.

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

[0047] In certain aspects, provided herein are ADCs, wherein the camptothecin analog comprises 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 Alkyl or C 1 ~C 6 fluoroalkyl, or R 1 and R 2 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy 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 ~C 6 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 6 cycloalkyl, 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 optionally substituted with 1 to 4 substituents selected from heterocycloalkyl, aryl, or heteroaryl; 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 (alkylene)-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 the moiety covalently attached to A, and A is -X 2 where R 1 is F and R 2 is -OMe, -BA is -NH 2 That's not possible.

[0049] In certain aspects, provided herein are ADCs, wherein the camptothecin analog comprises 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 Alkyl or C 1 ~C 6 fluoroalkyl, or R 1 and R 2 together with the carbon atom to which they are attached form a methylenedioxy or difluoromethylenedioxy 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- * -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 NHC(=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 - 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 comprises 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 NHC(=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 - and * 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 comprises any one of the following formulae:

[0054] [ka]

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

[0056] [ka]

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

[0058] [ka]

[0059] In a further aspect, provided herein is an ADC, wherein the camptothecin analog comprises Formula V:

[0060] [ka]

[0061] In a further aspect, provided herein is an ADC, wherein the camptothecin analog comprises Formula VI:

[0062] [ka]

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

[0064] [ka]

[0065] Self-destructive unit Self-immolation, or self-removal, of a portion of an ADC can be designed into the overall structure of the ADC. Self-immolation typically involves the activation of a trigger group, which then causes spontaneous elimination of the group itself, the self-immolative unit, by a chemical and / or biological reaction. The self-immolative unit can provide favorable properties to the ADC, such as providing space to reduce steric hindrance of cellular proteases reaching peptide cleavage sites within the ADC.

[0066] In some aspects 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, such that the trigger group is exposed after protease cleavage of the peptide unit of the ADC. In further aspects, the self-immolative unit is -NH-CH 2 -group, para-aminobenzyloxycarbonyl (PABC), ortho-aminobenzyl carbonate (OABC), or other self-immolative units known in the art.

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

[0068] Linker As disclosed herein, the payload can be conjugated to the Nectin-4 antibody by methods understood by those skilled in the art to form the Nectin-4 ADCs described herein. One example of such a conjugate includes connecting the payload described herein to the Nectin-4 antibody described herein via a linker.

[0069] The linker used in ADC is designed for stability in plasma to allow time for the ADC to localize to target cells. If the payload is released too early, it will reduce the therapeutic index of the ADC by damaging all kinds of non-target tissues. Once the ADC is internalized in the target cells, the linker should provide a mechanism for payload release so that the payload can function as designed.

[0070] Linkers known to those of skill in the art include, for example, cleavable and non-cleavable moieties. Thus, provided herein are ADCs in which a payload, e.g., a camptothecin analog, is conjugated to an antibody via a linker having a cleavable moiety or a linker having a non-cleavable moiety.

[0071] Any suitable linker known in the art can be used in preparing the ADCs of the disclosure. In certain aspects, the linker comprises a reactive group capable of conjugating both the antibody of the disclosure and the drug or cytotoxic agent. Examples include N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), V-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), bis-maleimide polyethylene glycol (BMPEO), BM(PEO). 2 , B.M. (P.E.O.) 3, N-(b-maleimidopropyloxy)succinimide ester (BMPS), g-maleimidobutyric acid N-succinimidyl ester (GMBS), e-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), 5-maleimidovaleric acid NHS, HBVS, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 4-(4-N-maleimidophenyl)-butyric acid hydrazide or HC1 salt (MPBH), N-sa Succinimidyl 3-(bromoacetamido)propionate (SBAP), N-succinimidyl iodoacetate (SIA), k-maleimido undecanoic acid N-succinimidyl ester (KMUA), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB), succinimidyl-6-(-maleimidopropionamido)hexanoate (SMPH), succinimidyl-(4-vinylsulfonyl)benzoate (SVSB), dithiobis-maleimidoethane (DTME), l,4-bis-maleimidobutane (BMB), l,4-bismaleimidyl-2,3-Dihydroxybutane (BMDB), bis-maleimidohexane (BMH), bis-maleimidoethane (BMOE), sulfosuccinimidyl 4-(N-maleimido-methyl)cyclohexane-l-carboxylate (sulfo-SMCC), sulfosuccinimidyl (4-iodo-acetyl)aminobenzoate (sulfo-SIAB), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBS or sGMBS), N-(e-maleimidocaproyloxy)sulfosuccinimide ester (sulfo-EMCS), N-(K-maleimidoundecanoyloxy)sulfosuccinimide ester (sulfo-KMUS), and sulfosuccinimidyl 4-(p-maleimidophenyloxy)sulfosuccinimide ester (sulfo-KMUS). succinimidyl) butyrate (sulfo-SMPB), succinimidyl 6-hydrazinonicotinamide acetone hydrazone (SANH), succinimidyl 4-hydrazido terephthalate hydrochloride (SHTH), succinimidyl hydrazinium nicotinate hydrochloride (SHNH), succinimidyl-p-formylbenzoate (SFB), and succinimidyl-p-formylphenoxyacetate (S FPA), V-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), V-succinimidyl-4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and V-succinimidyl-4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB).

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

[0073] In some embodiments of the disclosure, the ADCs comprise a linker that comprises a peptide unit that provides a site for protease cleavage.In some embodiments, the peptide units are -Gly-Gly-Gly-, -Ala-Val-, -Val-Ala-, -Val-Cit-, -Val-Lys-, -Lys-Val-, -Phe-Lys-, -Lys-Phe-, -Lys-Lys-, -Ala-Ly s-, -Lys-Ala-, -Phe-Cit-, -Cit-Phe,-Leu-Cit-, -Cit-Leu-, -Ile-Cit-, -Phe-Ala-, -Ala-Phe-, -Phe-Phe-Lys-, -Lys-Phe-Phe-, -Gly-Phe-L ys-, -Lys-Phe-Gly-, -Leu-Ala-Leu-, -Ile-Ala-Leu-, -Leu-Ala-Ile-, -Val-Ala-Val-, -Ala-Leu-Ala-Leu- (SEQ ID NO: 100), -Leu-Ala-Leu-Ala- Column number 101), -Gly-Phe-Leu-Gly- (SEQ ID NO: 103), -Gly-Leu-Phe-Gly- (SEQ ID NO: 104), -Val-Arg-, -Arg-Val-, -Arg-Arg-, -Ala-Ala-, -Ala-Met-, -Met-Ala -, -Thr-Thr-, -Thr-Met-, -Met-Thr-, -Leu-Ala-, -Ala-Leu-, -Cit-Val-, -Gln-Val-, -Val-Gln-, -Ser-Val-, -Val-Ser-, -Ser-Ala-, -Ser-Gly -, -Ala-Ser-, -Gly-Ser-, -Leu-Gln-, -Gln-Leu-, -Phe-Arg-, -Arg-Phe-, -Tyr-Arg-, -Arg-Tyr-, -Phe-Gln-, -Gln-Phe-, -Val-Thr-, -Thr-Val -, -Met-Tyr-, -Tyr-Met-, -Ala-Ala-, -Ala-Ala-Ala-, -Ala-Ala-Ala-Ala-(SEQ ID NO: 105), -Gly-Ala-Gly-Gly-(SEQ ID NO: 106), -Gly-Gly-Ala-Gly-(SEQ ID NO: 107), -Gly-Val-Gly-Gly-(SEQ ID NO: 108), -Gly-Gly-Val-Gly-(SEQ ID NO: 109), -Gly-Phe-Gly-Gly-(SEQ ID NO: 110), or -Gly-Gly-Phe-Gly-(SEQ ID NO: 102). Citrulline is represented by Cit.In an embodiment of the present disclosure, the peptide unit contains all naturally occurring amino acids in L-amino acid form. In a further embodiment, the peptide unit may include all D-amino acids or L-amino acids, or a combination thereof.

[0074] In some aspects, provided herein are ADCs further comprising a linker connecting the antibody to the cytotoxic agent. In further aspects, provided herein are linkers comprising peptide units. In further aspects, provided herein are peptide units comprising Ala-Ala-Ala, Val-Cit, or Gly-Gly-Phe-Gly (SEQ ID NO: 102). In some aspects, the peptide units comprise Ala-Ala-Ala. In some aspects, the peptide units comprise Val-Cit. In some aspects, the peptide units comprise Gly-Gly-Phe-Gly (SEQ ID NO: 102). In further aspects, the peptide units contain all naturally occurring amino acids in the L-amino acid form.

[0075] In some aspects of the disclosure, the ADCs comprise a linker comprising a spacer unit, referred to herein as spacer unit A, that connects a cysteine ​​of an antibody disclosed herein to a peptide unit and / or payload described herein. Some of the chemistries used in the art to connect cysteines include maleimide, succinimide, or bromoacetamide compounds and can be utilized in the ADCs of the disclosure. In further aspects of the disclosure, maleimide-type spacers such as maleimidocaproyl (mc) or maleimidomethylcyclohexane-1-carboxylate can be used.

[0076] In some aspects of the disclosure, provided herein are ADCs having a spacer unit A of formula VIII:

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

[0078] In some aspects of the disclosure, provided herein are ADCs having a spacer unit A of formula IX,

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

[0080] In one aspect of the disclosure, provided herein is an antibody-drug conjugate (ADC), the ADC having the formula:

[0081] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

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

[0083] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

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

[0085] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

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

[0087] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

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

[0089] [ka] In the formula, Ab is a Nectin-4 antibody disclosed herein, and n is about 1 to about 16.

[0090] In further embodiments, n is from about 2 to about 12. In another embodiment, n is from about 2 to about 8. In another 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 aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:2 and a LC comprising amino acids 2-215 of SEQ ID NO:3, where n is about 8. In certain aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:2 and a LC comprising amino acids 2-215 of SEQ ID NO:3, where n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:2 and a LC consisting of SEQ ID NO:3.

[0092] In certain aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:12 and a LC comprising amino acids 2-215 of SEQ ID NO:13, and n is about 8. In certain aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-444 of SEQ ID NO:12 and a LC comprising amino acids 2-215 of SEQ ID NO:13, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:12 and a LC consisting of SEQ ID NO:13.

[0093] In certain aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-443 of SEQ ID NO: 16 and a LC consisting of SEQ ID NO: 17, and n is about 8. In certain aspects disclosed herein, the Ab comprises a HC comprising amino acids 2-443 of SEQ ID NO: 16 and a LC consisting of SEQ ID NO: 17, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO: 16 and a LC consisting of SEQ ID NO: 17.

[0094] In certain aspects disclosed herein, the Ab comprises amino acids 2-447 of SEQ ID NO:26, the LC consists of SEQ ID NO:27, and n is about 8. In certain aspects disclosed herein, the Ab comprises amino acids 2-447 of SEQ ID NO:26, the LC consists of SEQ ID NO:27, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:26 and a LC consisting of SEQ ID NO:27.

[0095] In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:36, the LC consists of SEQ ID NO:37, and n is about 8. In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:36, the LC consists of SEQ ID NO:37, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:36 and a LC consisting of SEQ ID NO:37.

[0096] In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:46, the LC consists of SEQ ID NO:47, and n is about 8. In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:46, the LC consists of SEQ ID NO:47, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:46 and a LC consisting of SEQ ID NO:47.

[0097] In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:54, the LC consists of SEQ ID NO:55, and n is about 8. In certain aspects disclosed herein, the Ab comprises amino acids 2-446 of SEQ ID NO:54, the LC consists of SEQ ID NO:55, and n is about 4. In further aspects disclosed herein, the Ab comprises a HC consisting of SEQ ID NO:54 and a LC consisting of SEQ ID NO:55.

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

[0099] In certain aspects disclosed herein, the Ab comprises amino acids 2-450 of SEQ ID NO:74, the LC comprises amino acids 2-216 of SEQ ID NO:75, and n is about 8. In certain aspects disclosed herein, the Ab comprises amino acids 2-450 of SEQ ID NO:74, the LC comprises amino acids 2-216 of SEQ ID NO:75, and n is about 4. In further aspects disclosed herein, 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 Methods for conjugating the antibody disclosed herein to the payload disclosed herein are known in the art. In some methods, the antibody is conjugated to the linker in a first reaction, and then the antibody and linker are conjugated to the payload in a second reaction. In some methods, the antibody is conjugated to the payload or payload / linker in one reaction.

[0101] In some embodiments of the present disclosure, the Nectin-4 antibody described herein is covalently linked to the camptothecin analog described herein via the thiol group of one or more cysteine ​​residues located on the Nectin-4 antibody. In further embodiments, the cysteine ​​residues used in the conjugate are each interchain disulfide cysteine ​​residues. Methods are known in the art for controlling the reduction of interchain disulfides to allow conjugation to the involved cysteines. In other embodiments, the cysteine ​​residues used in the conjugate are engineered into the antibody apart from those used for the interchain disulfides.

[0102] In other aspects of the present disclosure, the Nectin-4 antibody described herein is covalently linked to the camptothecin analog described herein via the amino group of one or more lysine residues located on the Nectin-4 antibody.

[0103] In certain aspects, provided herein are ADCs wherein the attachment of the cytotoxic agent, cytotoxic agent-self-immolative spacer, or cytotoxic agent-self-immolative spacer-linker to the antibody occurs via a thiol group on one or more cysteines of the antibody. In further aspects, each of the one or more cysteines is a naturally occurring 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 represented by n, which is the number of drug molecules per antibody (also known as drug-to-antibody ratio or DAR). Depending on the context, the subscript n represents either the number of drug molecules bound to an individual antibody molecule (and thus is an integer value) or the average drug loading (and thus can be an integer or non-integer value). The average drug loading represents the average number of drug-linker molecules per antibody in the composition.

[0105] A higher DAR can generate more potent ADCs, but a higher DAR can also result in destabilization, aggregation, increased off-target toxicity, and enhanced drug clearance from the systemic circulation.

[0106] In embodiments of the present disclosure, the average drug loading, when referring to a composition comprising a population of ADCs, is about 1 to about 16, about 2 to about 12, or about 2 to about 10. In a further embodiment, the DAR is 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 a preparation can be characterized by conventional means known in the art using techniques such as mass spectrometry, HIC, ELISA assay, or HPLC.

[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 produce a reduced Nectin-4 antibody; (b) contacting the reduced Nectin-4 antibody with a compound of the present disclosure to produce a conjugate, wherein the compound comprises one or more of Formulae 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 easily prepared by a variety of procedures known to those skilled in the art, some of which are illustrated in the following preparations and examples. Those skilled in the art will recognize that the specific synthetic steps for each of the described routes can be combined in different ways or combined with steps from different schemes to prepare the conjugates of the present disclosure or salts thereof. The products of each step can be recovered by conventional methods well known in the art, including extraction, evaporation, precipitation, chromatography, filtration, trituration, and crystallization. All substituents are as previously defined unless otherwise indicated. 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 should in no way be construed as limiting the scope of the present disclosure.

[0109] therapeutic use In another aspect, provided herein is a method of treating cancer comprising administering to a patient in need of cancer treatment an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein. In a further aspect, provided herein is a method of treating cancer comprising administering to a patient in need of cancer treatment an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In a further aspect, the ADC or pharmaceutical composition described herein is administered in a perioperative, adjuvant, or neoadjuvant setting.

[0110] In a further aspect, a method of treating cancer is provided, wherein the cancer is bladder cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is urothelial cancer. In a further aspect, the urothelial cancer is metastatic urothelial cancer (muC). In a further aspect, a method of treating cancer is provided, wherein the cancer is breast cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is lung cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is gastric cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is colorectal 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 a further aspect, a method of treating cancer is provided, wherein the cancer is ovarian cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is cervical cancer. In a further aspect, a method of treating cancer is provided, wherein the cancer is prostate cancer.

[0111] In a further aspect, the patient has relapsed after receiving enfortumab vedotin or the patient has become refractory to enfortumab vedotin or standard of care. In a further aspect, the patient being treated with an ADC or pharmaceutical composition described herein is ineligible for treatment with enfortumab vedotin. In a further aspect, the patient being treated with an ADC or pharmaceutical composition described herein is naïve to treatment with enfortumab vedotin. In a further aspect, the patient being treated with an ADC or pharmaceutical composition described herein has previously received a programmed death receptor-1 (PD-1) or programmed death ligand 1 (PD-L1) inhibitor and a platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced or metastatic setting. In a further aspect, a patient being treated with an ADC or pharmaceutical composition described herein has become refractory or has relapsed to a combination of enfortumab and pembrolizumab, or a combination of nivolumab and ipilimumab, or a combination of atezolizumab and cisplatin / gemcitabine. In a further aspect, a patient being treated with an ADC or pharmaceutical composition described herein has become refractory or has relapsed to a combination of cisplatin or carboplatin and gemcitabine. In a further aspect, a patient being treated with an ADC or pharmaceutical composition described herein in combination with a PD-1 inhibitor or a PD-L1 inhibitor is ineligible for treatment with a cisplatin-containing chemotherapy.

[0112] In a further aspect, a method is provided that comprises administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with one or more anti-tumor agents.In a further aspect, a method is provided that comprises administering an effective amount of an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor.

[0113] In another embodiment, a method is provided herein that includes administering an effective amount of a Nectin-4 ADC or pharmaceutical composition described herein in combination with an FGFR compound, either simultaneously, separately, or sequentially. In a further embodiment, the cancer is urothelial carcinoma. In a further embodiment, the FGFR compound is erdafitinib, LOXO-435, futibatinib, bofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib.

[0114] In another aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in therapy. In a further aspect, provided is an ADC or pharmaceutical composition described herein for use in the treatment of cancer. In a further aspect, the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In a further aspect, the ADC or pharmaceutical composition described herein is for use in a perioperative, adjuvant, or neoadjuvant setting.

[0115] In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating bladder cancer. In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating urothelial cancer. In a further aspect, the urothelial cancer is metastatic urothelial carcinoma (muC). In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating breast cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating lung cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating gastric cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating colorectal cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating pancreatic cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating head and neck cancer. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of ovarian cancer.In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of cervical cancer.In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of prostate cancer.

[0116] In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating cancer, wherein the cancer has relapsed after treatment with enfortumab vedotin, or the cancer has become refractory to enfortumab vedotin. In a further aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein for use in treating cancer, wherein the cancer has relapsed after treatment with enfortumab vedotin, or the cancer has become refractory to standard of care. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating cancer, wherein prior use of enfortumab vedotin was contraindicated. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in treating cancer, wherein prior use of enfortumab vedotin has not been performed. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein for use in the treatment of cancer where prior use of a PD-1 or PD-L1 inhibitor and a platinum-containing chemotherapy has occurred, or where prior use of enfortumab in combination with pembrolizumab, nivolumab in combination with ipilimumab, or atezolizumab in combination with cisplatin / gemcitabine has occurred, in a neoadjuvant / adjuvant, locally advanced, or metastatic setting. In a further aspect, provided herein is an ADC or pharmaceutical composition described herein in simultaneous, separate, or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor for use in the treatment of cancer, where the cancer cannot be treated with a cisplatin-containing chemotherapy.

[0117] In a further aspect, provided herein is an ADC or pharmaceutical composition described herein in simultaneous, separate or sequential combination with one or more anti-tumor agents for use in the treatment of cancer. In a further aspect, the anti-tumor agent is a PD-1 inhibitor or a PD-L1 inhibitor.

[0118] In another aspect, provided herein is a Nectin-4 ADC or pharmaceutical composition described herein, combined simultaneously, separately, or sequentially with an FGFR compound for use in treating cancer. In a further aspect, the cancer is urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib.

[0119] In another aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein the cancer is bladder cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer.

[0120] In a further aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where the cancer has relapsed after treatment with enfortumab vedotin or the cancer has become refractory to enfortumab vedotin or standard of care. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where prior use of enfortumab vedotin was contraindicated. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where prior use of enfortumab vedotin has not occurred. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where prior use of a PD-1 or PD-L1 inhibitor and a platinum-containing chemotherapy in the neoadjuvant / adjuvant, locally advanced or metastatic setting. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where the cancer is not treatable with cisplatin-containing chemotherapy, and where such medicament is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0121] In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where such medicament is administered simultaneously, separately, or sequentially with one or more anti-tumor agents. In a further aspect, provided herein is the use of an ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, where such medicament is administered simultaneously, separately, or sequentially with a PD-1 inhibitor or a PD-L1 inhibitor.

[0122] In another aspect, provided herein is the use of a Nectin-4 ADC or pharmaceutical composition described herein in the manufacture of a medicament for the treatment of cancer, wherein such medicament is administered simultaneously, separately, or sequentially with an FGFR compound. In a further aspect, the cancer is urothelial carcinoma. In a further aspect, the FGFR compound is erdafitinib, LOXO-435, futibatinib, vofatamab, bemarituzumab, derazantinib, infigratinib, pemigatinib, rogaratinib, FGF401, or pemigatinib.

[0123] In a further aspect, the bladder cancer is urothelial carcinoma, squamous cell carcinoma, or adenocarcinoma. In a further aspect, 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. In a further aspect, the 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 carcinoma or lobular carcinoma. In a further aspect, the lung cancer is squamous non-small cell lung cancer (NSCLC) or non-squamous NSCLC. In a further aspect, the lung cancer is squamous cell carcinoma, adenocarcinoma, or small cell carcinoma. In a further aspect, the prostate cancer is metastatic castration-resistant prostate cancer. 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. In a further aspect, the ovarian cancer is fallopian tube cancer or peritoneal cancer.

[0124] In further embodiments, the antitumor agent may be a chemotherapy treatment agent, including platinum-containing chemotherapy, and / or may include cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-bound particle injection suspension, ixabepilone, capecitabine, FOLFOX (leucovorin, fluorouracil, and oxaliplatin), FOLFIRI (leucovorin, fluorouracil, and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed, methotrexate, vinblastine, and cetuximab. In further embodiments, the chemotherapy agent is a combination of cisplatin (or carboplatin) and gemcitabine, including for bladder cancer. In a further aspect, the anti-tumor agent may be a tumor immunotherapeutic agent, including those selected from the group consisting of nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat, and durvalumab.

[0125] Pharmaceutical Compositions and Methods of Administration The antibodies or ADCs described herein can be formulated as pharmaceutical compositions, which are administered by any route that renders the antibodies or ADCs bioavailable, such as, for example, oral, topical, or subcutaneous administration.

[0126] Also provided herein is a pharmaceutical composition comprising an antibody or ADC provided herein and one or more agents selected from the group consisting of physiologically acceptable carriers, diluents, excipients, and adjuvants.

[0127] The antibody or ADC of the present disclosure, or a pharmaceutical composition comprising the same, may be administered by parenteral routes (e.g., subcutaneous and intravenous). The antibody or ADC of the present disclosure may be administered alone to a patient in a single dose or multiple doses together with a pharma- ceutically acceptable carrier, diluent, or excipient. The pharmaceutical compositions described herein can be prepared by methods well known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22nd ed. (2012), A. Loyd et al., Pharmaceutical Press), and comprise the antibody or ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0128] Disclosed herein in certain aspects are pharmaceutical compositions comprising an antibody disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients. Disclosed herein in certain aspects are pharmaceutical compositions comprising an ADC disclosed herein and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0129] definition As used herein, the terms "a," "an," "the," and similar terms as used in the context of this disclosure (particularly in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise specified herein or clearly contradicted by context.

[0130] The term "bind," as used herein, unless otherwise specified, is intended to mean the ability of a protein or molecule to form a chemical bond or attractive interaction with another protein or molecule, resulting in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0131] As used herein, the term "effective amount" refers to the amount (duration and means of administration) necessary to achieve the desired therapeutic result. The effective amount of a protein or conjugate may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the protein or conjugate to induce a desired response in the individual. An effective amount is also an amount in which any toxic or harmful effects of the protein or conjugate are outweighed by the therapeutically beneficial effects.

[0132] As used herein, the terms "treat," "treatment," or "treating" refer to any process that may slow, control, retard, or halt the progression of a disorder or disease disclosed herein, or that may ameliorate the symptoms of a disorder or disease, but does not necessarily indicate the complete disappearance of all symptoms of the disorder or disease.

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

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

[0135] Example 1: Generation of Nectin-4 antibody The amino acid sequences of the CDRs, variable regions, complete heavy and light chains, and the nucleotide sequences encoding them, of Antibodies 1-8 are listed below in the section entitled "Amino Acid and Nucleotide Sequences." In addition, SEQ ID NOs for the CDRs, light chain, heavy chain, light chain variable region, and heavy chain variable region of Antibodies 1-8 are provided in Tables 1 and 2.

[0136] Anti-Nectin-4 antibodies of the present disclosure, including but not limited to antibodies 1-8, can be expressed and purified essentially as follows: Antibodies are expressed in suitable host cells, such as HEK293 or CHO, either transiently or stably transfected with an expression system for secreting the antibody using an optimal pre-defined HC:LC vector ratio, or with a single vector system encoding both the HC and LC. Expression plasmids contain cDNA versions of the antibody's LC and HC genes (e.g., as shown in Table 3) and are expressed from constructs commonly used and suitable for this purpose, such as those based on the human cytomegalovirus major immediate early promoter.

[0137] The medium into which the antibody of the present disclosure is secreted can be purified by conventional techniques, such as mixed-mode methods of ion exchange and hydrophobic interaction chromatography. For example, the medium can be applied to and eluted from a Protein A or Protein G column using conventional methods, and mixed-mode methods of ion exchange and hydrophobic interaction chromatography can also be used. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product can be immediately frozen, refrigerated, or lyophilized, for example at -70°C. Various methods of protein purification can be employed, which are known in the art and described, for example, in 30 Deutscher, Methods in Enzymology 182:83-89 (1990), and Scopes, Protein Purification: Principles and Practice, 3rd Edition, Springer, NY (1994). The antibodies may be immediately frozen at -70°C or stored at 2-8°C for several months, or lyophilized, 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. Essentially as provided in ACS Med. Chem. Lett. 2019, 10, 1386-1392 and WO2020219287, camptothecin analogs of the present disclosure, such as A1, can be synthesized as follows.

[0142] [ka]

[0143] Step 1: To a flask containing anhydrous 1,2-dichloroethane (50 mL), add 1M BCl in DCM. 3 (9.95 mL, 9.95 mmol) was added, followed by ice H 2 The mixture was cooled to 0 °C in an O bath. 3-Fluoro-4-methylaniline 1 (1.56 g, 12.4 mmol) was added in portions and then stirred at 0 °C for 10 min, after which 5-bromovaleronitrile 2 (1.72 mL, 14.9 mmol) was added, followed by AlCl 3 (2.16 g, 16.2 mmol) was added. The ice bath was removed and the reaction solution was allowed to warm gradually to room temperature. After stirring at room temperature for 10 min, the mixture was heated to reflux for 39 h. The solution was cooled to room temperature and diluted with cold H 2 25 mL of HO was slowly added followed by 5% aqueous HCl. After 30 min, the solution was diluted with DCM (50 mL). The organic layer was 2 Wash with anhydrous NaCl and brine. 2 SO 4The mixture was dried over 1000 ml of ethyl acetate and then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by reverse phase chromatography using the following conditions: Column: C18 (100 g), H 2 Elute with 25% ACN in O for 5 min, then H 2 25%–95% ACN in O for 15 min, followed by H 2 Switching to a gradient of 95% ACN in O over 5 min gave compound 3 as an off-white solid (1.42 g, 40%).

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

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

[0146] Synthesis scheme of linker + self-immolative unit + camptothecin analogue As essentially provided in ACS Med. Chem. Lett. 2019, 10, 1386-1392 and WO2020219287, the camptothecin analogs of the present disclosure can be conjugated to a linker (using a maleimide compound) and a self-immolative unit as follows:

[0147] [ka]

[0148] Step 4: A mixture of Fmoc-GGFG (1 g, 1.59 mmol, 1.5 equiv), A1 (0.48 mg, 1.06 mmol, 1 equiv) and 4 Å molecular sieves (3 g, 3×wt of A1) in NMP was treated with HCl in 1,4-dioxane (4 M, 4.5 equiv) at 20° C. The reaction was quenched by adding HO. The solids were removed by filtration and the filtrate was extracted with EtOAc. The organic layer was washed with Na 2 SO 4 The mixture was dried over 1000 ml of ethyl acetate, filtered and concentrated under reduced pressure. The residue was purified by normal phase chromatography (SiO 2 , 0-10% MeOH in DCM) to give Fmoc-GGFF-A1 (0.75 g, yield 69%).

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

[0150] Step 6: The crude mixture of GGFG-A1 (0.73 mmol, 1 equiv) and TsOH in DMF was basified to pH 7 with NMM at 0° C., followed by the addition of additional NMM (0.16 mL, 1.47 mmol, 2 equiv) and compound 6 (0.283 g, 0.917 mmol, 1.25 equiv). The resulting mixture was stirred at 0° C. for 4-6 h, then quenched and purified by normal phase chromatography (SiO2, 0-10% MeOH in DCM) to give mc-GGFG-A1 (0.51 g, 70% yield).

[0151] Camptothecin analogs of the present disclosure can be conjugated to a linker (using a bromoacetamide compound) and a self-immolative unit as follows:

[0152] [ka]

[0153] Step 1: To a solution of Fmoc-GGFG-A1 (0.5 g, 0.489 mmol) in DMF (5 mL) was added Et 2 NH (0.025 g, 0.34 mmol) was added. The mixture was stirred at 15-25 °C for 1 h. The reaction was then quenched by the addition of THF and then concentrated under reduced pressure. The residue was purified by reversed phase chromatography using the following conditions: Column, C18, 150 × 30 mm × 5 μm, H 2 Elution with 22–45% ACN in O (0.225% FA) afforded GGF-A1 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 was added compound 8 (2.9 g, 12.29 mmol) at 0° C. The reaction mixture was allowed to warm to 20° C. Stirring was continued at 20° C. for 18 h, after which additional compound 8 (0.5 g, 3.06 mmol) was added. After stirring at 20° C. for an additional 4 h, the reaction was concentrated under reduced pressure. The residue was purified by reverse phase chromatography. Column: C18 column, 150×30 mm×5 μm, H 2Elution with 22–45% ACN in O (0.225% FA) afforded compound 9 (1.15 g, 33% yield).

[0155] Step 3: A mixture of compound 9 (430 mg, 1.51 mmol) and DMTMMT (340 mg, 1.04 mmol) in DMAC (11 mL) was cooled to 0° C. A mixture of GGFG-A1 (0.500 g, 0.63 mmol) and DIPEA (0.090 g, 0.70 mmol) in DMAC (4 mL) was added dropwise. Stirring was continued at 0° C. for 0.5 h, followed by the addition of DCM (300 mL). The mixture was washed with 10% aqueous NaBr (3×50 mL). The organic layer was extracted with Na 2 SO 4 The mixture was dried at 40° C., filtered and concentrated under reduced pressure at 0-10° C. The residue was purified by reverse phase chromatography. Column: C18 column, 150×30 mm×5 μm, H 2 Elution with 15–45% ACN in O (0.225% FA) gave Br-GGFG-A1 (0.23 g, 34.7% yield).

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

[0157] Part II. Preparation of ADCs with bromoacetyl linker-payloads To prepare antibody-drug conjugates with eight drugs per antibody, IgG1 antibodies are fully reduced using 6-8 molar equivalents of a reducing reagent such as DTT or TCEP for 2 hours at 37°C. The reduced antibodies are then buffer exchanged using a PD-10 desalting column with 50 mM HEPES (pH 7.4) containing 2 mM EDTA, and the eluate is adjusted to a protein concentration of 5-10 mg / mL with HEPES buffer. An excess of linker payload (e.g., 12 molar equivalents) is added over 2-3 hours, and the conjugation reaction may be stopped by adding a substantial excess of L-cysteine ​​(e.g., 10 molar equivalents). The resulting mixture of ADCs may be purified on a PD-10 desalting column equilibrated with 25 mM histidine, 9% sucrose (pH 5.5), followed by three centrifugation cycles using a 30 kDa MWCO centrifuge unit to remove unreacted linker-payload related species. Finally, the resulting ADC can be sterile filtered through a 0.2 μM filter and stored at 4° C. or −80° C. for future 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 A Biacore 8K+ instrument (Cytiva, Marlborough, Mass.) was used to determine the kinetic and affinity parameters of the binding interactions of Nectin-4 ADCs (exemplified herein by each of antibodies 1-8 conjugated as in Formula XI, with a DAR of 8) with recombinant HIS-tagged human (Acro Biosystems, Catalog No. NE4-H52H3, Newark, Del.), cynomolgus monkey (Acro Biosystems, Catalog No. NE4-C52H4), and rat (R&D Systems, Minneapolis, Minn., Catalog No. 9997-N4-050).

[0159] An anti-human Fc sensor surface was prepared by amine coupling of goat anti-human IgG Fc (Southern Biotech, catalog number 2014-01, Birmingham, AL) to a Biacore Series S CM4 (Cytiva, catalog number BR-100534) sensor surface at 25 °C. A running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% Tween-20, pH 7.4 was used for immobilization. All eight channels of flow cells 1 and 2 were activated with a 1:1 (v / v) mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 100 nM N-hydroxysuccinimide (NHS) at a flow rate of 10 μL / min for 7 min. Anti-human IgG Fc capture reagent was then bound to the sensor surface (diluted to 50 μg / mL in 10 mM acetate pH 4.5 buffer) by injecting all flow cells and channels at a flow rate of 10 μL / min for 7 min. Remaining active groups were blocked by injecting 100 mM ethylenediamine (in 200 mM borate buffer, pH 8.5) at a flow rate of 10 μL / min for 7 min. All channels and flow cells were then preconditioned by injecting 75 mM phosphoric acid three times consecutively for 1 min at 10 μL / min.

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

[0161] In each analysis cycle, a different ADC was captured on flow cell 2 of each channel by injecting at 5 μg / mL and 10 μL / min. After capture, the same analyte was injected on flow cells 1 and 2 of all eight channels at 30 μL / min for 2 min and dissociation was monitored for 10 min. After dissociation, all surfaces were regenerated by injecting 75 mM phosphoric acid three times at 10 μL / min for 1 min. The ADC capture and analyte cycles were repeated to obtain analyte binding for each ADC at concentrations of 0, 2.5, 7.4, 22, 67, 200 and 600 nM Nectin-4.

[0162] The sensorgram data were globally fitted using the default 1:1 binding model in Biacore Insight Evaluation Software v3.0.12.15655. 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 and the results fit poorly to a 1:1 binding model. 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 (Calculated using the ELISA kit), where n is the number of replicates. For n=1 or 2, replicates are shown. For n=3, the mean ± standard deviation is shown.

[0164] Cell surface binding of Nectin-4 antibodies in cell lines expressing the Nectin-4 receptor A panel of nine Nectin-4 antibodies was tested for cell surface binding to two Nectin-4 expressing tumor cell lines. Cell lines were selected to exhibit high and low receptor density, with SUM190PT tumor cells exhibiting high endogenous expression and NCI-H1781 tumor cells exhibiting lower endogenous expression. SUM190PT and NCI H1781 cells were determined to have antibody binding capacities of 108,000 and 20,000, respectively (using MESF quantification kit, Bangs Laboratories). T24 parental cells were selected as a Nectin-4 negative cell line. Antibody binding was quantified by flow cytometry and the EC of the binding curves were calculated. 50 Both the mean binding MFI and the maximum binding MFI for each antibody were recorded.

[0165] Cells were dissociated using non-enzymatic dissociation buffer for 5 min at 37°C. Cells were counted and plated at 10 5Cells / well were dispensed. Cells were centrifuged at 1800 rpm×5 min and the supernatant was discarded. An 11-point antibody dilution series was prepared starting at 300 nM and diluted 1:4 in assay buffer (1×PBS containing 1% BSA and 0.09% sodium azide). The dilution series was added to the cells at 100 μL / well and mixed by pipetting. Several untreated control wells / cell lines were prepared with assay buffer only. Cells and antibodies were incubated for 1 hour at 4° C. on an orbital shaker. After incubation, the assay plate was centrifuged and washed twice with 300 μL / well of assay buffer. The cell pellet was then stained with a 1:500 dilution (100 μL / well) of Alexa647-conjugated mouse anti-human IgG secondary antibody in assay buffer. The assay plate was incubated with shaking at 4° C. in the dark for 1 hour. After incubation, the plates were centrifuged and the cells were washed twice with 300 μL / well of assay buffer. A 1:5000 solution of Zombie Green live / dead marker (BioLegend) in 1×PBS was dispensed onto the cells at 100 μL / well and the plates were incubated at 4° C. in the dark with shaking for 10 min. The cell pellets were then washed once with assay buffer and fixed with 200 μL / well of 4% paraformaldehyde in 1×PBS for 15 min at room temperature in the dark. The cells were centrifuged and washed once before being resuspended in 65 μL of assay buffer and acquired on a Sartorious iQue HTFC Cytometer.

[0166] Cells were acquired on a Sartorious iQue HTFC Cytometer and FCS files were generated using ForeCyt standard edition (v.6.2.6652). FCS files were then analyzed with FlowJo (v10.8.1). Debris was excluded from the analysis by forward scatter (FSC) versus side scatter (SSC) gating and single cells were selected by forward scatter area (FSC-A) versus height (FSC-H) gating. Finally, dead cells staining positive with Zombie Green were excluded and the MFI of Alexa647 positive live cells was quantified. Mean unstained cell autofluorescence was subtracted from all samples. Data were graphed and analyzed with GraphPad Prism (v9.5.1). EC 50 was determined via agonist vs. response-variable slope (4 parameter) curve fitting and % maximum binding of enfortumab was calculated by setting the average of maximum enfortumab MFI in each cell line to 100%.

[0167] The nine Nectin-4 antibodies did not bind to the Nectin-4 negative T24 parental line. As shown in Table 5, the antibodies showed good binding to both high and low Nectin-4 expressing tumor cells.

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

[0169] Characterization of Nectin-4 antibody and ADC binding to normal human epidermal keratinocytes using flow cytometry To characterize the binding of the disclosed Nectin-4 antibodies and ADCs to normal human epidermal keratinocytes, HEKα cells were plated at 1 million cells per well in complete culture medium (Dermal Cell Basal Medium+Keratinocytes Growth Kit, ATCC) in 150×25 mm tissue culture dishes (Corning Inc.) and became confluent within 24 hours. The differentiation process was allowed to proceed for an additional 5–10 days. After induction of differentiation, cells were dissociated at 37°C using 2 mg / mL Clostridium histolyticum collagenase, Type XI (Sigma-Aldrich). Cells were counted and plated at 10 5 1000μL / well of cells / well were dispensed. Antibodies and ADCs were added to cells in serial dilutions of 1:4 starting at 300nM in assay buffer (1xDPBS containing 2% FBS, Gibco). Cells were incubated for 1h at 4°C on a microplate shaker in the dark. After incubation, cells were washed twice with 200μL / well of assay buffer and stained with 1:1,000 diluted Alexa Fluor 647-conjugated AffiniPure F(ab')2 fragment goat anti-human IgG(H+L) (Jackson ImmunoResearch). After 1h incubation at 4°C, cells were washed twice with 200μL / well of assay buffer and stained with 1:2000 diluted Zombie Green fixable viability dye (BioLegend). Cells were incubated for 20min at 4°C on a microplate shaker. Cells were washed with assay buffer and fixed with 200 μL / well of BD Cytofix fixation buffer for 20 min at 4° C. Cells are resuspended in assay buffer for acquisition on an Attune CytPix flow cytometer (Thermo Fisher Scientific). Cells were determined to have an antibody binding capacity of 17,000 (using the MESF quantification kit, Bangs Laboratories).

[0170] As shown in Table 6, the tested Nectin-4 antibodies and ADCs (conjugated as per Formula XI, with a DAR of 8) showed similar or higher EC values ​​than Enfortumab and Enfortumab ADC (exemplified herein with each Ab conjugated as per Formula XI, with a DAR of 8). 50 Ab1 binds to normal human epidermal keratinocytes at 100-fold higher than enfortumab. Ab1 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 Characterization of the internalization ability of Nectin-4 antibodies and ADCs in human Nectin-4 positive cells using fluorescence imaging The T24 cell line was engineered to express human nectin-4-eGFP and clonally selected for high expression. T24-human nectin-4-eGFP clone 3 was determined to have an antibody binding capacity of 379,000 (using MESF quantification kit, Bangs Laboratories). T24 human nectin-4-eGFP clone 3 cells were seeded at 12,000 cells per well in black clear bottom CellCarrier Ultra 384-well microplates (Perkin Elmer) in complete culture medium (modified McCoy's 5A + 10% FBS + Glutamax + 400 μg / mL G418 + Pen / Strep). Plates were covered with AeraSeal™ sealing films and incubated at 37°C, 5% CO 2 The plates were incubated overnight at 37°C for 24 hours. Antibodies and ADCs were added to the cells the next day at a 1:3 dilution starting at 300 nM. Plates were covered with AeraSeal™ and placed in an incubator for imaging on a PerkinElmer Opera Phenix Screening System for 24 hours. Data was processed and analyzed in Harmony and Microsoft Excel, and graphed in GraphPad Prism.

[0173] As shown in Table 7, Nectin-4 antibodies and Nectin-4 ADCs (exemplified herein by antibodies 1-8, respectively, conjugated as in Formula XI, with a DAR of 8) induce degradation of the Nectin-4-eGFP signal. As shown in Table 7, the Nectin-4 ADCs tested had equal or greater internalization capacity than the enfortumab antibody in the same ADC format as antibodies 1-8.

[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 NCI-H1781 cells, a low expressing cell line, were seeded in white clear bottom 96-well tissue culture plates in culture medium (RPMI 1640 + 1x GlutaMAX + 10% heat inactivated fetal bovine serum + 1 mM sodium pyruvate). The cells were incubated at 37°C, 5% CO 2 The plates were then incubated at 37° C. for 24 hours at 4° C. for 30 minutes. The following day, ADCs were added at 1:3 serial dilutions in culture medium, starting from a final working concentration of 100 nM. The plates were covered with Breathe-Easy® sealing membrane and incubated at 37° C. for 30 minutes at 4° C. for 30 minutes at 4° C. 2 Plates were incubated with 0.5% ethanol at 37 °C for 10 min. After 5 days of treatment, plates were read using the CellTiter-Glo Luminescent Cell Viability Assay. 100 μL / well of CellTiter-Glo reagent was incubated in the plates for 10 min at room temperature. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percent cell death was calculated relative to 0% untreated. Data was graphed and analyzed using GraphPad Prism version 9.5.1. IC 50was determined by log(inhibitor) versus response-variable slope (4 parameter) curve fitting.

[0176] The T24 cell line was engineered to express human nectin-4 and clonally selected. A high expressing clonal cell line, T24-human nectin-4 clone 108, was seeded in culture medium (McCoy's 5A+1×GlutaMAX+10% heat inactivated fetal bovine serum+400 μg / mL G418). ADCs were added for 5 days at a final working concentration of 200 nM, serially diluted 1:4 in culture medium.

[0177] T24-human nectin-4 clone 108 and NCI H1781 cells were determined to have antibody binding capacities of 56,000 and 20,000, respectively (using the MESF quantification kit, Bangs Laboratories).

[0178] As shown in Table 8, selected exemplary Nectin-4 ADCs (exemplified herein with each of antibodies 1-8 conjugated as in Formula XI with a DAR of 8) showed similar potent maximal cell killing in Nectin-4 cell lines with differential expression levels as the Enfortumab antibody in the same ADC format as antibodies 1-8 (Enfortumab with an effector null mutation, conjugated to Formula XI with a DAR of 8). IC 50 The values ​​suggest similar potency across ADCs tested in T24-human nectin-4 clone 108 cells and variable potency in ADCs tested in NCI H1781 cells. No nonspecific cytotoxicity was observed in T24 nectin-4 negative cells tested.

[0179] [Table 10]

[0180] Characterization of cytotoxicity of Nectin-4 ADC in MMAE-resistant cell lines Two Nectin-4 ADCs described herein and Enfortumab Vedotin were tested for activity in T24 Nectin-4 cells, which are resistant to MMAE. T24-hNectin-4 clone 14 cells were seeded at 500 cells per well in white clear-bottom 96-well tissue culture plates in 100 μL of culture medium and incubated at 37° C., 5% CO 2 The plates were incubated at 37° C., 5% CO. The following day, each ADC was serially diluted 1:4 in culture medium from a starting concentration of 400 nM. 100 μL of each ADC dilution was added per well, the plates were covered with Breathe-Easy® sealing membrane and incubated at 37° C., 5% CO. 2 After 5 days of treatment, plates were removed from the incubator and placed at room temperature for 15 minutes, and 100 μL of medium per well was removed. 100 μL of CellTiter-Glo reagent per well was added, and plates were incubated at room temperature for 10 minutes. RLU (relative luminescence units) were obtained by SpectraMax M5e using SoftMax Pro 5.4. Percentage of cell death was calculated as 0% for DMSO only (free payload) or no treatment (ADC). Data were graphed and analyzed using Graphpad Prism version 9.5.1. IC50 was determined by log(inhibitor) vs. response-variable slope (4 parameter) curve fitting.

[0181] ADCs made with Ab1 and Ab2 (conjugated with Formula XI, with a DAR of 8) retained activity against MMAE-resistant cells, whereas enfortumab vedotin lost efficacy.

[0182] Bystander activity of enfortumab with exemplified linkers / payloads compared to enfortumab vedotin The UMUC3 cell line was engineered to express human nectin-4, and two clonal populations with different expression levels were selected. In a white clear-bottom 96-well plate, UMUC3-hNectin-4 clone F7 / UMUC3-Luc-GFP cells were mixed and seeded at a total of 1500 cells / well / 100 μL in a 4:1 ratio in assay medium (MEM+1×GlutaMAX+10% heat-inactivated fetal bovine serum+1 mM sodium pyruvate). UMUC3-hNectin-4 clone E3 / UMUC3-Luc-GFP cells were mixed and seeded at a total of 2100 cells / well / 100 μL in assay medium in a 6:1 ratio. UMUC3-Luc-GFP cells were negative for nectin-4 expression. Plates were incubated at 37°C, 5% CO 2 The plates were incubated overnight at 37° C. The next day, ADCs were added in 1:3 serial dilutions in assay medium from a final working concentration of 100 nM. To assess the sensitivity of cell lines to free payload, free payload was added in 1:3 serial dilutions in assay medium from a final working concentration of 200 nM. Plates were covered with Breathe-Easy® sealing membranes and incubated at 37° C., 5% CO 2 After 5 days of treatment, plates were read using the ONE-Glo™ Luciferase Assay System. 100 μL / well of ONE-Glo™ Assay Reagent was incubated in the plate for 10 minutes at room temperature. Luminescence was read on a SpectraMax M5e. RLU (Relative Luminescence Units) were obtained using SoftMax Pro 5.4. Percentage of UMUC3-Luc-GFP death was calculated relative to 0% untreated. Data was graphed and analyzed using Graphpad Prism version 9.5.1. IC 50 was determined by log(inhibitor) vs. response (3-parameter) curve fitting for bystander activity and log(inhibitor) vs. response (4-parameter) curve fitting for free payload cytotoxicity.

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

[0184] As shown in Table 9, enfortumab conjugated as Formula XI with a DAR of 8 showed a more potent bystander effect on UMUC3-Luc-GFP cells compared to enfortumab vedotin. Notably, Table 10 shows that UMUC3-Luc-GFP cell lines were similarly sensitive to free payload Formula XI and MMAE. Higher expression on positive cell lines results in higher bystander activity with enfortumab vedotin, but its potency remains lower than enfortumab conjugated as Formula XI.

[0185] [Table 11]

[0186] [Table 12]

[0187] In an experiment performed essentially as described herein, the UMUC3-hNectin-4 clone F7 / UMUC3-luciferase-GFP cell pair was used to evaluate the bystander effect of the four Nectin-4 ADCs described herein and Enfortumab vedotin. 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 ADCs, luciferase luminescence was monitored using the ONE-Glo™ Luciferase Assay System. ADCs made with antibodies 1, 1a, 2, and 7 (conjugated with Formula XI, with a DAR of 8) showed strong bystander effects, while Enfortumab vedotin showed a much weaker bystander effect.

[0188] Characterization of the bystander effect of nectin-4 ADC in the cell line T24 nectin-4 negative cells The T24 cell line was engineered to express mScarlet and clonally selected for testing in the bystander assay. T24-mScarlet clone 5 and T24-human nectin4 clone 108 were mixed and plated in a clear flat-bottom 96-well plate at a 9:1 ratio in culture medium (McCoy's 5A + 1x GlutaMAX + 10% heat-inactivated fetal bovine serum + 400 μg / mL G418) for a total of 1000 cells / well / 100 μL. T24 mScarlet clone 5 cells were negative for nectin-4 expression. Plates were incubated at 37°C, 5% CO 2 The plates were incubated overnight at 4°C for 12 h. The following day, ADCs were added at a 1:4 serial dilution in culture medium from a final working concentration of 50 nM. Plates were covered with a Breathe-Easy® sealing membrane, placed in a BioSpa, and scanned daily for 5 days on a Cytation5.

[0189] Images of T24-mScarlet cells were acquired and analyzed by Cytation5 using Gen5 Image Prime 3.11. The percentage of T24-mScarlet cell death was calculated by the reduced integrated intensity (area x mean intensity) and corrected for time 0 and no treatment. Data were graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 was determined by log(inhibitor) versus response-variable slope (4 parameter) curve fitting.

[0190] As shown in Table 11, certain Nectin-4 ADCs of the disclosure (exemplified herein by each of antibodies 1-8 conjugated as Formula XI with a DAR of 8) demonstrated similar potency and strength of bystander effect to enfortumab in the same ADC format as antibodies 1-8 (enfortumab conjugated to Formula XI with a DAR of 8) in T24 mScarlet clone 5 Nectin-4 negative cells.

[0191] [Table 13]

[0192] Characterization of the activity of Nectin-4 ADCs with other payloads and other linker compounds in low and high Nectin-4 expressing cell lines NCI-H1781 cells were seeded in white clear-bottom 96-well tissue culture plates in culture medium (RPMI 1640 + 1xGlutaMax + 10% heat-inactivated fetal bovine serum + 1 mM sodium pyruvate). Cells were incubated at 37°C, 5% CO 2 The plates were then incubated at 37° C. for 24 hours at 4° C. for 10 minutes. The following day, ADCs were added at 1:4 serial dilutions in culture medium, starting from a final working concentration of 100 nM. The plates were covered with Breathe-Easy® sealing membrane and incubated at 37° C. for 12 hours at 4° C. for 14 hours at 5% CO. 2 Plates were incubated with 0.5% ethanol at 37 °C for 10 min. After 5 days of treatment, plates were read using the CellTiter-Glo Luminescent Cell Viability Assay. 100 μL / well of CellTiter-Glo reagent was incubated in the plates for 10 min at room temperature. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percent cell death was calculated relative to 0% untreated. Data was graphed and analyzed using GraphPad Prism version 9.5.1. IC 50 was determined by log(inhibitor) versus response-variable slope (4 parameter) curve fitting.

[0193] UMUC3 cell line was engineered to express human nectin4 and clonally selected for high expression. UMUC3-human nectin4 clone F7 modified cell line was seeded in culture medium (MEM+1×GlutaMAX+10% heat-inactivated fetal bovine serum+1 mM sodium pyruvate+500 μg / mL G418). ADC was added for 6 days at a final working concentration of 100 nM, serially diluted 1:4 in culture medium.

[0194] NCI H1781 and UMUC3-hNectin4 clones F7 and cells were determined to have antibody binding capacities of 20,000 and 113,000, respectively (using the MESF quantification kit, Bangs Laboratories).

[0195] As shown in Table 12, both the Ab2 ADC conjugated as in Formula XI with a DAR of 8 and the Ab2 ADC in the PEG8-VA-exatecan, DAR8 format show potent cytotoxic effects against NCI-H1781 and UMUC3-human nectin-4 clone F7 cell lines.

[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 nectin4 and clonally selected for high expression. T24-human nectin4 clone 147 was determined to have an antibody binding capacity of 411,000 (using MESF quantification kit, Bangs Laboratories). Target cells, T24-human nectin4 clone 147, were added to clear tissue culture 96-well plates in test medium (IMDM + 1xGlutaMax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100U / mL-100μg / mL) and incubated at 37°C, 5% CO 2 The following day, 40 μL / well of antibody was added, serially diluted 1:4 in test medium from a final working concentration of 200 nM. Antibodies were incubated at 37° C. and 5% CO 2 Then, effector cells, Jurkat-Lucia NFAT-CD16, were added at 200k / 80μL / well and incubated at 37℃, 5% CO 2After 23 hours, 20 μL of supernatant and 50 μL of pre-prepared QUANTI-Luc / well were mixed in a white opaque plate. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0198] Unlike the enfortumab antibody, which has a wild-type IgG1 Fc, antibodies 1 to 8 were effector null antibodies and did not confer 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 clonally selected. Target cells, T24-human nectin4 eGFP clone 3, were seeded into clear tissue culture 96-well plates in test medium (IMDM + 1xGlutaMax + 10% heat-inactivated fetal bovine serum + Pen-Strep 100U / mL-100μg / mL) and incubated at 37°C, 5% CO 2 The following day, 40 μL / well of mAb was added, serially diluted 1:4 in test medium from a final working concentration of 200 nM, and incubated at 37°C, 5% CO 2 Then, effector cells, Jurkat-Lucia NFAT-CD32, were added at 200k / 80μL / well and incubated at 37℃, 5% CO 2 After 23 hours, 20 μL of supernatant and 50 μL of pre-prepared QUANTI-Luc / well were mixed in a white opaque plate. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4 and plotted on the Y-axis against compound concentration on the X-axis using GraphPad Prism version 9.5.1.

[0200] Unlike the enfortumab antibody, which has a wild-type IgG1 Fc, antibodies 1 to 8 were effector null antibodies and did not confer 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 plated in white clear-bottom 96-well tissue culture plates in assay medium (McCoy's 5A + 1xGlutaMax + 10% heat-inactivated fetal bovine serum) and incubated at 37°C, 5% CO 2 The next day, 50 μL / well of antibody was added, serially diluted 1:3 in assay medium, from a final working concentration of 200 nM, and incubated at 37°C, 5% CO 2 Diluted human serum complement (1:3) was then added at 50 μL / well in assay medium and incubated at 37° C., 5% CO 2 Plates were incubated at 4°C for 3 hours. Plates were read using the CellTiter-Glo Luminescent Cell Viability Assay. 100 μL / well of CellTiter-Glo reagent was incubated in the plates for 10 minutes at room temperature. Luminescence was read on a SpectraMax M5e. RLU (relative luminescence units) were obtained using SoftMax Pro 5.4. Percent cell death was calculated relative to untreated. Data was graphed and analyzed using GraphPad Prism version 9.5.1.

[0202] As a positive control, Jeko-1 cells were treated as above with anti-CD20 antibody in assay medium (RPMI1640 + 1x GlutaMAX + 10% heat-inactivated fetal bovine serum).

[0203] Similar to enfortumab, antibodies 1-8 did not show CDC capability in the nectin-4 expressing T24 cell line.Anti-CD20 control antibody resulted in CDC activity and was used as a positive control in CD20 expressing Jeko-1 cells.

[0204] Example 7: Efficacy of Nectin-4 ADC in tumor xenograft models To test the efficacy of the Nectin-4 ADC of the present disclosure with effector null mutations in the Fc region and compare it to the Enfortumab antibody in an ADC with wild-type Fc and the same payload background as that of the present disclosure, two tumor xenograft models with high or moderate Nectin-4 expression were tested as described. UM-UC-3 Nectin-4 clone F7 cells with high Nectin-4 expression were unilaterally implanted into the right flank of immunodeficient female mice (nu / nu) 5-8 weeks of age and weighing 18-20 grams. Tumors were approximately 150-250 mm 3 When tumor volume reached 100 mg / kg, animals were divided into treatment or control groups according to tumor volume and dosing was initiated (day 0, n=8 per group). Test articles were formulated in 5% dextrose and given as a single dose (2 mg / kg). Tumors were measured biweekly until day 40.

[0205] In another study, 5- to 6-week-old female NSG mice were subcutaneously injected with MDA-MB-468 cells, which have moderate Nectin-4 expression. 3 When tumor volume reached 100 mg / kg, animals were divided into treatment or control groups according to tumor volume and dosing was initiated (day 0, n=8 per group). Test articles were formulated in 5% dextrose and given as a single dose (2 mg / kg). Tumors were measured biweekly until day 71.

[0206] In both models, a 2 mg / kg single dose treatment of either a Nectin-4 ADC (exemplified herein with each of antibodies 1-8 conjugated as Formula XI with a DAR of 8) or a benchmark Enfortumab ADC (Enfortumab conjugated to Formula XI with a DAR of 8) was used. Tumor growth was measured on day 40 for UMUC3 Nectin-4 clone F7 and on day 71 for MDAMB468 xenografts. As shown in Table 13, treatment with Nectin-4 ADC using effector null antibodies 1-8 resulted in tumor growth inhibition with equal or, for certain ADCs, better efficacy than the benchmark Enfortumab ADC with a wild-type Fc region.

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

[0208] In a xenograft study performed similarly as described using UM-UC-3 Nectin-4 clone F7 cells, the Nectin-4 ADC described herein was administered in combination with cisplatin and gemcitabine at 0.5 mg / kg, 1 mg / kg, and 2 mg / kg. Increased antitumor activity was observed when SOC agents were combined with the Nectin-4 ADC at lower doses of 0.5 and 1 mg / kg. The present invention also includes the following aspects. (1) An antibody that binds to human Nectin-4, the antibody comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; a) the HCDR1 comprises SEQ ID NO: 4, the HCDR2 comprises SEQ ID NO: 5, the HCDR3 comprises SEQ ID NO: 6, the LCDR1 comprises SEQ ID NO: 7, the LCDR2 comprises SEQ ID NO: 8 and the LCDR3 comprises SEQ ID NO: 9; b) the HCDR1 comprises SEQ ID NO: 18, the HCDR2 comprises SEQ ID NO: 19, the HCDR3 comprises SEQ ID NO: 20, the LCDR1 comprises SEQ ID NO: 21, the LCDR2 comprises SEQ ID NO: 22 and the LCDR3 comprises SEQ ID NO: 23; c) the HCDR1 comprises SEQ ID NO: 28, the HCDR2 comprises SEQ ID NO: 29, the HCDR3 comprises SEQ ID NO: 30, the LCDR1 comprises SEQ ID NO: 31, the LCDR2 comprises SEQ ID NO: 32 and the LCDR3 comprises SEQ ID NO: 33; d) the HCDR1 comprises SEQ ID NO: 38, the HCDR2 comprises SEQ ID NO: 39, the HCDR3 comprises SEQ ID NO: 40, the LCDR1 comprises SEQ ID NO: 41, the LCDR2 comprises SEQ ID NO: 42 and the LCDR3 comprises SEQ ID NO: 43; e) the HCDR1 comprises SEQ ID NO: 48, the HCDR2 comprises SEQ ID NO: 49, the HCDR3 comprises SEQ ID NO: 50, the LCDR1 comprises SEQ ID NO: 41, the LCDR2 comprises SEQ ID NO: 42 and the LCDR3 comprises SEQ ID NO: 51; f) the HCDR1 comprises SEQ ID NO: 56, the HCDR2 comprises SEQ ID NO: 57, the HCDR3 comprises SEQ ID NO: 58, the LCDR1 comprises SEQ ID NO: 59, the LCDR2 comprises SEQ ID NO: 60 and the LCDR3 comprises SEQ ID NO: 61; g) the HCDR1 comprises SEQ ID NO: 66, the HCDR2 comprises SEQ ID NO: 67, the HCDR3 comprises SEQ ID NO: 68, the LCDR1 comprises SEQ ID NO: 69, the LCDR2 comprises SEQ ID NO: 70 and the LCDR3 comprises SEQ ID NO: 71, or h) An antibody, wherein the HCDR1 comprises SEQ ID NO: 76, the HCDR2 comprises SEQ ID NO: 77, the HCDR3 comprises SEQ ID NO: 78, the LCDR1 comprises SEQ ID NO: 69, the LCDR2 comprises SEQ ID NO: 70 and the LCDR3 comprises SEQ ID NO: 79. (2) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 4, the HCDR2 comprises SEQ ID NO: 5, the HCDR3 comprises SEQ ID NO: 6, the LCDR1 comprises SEQ ID NO: 7, the LCDR2 comprises SEQ ID NO: 8, and the LCDR3 comprises SEQ ID NO: 9. (3) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 18, the HCDR2 comprises SEQ ID NO: 19, the HCDR3 comprises SEQ ID NO: 20, the LCDR1 comprises SEQ ID NO: 21, the LCDR2 comprises SEQ ID NO: 22, and the LCDR3 comprises SEQ ID NO: 23. (4) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 28, the HCDR2 comprises SEQ ID NO: 29, the HCDR3 comprises SEQ ID NO: 30, the LCDR1 comprises SEQ ID NO: 31, the LCDR2 comprises SEQ ID NO: 32, and the LCDR3 comprises SEQ ID NO: 33. (5) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 38, the HCDR2 comprises SEQ ID NO: 39, the HCDR3 comprises SEQ ID NO: 40, the LCDR1 comprises SEQ ID NO: 41, the LCDR2 comprises SEQ ID NO: 42, and the LCDR3 comprises SEQ ID NO: 43. (6) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 48, the HCDR2 comprises SEQ ID NO: 49, the HCDR3 comprises SEQ ID NO: 50, the LCDR1 comprises SEQ ID NO: 41, the LCDR2 comprises SEQ ID NO: 42, and the LCDR3 comprises SEQ ID NO: 51. (7) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 56, the HCDR2 comprises SEQ ID NO: 57, the HCDR3 comprises SEQ ID NO: 58, the LCDR1 comprises SEQ ID NO: 59, the LCDR2 comprises SEQ ID NO: 60, and the LCDR3 comprises SEQ ID NO: 61. (8) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 66, the HCDR2 comprises SEQ ID NO: 67, the HCDR3 comprises SEQ ID NO: 68, the LCDR1 comprises SEQ ID NO: 69, the LCDR2 comprises SEQ ID NO: 70, and the LCDR3 comprises SEQ ID NO: 71. (9) The antibody of claim 1, wherein the HCDR1 comprises SEQ ID NO: 76, the HCDR2 comprises SEQ ID NO: 77, the HCDR3 comprises SEQ ID NO: 78, the LCDR1 comprises SEQ ID NO: 69, the LCDR2 comprises SEQ ID NO: 70, and the LCDR3 comprises SEQ ID NO: 79. (10) 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 The antibody of claim 1, wherein i) the HCVR comprises sequence number 80 and the LCVR comprises sequence number 81. (11) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 10 and an LCVR comprising SEQ ID NO: 11. (12) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 14 and an LCVR comprising SEQ ID NO: 15. (13) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO:24 and an LCVR comprising SEQ ID NO:25. (14) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 34 and an LCVR comprising SEQ ID NO: 35. (15) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 44 and an LCVR comprising SEQ ID NO: 45. (16) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO:52 and an LCVR comprising SEQ ID NO:53. (17) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 62 and an LCVR comprising SEQ ID NO: 63. (18) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 72 and an LCVR comprising SEQ ID NO: 73. (19) The antibody of claim 10, wherein the antibody comprises an HCVR comprising SEQ ID NO: 80 and an LCVR comprising SEQ ID NO: 81. (20) The antibody according to any one of claims 1 to 19, wherein the antibody has a human IgG1 or IgG4 isotype. (21) The antibody of claim 20, wherein the antibody has a human IgG1 isotype. (22) 22. The antibody of claim 21, comprising alanines at residues 234 and 235 (according to EU index numbering). (23) 23. The antibody of claim 22, further comprising a serine at position 265 (according to EU index numbering). (24) the antibody comprises a heavy chain (HC) and a light chain (LC); a) the HC comprises amino acids 2 to 444 of SEQ ID NO: 2, and the LC comprises amino acids 2 to 215 of SEQ ID NO: 3; b) the HC comprises amino acids 2 to 444 of SEQ ID NO: 12, and the LC comprises amino acids 2 to 215 of SEQ ID NO: 13; c) the HC comprises amino acids 2 to 443 of SEQ ID NO: 16, and the LC consists of SEQ ID NO: 17; d) the HC comprises amino acids 2 to 447 of SEQ ID NO: 26, and the LC consists of SEQ ID NO: 27; e) the HC comprises amino acids 2 to 446 of SEQ ID NO: 36, and the LC consists of SEQ ID NO: 37; f) the HC comprises amino acids 2 to 446 of SEQ ID NO: 46, and the LC consists of SEQ ID NO: 47; g) the HC comprises amino acids 2 to 446 of SEQ ID NO: 54, and the LC consists of SEQ ID NO: 55; h) the HC comprises amino acids 2 to 450 of SEQ ID NO: 64 and the LC comprises amino acids 2 to 216 of SEQ ID NO: 65; or i) The antibody according to claim 1, wherein the HC comprises amino acids 2 to 450 of SEQ ID NO: 74, and the LC comprises amino acids 2 to 216 of SEQ ID NO: 75. (25) a) said HC consists of SEQ ID NO:2 and said 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 25. The antibody of claim 24, wherein i) the HC consists of sequence number 74 and the LC consists of sequence number 75. (26) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 2 and the LC consists of SEQ ID NO: 3. (27) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 12 and the LC consists of SEQ ID NO: 13. (28) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 16 and the LC consists of SEQ ID NO: 17. (29) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 26 and the LC consists of SEQ ID NO: 27. (30) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 36 and the LC consists of SEQ ID NO: 37. (31) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 46 and the LC consists of SEQ ID NO: 47. (32) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 54 and the LC consists of SEQ ID NO: 55. (33) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 64 and the LC consists of SEQ ID NO: 65. (34) 26. The antibody of claim 24 or 25, wherein the HC consists of SEQ ID NO: 74 and the LC consists of SEQ ID NO: 75. (35) 35. An antibody-drug conjugate (ADC) comprising the antibody of any one of claims 1 to 34 conjugated to a cytotoxic agent. (36) 36. The ADC of claim 35, wherein the cytotoxic agent is selected from the group consisting of a microtubule inhibitor, a topoisomerase I inhibitor, a DNA damaging agent, a DNA alkylating agent, and a DNA minor groove binding agent. (37) 37. The ADC of claim 35 or 36, wherein the cytotoxic agent is a topoisomerase I inhibitor. (38) 38. The ADC of claim 37, wherein the topoisomerase I inhibitor is a camptothecin analogue. (39) The camptothecin analog comprises the formula: XY, During the ceremony, Y is of the formula: JPEG0007689606000037.jpg28141 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 NHC(=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 - and * is the moiety covalently attached to Y, and R 1 is benzyl. (40) 40. The ADC of claim 38 or 39, wherein the camptothecin analog comprises any one of the following formulas: JPEG0007689606000038.jpg82141 (41) 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: JPEG0007689606000039.jpg41141 (42) 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: JPEG0007689606000040.jpg39141 (43) 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: JPEG0007689606000041.jpg39141 (44) 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: JPEG0007689606000042.jpg39141 (45) 41. The ADC of claim 40, wherein the camptothecin analog comprises the formula: JPEG0007689606000043.jpg43141 (46) The ADC of any one of claims 35 to 45, wherein the ADC further comprises a self-immolative spacer. (47) The self-immolative spacer is -NH-CH 2 47. The ADC of claim 46, wherein said ADC is - or absent. (48) The ADC of any one of claims 35 to 47, further comprising a linker connecting the antibody to the cytotoxic agent. (49) 49. The ADC of claim 48, wherein the linker comprises a peptide unit. (50) 50. The ADC of claim 49, wherein the peptide unit comprises Ala-Ala-Ala, Val-Cit, or Gly-Gly-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) 51. The ADC of claim 50, wherein the peptide unit comprises Gly-Gly-Phe-Gly (SEQ ID NO: 102). (54) 54. The ADC of any one of claims 48 to 53, wherein the linker further comprises a spacer unit A between the antibody and the peptide unit. (55) Spacer unit A is of the formula: JPEG0007689606000044.jpg16141 The ADC of claim 54, wherein z is 1 to 5. (56) Spacer unit A is of the formula: JPEG0007689606000045.jpg25141 The ADC of claim 54, wherein z is 1 to 5. (57) An antibody-drug conjugate (ADC), the ADC having one of the following formulas: JPEG0007689606000046.jpg239150 and During the ceremony, Ab is an antibody according to any one of claims 1 to 34, n is about 1 to 16, ADC. (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: TIFF0007689606000047.tif45128 (61) 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: TIFF0007689606000048.tif57128 (62) 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: TIFF0007689606000049.tif47128 (63) 60. The ADC of any one of claims 57 to 59, wherein the ADC is of the formula: JPEG0007689606000050.jpg59141 (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) 68. The ADC of any one of claims 57 to 67, wherein the connection to the antibody occurs via a thiol group of one or more cysteines of the antibody. (69) 69. The ADC of claim 68, wherein each of the one or more cysteines is a native cysteine ​​in the hinge region of the antibody. (70) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises an HC comprising amino acids 2 to 444 of SEQ ID NO: 2 and an LC comprising amino acids 2 to 215 of SEQ ID NO: 3, and n is about 8. (71) The ADC of claim 70, wherein the Ab comprises a HC consisting of SEQ ID NO:2 and a LC consisting of SEQ ID NO:3. (72) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises an HC comprising amino acids 2 to 444 of SEQ ID NO: 12 and an LC comprising amino acids 2 to 215 of SEQ ID NO: 13, and n is about 8. (73) The ADC of claim 72, wherein the Ab comprises a HC consisting of SEQ ID NO: 12 and a LC consisting of SEQ ID NO: 13. (74) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises an HC comprising amino acids 2 to 443 of SEQ ID NO: 16 and an LC consisting of SEQ ID NO: 17, and n is about 8. (75) The ADC of claim 74, wherein the Ab comprises a HC consisting of SEQ ID NO: 16 and a LC consisting of SEQ ID NO: 17. (76) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises amino acids 2 to 447 of SEQ ID NO: 26 and an LC consisting of SEQ ID NO: 27, and n is about 8. (77) The ADC of claim 76, wherein the Ab comprises a HC consisting of SEQ ID NO:26 and a LC consisting of SEQ ID NO:27. (78) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises amino acids 2 to 446 of SEQ ID NO: 36 and an LC consisting of SEQ ID NO: 37, and n is about 8. (79) The ADC of claim 78, wherein the Ab comprises a HC consisting of SEQ ID NO:36 and a LC consisting of SEQ ID NO:37. (80) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises amino acids 2 to 446 of SEQ ID NO: 46 and an LC consisting of SEQ ID NO: 47, and n is about 8. (81) The ADC of claim 80, wherein the Ab comprises a HC consisting of SEQ ID NO: 46 and a LC consisting of SEQ ID NO: 47. (82) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises amino acids 2 to 446 of SEQ ID NO: 54 and an LC consisting of SEQ ID NO: 55, and n is about 8. (83) The ADC of claim 82, wherein the Ab comprises a HC consisting of SEQ ID NO:54 and a LC consisting of SEQ ID NO:55. (84) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises an LC comprising amino acids 2 to 450 of SEQ ID NO: 64 and amino acids 2 to 216 of SEQ ID NO: 65, and n is about 8. (85) The ADC of claim 84, wherein the Ab comprises a HC consisting of SEQ ID NO:64 and a LC consisting of SEQ ID NO:65. (86) 70. The ADC of any one of claims 57 to 63 or 68 to 69, wherein the Ab comprises an LC comprising amino acids 2 to 450 of SEQ ID NO: 74 and amino acids 2 to 216 of SEQ ID NO: 75, and n is about 8. (87) The ADC of claim 86, wherein the Ab comprises a HC consisting of SEQ ID NO: 74 and a LC consisting of SEQ ID NO: 75. (88) 35. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 34 and one or more pharma- ceutically acceptable carriers, diluents or excipients. (89) 88. A pharmaceutical composition comprising the ADC of any one of claims 35 to 87 and one or more pharma- ceutically acceptable carriers, diluents, or excipients. (90) A method for treating cancer, comprising administering to a patient in need thereof an effective amount of the ADC of any one of claims 35 to 87. (91) 91. The method of claim 90, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. (92) 92. The method of claim 90 or 91, wherein the cancer is urothelial cancer. (93) The method of any one of claims 90 to 92, wherein the patient being treated has relapsed after receiving enfortumab vedotin or has become refractory to enfortumab vedotin. (94) The method of any one of claims 90 to 92, wherein the patient is ineligible for treatment with enfortumab vedotin. (95) 95. The method of any one of claims 90 to 94, further comprising administering simultaneously, separately or sequentially a PD-1 inhibitor or a PD-L1 inhibitor. (96) 88. The ADC according to any one of claims 35 to 87, for use in treatment. (97) The ADC according to any one of claims 35 to 87, for use in treating cancer. (98) 98. The ADC for use according to claim 97, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. (99) 99. The ADC for use of claim 98, wherein the cancer is urothelial carcinoma. (100) 100. The ADC for use according to any one of claims 97 to 99, wherein the cancer has relapsed following treatment with enfortumab vedotin or the cancer has become refractory to enfortumab vedotin. (101) 100. The ADC for use according to any one of claims 97 to 99, wherein prior use of enfortumab vedotin was contraindicated. (102) 102. The ADC for use according to any one of claims 97 to 101, wherein the ADC is administered in simultaneous, separate or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor. (103) 88. A pharmaceutical composition for use in treating cancer, comprising an effective amount of the ADC according to any one of claims 35 to 87. (104) 104. The composition for use of claim 103, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. (105) 105. The composition of claim 103 or 104, administered in simultaneous, separate or sequential combination with a PD-1 inhibitor or a PD-L1 inhibitor. (106) 88. Use of the ADC of any one of claims 35 to 87 for the manufacture of a medicament for the treatment of cancer. (107) 107. The use of claim 106, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer. (108) 108. The use of claim 106 or 107, wherein the medicament further comprises a PD-1 inhibitor or a PD-L1 inhibitor. (109) 35. A method for producing an ADC, comprising contacting an antibody according to any one of claims 1 to 34 with a compound of the formula: JPEG0007689606000051.jpg192141 (110) The method of claim 109, further comprising reducing the antibody with a reducing agent prior to said contacting to produce a reduced Nectin-4 antibody. (111) 111. The method of claim 110, wherein the reducing agent is DTT or TCEP.

[0209] Amino acid and nucleotide sequences SEQ ID NO:1 (human nectin-4) MPLSLGAEMWGPEAWLLLLLLLASFTGRCPAGELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARVDAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYEC RVSTFPAGSFQARLRLRVLVPPLPSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLED QNLWHIGREGAMLKCLSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVVLMSRYHRRKAQQM TQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRAEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV SEQ ID NO:2 (HC of Ab1) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREDWDFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:3 (LC of Ab1) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREDWDFDYWGQGTLVTVSS SEQ ID NO: 11 (LCVR of Ab1) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPITFGQGTKVEIK SEQ ID NO: 12 (HC of Ab1a) QVQLVQSGAQVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCAREDWDFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 13 (LC of Ab1a) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQKPGQAPRLLIYGASNRATDIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQQYGSSPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 (HCVR of Ab1a) QVQLVQSGAQVKKPGASVKVSCKASGYTFTGYYIHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYFCAREDWDFDYWGQGTLVTVSS SEQ ID NO: 15 (LCVR of Ab1a) EIVLTQSPGTLSLSPGERATLSCRTSQSVSSSYLAWYQQKPGQAPRLLIYGASNRATDIPDRFSGSGSGTDFTLTINRLEPEDFAVYYCQQYGSSPITFGQGTRLEIK SEQ ID NO: 16 (HC of Ab2) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSAIGTVGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREWNGMDVWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTH TCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17 (LC of Ab2) DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYTTPYSFGQGTKV EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMHWVRQATGKGLEWVSAIGTVGDTYYPGSVKGRFTISRENAKNSLYLQMNSLRAGDTAVYYCAREWNGMDVWGQGTTVTVSS SEQ ID NO:25 (LCVR of Ab2) DIVMTQSPDSLAVSLGERATINCKSSQSVLYNSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQFYTTPYSFGQGTKVEIK SEQ ID NO:26 (HC of Ab3) EVQLVESGGGLVKPGGSLRLSCAASGFNLNHYNMNWVRQAPGKGLEWVSSVSSGGGFRYYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAVFHDAFDIWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:27 (LC of Ab3) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQINSYPFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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) EVQLVESGGGLVKPGGSLRLSCAASGFNLNHYNMNWVRQAPGKGLEWVSSVSSGGGFRYYADSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAVFHDAFDIWGQGTLVTVSS SEQ ID NO: 35 (LCVR of Ab3) DIQLTQSPSFLSASVGDRVTITCRASQDISSYLAWYQQKPGKAPKLLIYVASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQINSYPFTFGQGTKVEIK SEQ ID NO: 36 (HC of Ab4) QVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGIFFDAFDIWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37 (LC of Ab4) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQFNSYPWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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) QVQLQESGPGLVKPSETLSLTCTVSGGSISGYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARLGIFFDAFDIWGQGTLVTVSS SEQ ID NO: 45 (LCVR of Ab4) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQFNSYPWTFGQGTKVEIK SEQ ID NO: 46 (HC of Ab5) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYVHWVRQAPGQGLEWMGIINPSIISTSYAQKFQARVTMTRDTTSTSTVYMELSSLRSEDTAVYYCARGLNFDAFDIWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 47 (LC of Ab5) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPFTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 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) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVHWVRQAPGQGLEWMGIINPSIISTSYAQKFQARVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGLNFDAFDIWGQGTLVTVSS SEQ ID NO:53 (LCVR of Ab5) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNNYPFTFGQGTKVEIK SEQ ID NO:54 (HC of Ab6) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRLGTYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:55 (LC of Ab6) SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSSDQVVFGGGTKLTV LGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 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) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGIINPSGGSTTYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRLGTYFDYWGQGTLVTVSS SEQ ID NO: 63 (LCVR of Ab6) SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDRSSDQVVFGGGTKLTVL SEQ ID NO: 64 (HC of Ab7) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSSGSASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGVGRDILQAFDIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 65 (LC of Ab7) QSVLTQPPSVSEAPRQRVTISCSSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGHVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 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) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYYMHWVRQAPGQGLEWMGIINPSSGSASYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAREGVGRDILQAFDIWGQGTLVTVSS SEQ ID NO: 73 (LCVR of Ab7) QSVLTQPPSVSEAPRQRVTISCSSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGHVFGGGTKLTVL SEQ ID NO: 74 (HC of Ab8) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGIINPISGRTSSAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEGVGGELLRAFDIWG QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVSVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 75 (LC of Ab8) QSVLTQPPSVSEAPRQRVTISCSSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGFVFGGGTKLT VLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 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) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYIHWVRQAPGQGLEWMGIINPISGRTSSAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKEGVGGELLRAFDIWGQGTLVTVSS SEQ ID NO: 81 (LCVR of Ab8) QSVLTQPPSVSEAPRQRVTISCSSGSSSNIGNNAVNWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGFVFGGGTKLTVL SEQ ID NO: 82 (DNA of HC for Ab1) SEQ ID NO: 83 (DNA of LC for Ab1) GAGATCGTGCTGACCCAGAGTCCAGGAACACTCAGCCTGTCCCCCGGAGAACGGGCTACTTTGTCATGCCGTACGAGCCAGAGCGTGTCCTCTTCTTATCTGGCTTGGTACCAGCAAAAGCCTGGACAAGCGCCTCGATTACTTATCTATGGTGCCTCTAACCGCGCCACAGGCATTCCAGACAGATTCTCAGGGTCTGGCAGTGGCACCGACTTTACACTAACCATTTCCAGGCTCGAACCCGAGGATTTCGCAGTCTACTACTGTCAGCAGTATGGGTCGAGCCCGATTACTTTTGGCCAGGGTACCAAGGTTGAGATCAAAagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 84 (DNA of HC for Ab1a) SEQ ID NO: 85 (DNA of LC against Ab1a) GAAATCGTGCTTACCCAATCCCCTGGTACTCTCTCCCTCAGCCCCGGCGAACGGGCCACCCTCTCCTGCCGAACAAGCCAAAGTGTGTCTAGCTCCTACCTCGCCTGGTATCAACAGAAACCCGGCCAAGCACCACGACTTCTTATCTATGGTGCAAGCAACAGGGCTACTGACATTCCAGATCGCTTCAGTGGCTCTGGCTCAGGAACAGACTTCACTCTGACTATCAACAGGTTGGAACCTGAGGATTTTGCCGTATATTATTGCCAACAATACGGCTCCTCTCCTATAACCTTTGGTCAAGGTACACGCCTGGAAATAAAGagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 86 (DNA of HC against Ab2) SEQ ID NO: 87 (DNA of LC against Ab2) GACATTGTCATGACCCAGTCACCTGACTCCTTGGCGGTGAGCCTGGGAGAAAGAGCAACAATTAACTGCAAATCCAGTCAATCGGTCCTCTACAACTCCAATAACAAGAATTACCTGGCTTGGTATCAGCAGAAGCCCGGCCAGCCACCTAAGCTCCTGATCTATTGGGCTAGTACTCGGGAGTCCGGAGTTCCCGATAGGTTCTCTGGTTCAGGGTCTGGCACCGACTTTACCCTTACAATAAGCAGCCTACAGGCCGAAGATGTAGCCGTGTATTACTGTCAACAGTTCTATACAACGCCATACTCTTTTGGCCAGGGGACTAAAGTGGAGATCAAGagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 88 (DNA of HC against Ab3) SEQ ID NO: 89 (DNA of LC for Ab3) GATATCCAACTGACCCAGTCTCCAAGTTTCCTGTCTGCTTCAGTGGGCGATAGGGTCACAATCACCTGTCGGGCCTCCCAAGACATCTCCTCGTACCTGGCGTGGTACCAGCAGAAGCCTGGCAAGGCTCCCAAATTGCTCATATACGTTGCAAGTACCCTTCAGAGCGGAGTGCCATCCAGATTCTCAGGGTCCGGAAGCGGGACTGAATTTACACTAACGATCAGCAGCCTCCAGCCCGAGGACTTCGCCACCTATTATTGCCAGCAGATTAACTCTTATCCTTTTACATTTGGCCAGGGTACTAAAGTAGAGATTAAGagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 90 (DNA of HC for Ab4) SEQ ID NO: 91 (DNA of LC for Ab4) GATATACAACTAACACAGTCTCCTTCCTTCTTGTCTGCTAGTGTGGGCGACAGGGTCACAATCACTTGCCGGGCAAGCCAGGGAATTTCCTCCTACCTGGCATGGTATCAGCAGAAGCCCGGGAAAGCTCCCAAGCTCCTGATCTATGCCGCCAGCACGCTTCAGAGCGGGGTGCCATCCAGATTTTCGGGTAGCGGCTCTGGCACTGAGTTTACCCTCACAATTAGTTCACTGCAGCCTGAAGACTTTGCCACCTACTACTGTCAACAGTTCAACTCATATCCATGGACCTTCGGTCAGGGAACCAAAGTTGAGATCAAGagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 92 (DNA of HC for Ab5) SEQ ID NO: 93 (DNA of LC for Ab5) GACATCCAGTTAACTCAGTCTCCTAGCTTCCTGAGCGCTTCTGTTGGAGATAGAGTCACAATTACATGTAGGGCCTCCCAGGGGATTTCATCATATCTGGCTTGGTATCAACAGAAGCCTGGCAAAGCACCAAAGCTCCTGATCTATGCCGCATCTACCCTACAATCGGGTGTGCCCTCCCGGTTCAGTGGCTCCGGGAGTGGAACGGAATTTACCCTTACCATCTCCAGCTTGCAGCCCGAGGACTTCGCCACATACTACTGCCAGCAGCTCAATAACTACCCATTTACTTTTGGTCAGGGCACCAAAGTGGAGATAAAGagaactgtggcggcgccatctgtcttcatcttcccgccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgc SEQ ID NO: 94 (DNA of HC for Ab6) SEQ ID NO: 95 (DNA of LC for Ab6) AGCTACGTCCTTACTCAGCCACCTAGTGTGTCAGTAGCGCCTGGGCAAACCGCTCGCATAACTTGCGGGGGAAACAACATCGGCTCTAAATCCGTCCACTGGTACCAGCAGAAGCCCGGCCAGGCACCCGTATTGGTTGTTTACGACGATAGCGACAGACCGTCCGGTATCCCAGAGCGGTTTTCCGGTAGTAATTCTGGAAATACAGCTACCCTGACAATTTCTAGGGTGGAAGCCGGGGATGAGGCCGACTATTATTGTCAAGTGTGGGATCGATCAAGCGACCAGGTGGTCTTCGGCGGAGGCACGAAGCTGACCGTGCTCggccagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagacaaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca SEQ ID NO: 96 (DNA of HC for Ab7) SEQ ID NO: 97 (DNA of LC against Ab7) CAGTCTGTTCTCACACAACCACCCTCAGTAAGCGAGGCTCCCCGGCAGAGAGTCACAATCTCCTGTAGCGGAAGTAGTTCAAACATAGGGAATAATGCGGTGAACTGGTATCAGCAGTTGCCCGGTAAAGCCCCTAAACTGCTCATTTACTACGACGACTTACTACCTTCCGGCGTGAGCGACAGGTTTTCAGGAAGCAAGTCTGGCACTTCGGCCTCCCTGGCAATCTCTGGACTTCAGAGTGAGGATGAAGCTGACTATTACTGCGCAGCCTGGGATGATTCCTTGAACGGCCACGTCTTCGGTGGCGGGACCAAGTTGACCGTGCTGggccagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcgggagtggagacaaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca SEQ ID NO: 98 (DNA of HC against Ab8) SEQ ID NO: 99 (DNA of LC against Ab8) CAGAGTGTGCTGACCCAGCCCCCAAGCGTCTCCGAGGCACCCAGACAGAGGGTGACAATCTCCTGCTCCGGAAGCAGTTCAAACATAGGCAACAACGCCGTAAATTGGTACCAGCAGCTGCCAGGTAAGGCCCCTAAACTTCTGATCTATTACGATGATTTG CTCCCTTCAGGTGTCTCTGACCGCTTTTCCGGAAGCAAATCTGGGACCAGCGCTTCTTTGGCAATTTCTGGCCTGCAATCGGAAGACGAGGCTGATTACTATTGTGCCGCTTGGGACGACAGTTTAAATGGGTTCGTTTTCGGCGGCGGAACAAAGCTAACT GTGCTCggccagcccaaggctgccccctcggtcactctgttcccgccctcctctgaggagcttcaagccaacaaggccacactggtgtgtctcataagtgacttctacccgggagccgtgacagtggcctggaaggcagatagcagccccgtcaaggcggga gtggagacaaccacaccctccaaacaaagcaacaacaagtacgcggccagcagctatctgagcctgacgcctgagcagtggaagtcccacagaagctacagctgccaggtcacgcatgaagggagcaccgtggagaagacagtggcccctacagaatgttca SEQ ID NO: 100 - Ala-Leu-Ala-Leu- (also known as ALAL in the one-letter code) SEQ ID NO: 101 - Leu-Ala-Leu-Ala- (also known as LALA in the one-letter code) SEQ ID NO: 102 - Gly-Gly-Phe-Gly- (also known as GGFG in the one-letter code) SEQ ID NO: 103 - Gly-Phe-Leu-Gly- (also known as GFLG in the one-letter code) SEQ ID NO: 104 - Gly-Leu-Phe-Gly- (also known as GLFG in the one-letter code) SEQ ID NO:105 - Ala-Ala-Ala-Ala- (also known as AAAA in one-letter notation) SEQ ID NO: 106 - Gly-Ala-Gly-Gly-(also known as GAGG in the one-letter code) SEQ ID NO: 107 - Gly-Gly-Ala-Gly- (also known as GGAG in the one-letter code) SEQ ID NO: 108 - Gly-Val-Gly-Gly- (also known as GVGG in the one-letter code) SEQ ID NO: 109 - Gly-Gly-Val-Gly- (also known as GGVG in the one-letter code) SEQ ID NO: 110 - Gly-Phe-Gly-Gly- (also known as GFGG in the one-letter code)

Claims

1. An antibody-drug conjugate (ADC) comprising an antibody conjugated to a cytotoxic agent, the cytotoxic agent having the formula: and the antibody binds to human Nectin-4 and comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR), the HCVR comprising heavy chain complementarity determining regions (HCDRs) HCDR1, HCDR2, and HCDR3, and the LCVR comprising light chain complementarity determining regions (LCDRs) LCDR1, LCDR2, and LCDR3; Where: a) the HCDR1 comprises SEQ ID NO:4, the HCDR2 comprises SEQ ID NO:5, the HCDR3 comprises SEQ ID NO:6, the LCDR1 comprises SEQ ID NO:7, the LCDR2 comprises SEQ ID NO:8, and the LCDR3 comprises SEQ ID NO:9, or b) An ADC, wherein the HCDR1 comprises SEQ ID NO: 18, the HCDR2 comprises SEQ ID NO: 19, the HCDR3 comprises SEQ ID NO: 20, the LCDR1 comprises SEQ ID NO: 21, the LCDR2 comprises SEQ ID NO: 22, and the LCDR3 comprises SEQ ID NO:

23.

2. a) the HCVR comprises SEQ ID NO: 10 and the LCVR comprises SEQ ID NO: 11, or b) the HCVR comprises SEQ ID NO:24 and the LCVR comprises SEQ ID NO:25; 2. The ADC of claim 1.

3. 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 2-215 of SEQ ID NO:3; or b) The ADC of claim 1, wherein the HC comprises amino acids 2-443 of SEQ ID NO: 16 and the LC consists of SEQ ID NO:

17.

4. a) the HC consists of SEQ ID NO:2 and the LC consists of SEQ ID NO:3, or b) the HC consists of SEQ ID NO: 16 and the LC consists of SEQ ID NO: 17; The ADC of claim 3.

5. The ADC of claim 1, further comprising a linker connecting the antibody to the cytotoxic agent, the linker comprising a peptide unit of Ala-Ala-Ala, or Gly-Gly-Phe-Gly (SEQ ID NO: 102).

6. The linker according to claim 1, further comprising a spacer unit A between the antibody and the peptide unit, the spacer unit A having the formula: 【Chemistry 8】 (wherein z is 1 to 5).

7. The ADC has the following formula: 【Chemistry 10】 and where Ab is an antibody; 2. The ADC of claim 1, wherein n is about 1 to 16.

8. 8. The ADC of claim 7, wherein the ADC is of the formula: 【Chemistry 11】

9. 8. The ADC of claim 7, wherein the ADC is of the formula: 【Chemistry 12】

10. 8. The ADC of claim 7, wherein n is about 4.

11. 8. The ADC of claim 7, wherein n is about 8.

12. 8. The ADC of claim 7, wherein the connection to the antibody occurs through a thiol group of one or more cysteines of the antibody.

13. 13. The ADC of claim 12, wherein each of the one or more cysteines is a naturally occurring cysteine ​​in the hinge region of the antibody.

14. The Ab is a) a HC comprising amino acids 2-444 of SEQ ID NO:2 and a LC comprising amino acids 2-215 of SEQ ID NO:3; or The ADC of claim 7, comprising: b) a HC comprising amino acids 2-443 of SEQ ID NO: 16; and a LC consisting of SEQ ID NO:

17.

15. 13. A pharmaceutical composition comprising the ADC of claim 1 and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

16. A pharmaceutical composition for use in treating cancer, comprising an effective amount of the ADC of claim 1.

17. 17. The composition of claim 16, wherein the cancer is urothelial cancer, breast cancer, lung cancer, gastric cancer, colorectal cancer, pancreatic cancer, head and neck cancer, ovarian cancer, or prostate cancer.

18. The composition of claim 16, which is administered in combination with a PD-1 inhibitor or a PD-L1 inhibitor simultaneously, separately or sequentially.

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