Anti-nectin-4 antibodies and uses of the same

Abstract

Provided are novel anti-nectin-4 antibodies and uses thereof. The novel anti-nectin-4 antibodies of the present disclosure have appropriately adjusted affinity for nectin-4 and reduced cytotoxicity against normal cells as compared to cytotoxicity against cancer cells, and thus the antibodies and antibody-drug conjugates including the antibodies may exhibit excellent therapeutic effects while significantly reducing side effects.

Description

ANTI-NECTIN-4 ANTIBODIES AND USES OF THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of Korean Patent Application No. 10-2024-0077761, filed June 14, 2024, which is hereby incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates to novel anti-nectin-4 antibodies and uses thereof.BACKGROUND

[0003] Nectins are cell adhesion molecules that assist construction of epithelial and endothelial junctions and serve as receptors for entry of herpes simplex virus, measles virus, and poliovirus.

[0004] Nectin-4 (also referred to as nectin cell adhesion molecule 4) is a type 1 transmembrane protein and belongs to the nectin family of cell-adhesion proteins. Adhesive proteins of the nectin family are structurally related and exhibit three conserved immunoglobulin-like domains (V, C, and C) in an extracellular domain. A molecular weight of nectin-4 is about 55 kDa, and a molecular weight of the extracellular domain is about 36 kDa. Nectin-4 regulates various cellular activities such as migration, proliferation, differentiation, polarization, and virus entry. While other nectin family members are widely expressed in adult tissues, nectin-4 is mainly expressed in the embry o and placenta and expression thereof is reduced and very' limited in adult tissues. In addition, nectin-4 is overexpressed in various solid tumors such as ovarian cancer, ductal breast carcinoma, lung adenocarcinoma, and pancreatic cancer. Nectin-4 is emerging as a metastasis-associated protein and may be related to the progression and bad prognosis of a disease.

[0005] Meanwhile, enfortumab vedotin. which is an antibody-drug conjugate including enfortumab as an anti-nectin-4 antibody, was approved as a therapeutic agent for urothelial carcinoma. However, it is know n that side effects of skin toxicity7occur in about 40 % of patients using the same, and there is a disadvantage of deterioration in patient lives administered with enfortumab vedotin due to such skin toxicity.

[0006] Therefore, the present inventors have developed a novel anti-nectin-4 antibody having adjusted affinity to reduce skin toxicity' and maintain efficacy against cancer cells.SUMMARY

[0007] In one aspect, provided herein is an antibody or antigen-binding fragment thereof having a binding specificity to human nectin cell adhesion molecule 4 (nectin-4), wherein the antibody or the antigen-binding fragment thereof binds to a cancer cell with relative maximal binding level of about 97%-l 10% relative to a reference antibody, while the antibody or antigenbinding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 90% or less relative to the reference antibody, wherein the reference antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO.: 11, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO.: 12.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof binds to nectin- 4 with a KD value of 2.1 x 10'8M or greater. In some embodiments, the antibody or antigenbinding fragment thereof binds to nectin-4 with a KD value of two folds or greater compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 on a cancer cell with binding affinity of about 50% to about 90% compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof binds to a non-cancer cell with binding affinity of about 80% to about 90% relative to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof exhibits B-value (slope) between about 0.70 or greater for dose dependent 4-parameter curve for binding to a cancer cell under full curve binding FACS assay. In some embodiments, the antibody or antigen-binding fragment thereof shows greater internalization activity compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 1 , and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0009] In another aspect, provided herein is an antibody or antigen-binding fragment thereof having a binding specificity to human nectin cell adhesion molecule 4 (nectin-4), wherein theantibody or the antigen-binding fragment thereof binds to a cancer cell with EC50 of 0.25 nM or greater, and binds to a non-cancer cell with EC50 of 1.0 nM or greater.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof binds to the cancer cell with EC 50 of about 0.25-2.5 nM and / or the antibody or antigen-binding fragment thereof binds to the non-cancer cell with EC50 of about 1.0-4.0 nM. The EC50 may be measured by FACS.

[0011] In some embodiments, the cancer cell is a solid cancer cell. The cancer cell may be MDA-MB-468 cell. In some embodiments, the non-cancer cell is keratinocyte. In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having one or more amino acid substitution relative to the amino acid sequence of SEQ ID NO: 11 ; or a light chain variable region having one or more amino acid substitution relative to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDR) HCDR1. HCDR2. and HCDR3. and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and wherein one of the LCDR sequence is selected from the group consisting of SEQ ID NOs: 7, 8, 9, and 10.

[0012] In another aspect, provided herein is an antibody or antigen-binding fragment thereof having a binding specificity to human nectin-4 antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDR) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and wherein: the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 2; the HCDR3 comprises an amino acid sequence of SEQ ID NO: 3 or an ammo acid sequence having one. two, or three amino acid substitution relative to SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence ofSEQ ID NO: 6 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 6, wherein at least one of HCDR1, HCDR2, HCDR3, LCDR1. LCDR2, LCDR3 has one or more amino acid substitutions relative to SEQ ID Nos: 1, 2. 3, 4, 5. or 6, respectively.

[0013] In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 6. In some embodiments, the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 ; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or 10; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or 7; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, 8 or 9. In some embodiments, (a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 10; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6; (c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence ofSEQ ID NO: 8; or (d) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, (a) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 13, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13; (b) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity' to the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 14, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14; (c) the VH comprises the amino acid sequence of SEQ ID NO: 1 1 , or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:11 and the VL comprises the amino acid sequence of SEQ ID NO: 15, or a peptide having at least 90% sequence identity' to the amino acid sequence of SEQ ID NO: 15; or (d) the VH comprises the amino acid sequence of SEQ ID NO: 11 , or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 16.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof binds to a cancer cell with relative maximal binding level of about 97%-l 10% relative to a reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, while binds to a non-cancer cell with relative maximal binding level of about 90% or less relative to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. The antibody or the antigen-binding fragment thereof may bind to nectin-4 with a KD of 2. 1 x 10‘8M or greater. In some embodiments, the antibody or antigenbinding fragment thereof binds to nectin-4 with a KD of two folds or greater compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity with about 50% to about 90% compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, under full curve binding FACS assay against a cancer cell. In some embodiments, the antibody or antigen-binding fragment thereof has binding affinity to non-cancer cell of about 80% to about 90% relative to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the antibody or antigen-binding fragment thereof exhibits B-value (slope) between about 0.70 or greater for dose dependent 4- parameter curve for binding to MDA-MB-468 under full curve binding FACS assay. In some embodiments, the antibody or antigen-binding fragment thereof binds to a cancer cell with ECso of 0.25 nM or greater, and binds to a non-cancer cell with ECso of 1 .0 nM or greater. In some embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity to a cancer cell with ECso of about 0.25-2.5 nM. In some embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity to a non-cancer cell with ECso of about 1.0-4.0 nM. In some embodiments, the EC50 is measured by FACS. In some embodiments, the antibody or antigen-binding fragment thereof shows greater internalization activity compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the cancer cell is a solid cancer cell. In some embodiments, the cancer cell is MDA-MB-468 cell. In some embodiments, wherein the non-cancer cell is keratinocyte.

[0015] In the preceding paragraphs, the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, may be enfortumab.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof is a F(ab')2. a F(ab)2, a Fab', a Fab, a Fv, or a scFv. In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or the combination thereof. In some embodiments, the antibody or antigenbinding fragment thereof is of an isotype of IgGl. IgG2, IgG3 or IgG4. In some embodiments.the antibody or antigen-binding fragment thereof is of an isotype of human IgGl. In some embodiments, a constant region of the antibody or antigen-binding fragment has L234A and L235A mutations.

[0017] In another aspect, provided herein is a nucleic acid encoding the antibody or antigenbinding fragment of any preceding paragraph, and a vector comprising the same. In another aspect, provided herein is a host cell comprising the nucleic acid or the vector. In another aspect, provided herein is a method of preparing an anti-nectin-4 antibody or antigen-binding fragment thereof, the method comprising expressing the antibody or antigen-binding fragment thereof by culturing the host cell.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any preceding paragraph, and a pharmaceutically acceptable carrier.

[0019] In another aspect, provided herein is a method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or antigen-binding fragment thereof of any preceding paragraph.

[0020] In some embodiments, the cancer is solid cancer. The cancer may be selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer. In some embodiments, the cancer is selected from a group consisting of TNBC, NSCLC. and bladder cancer. In some embodiments, the cancer expresses nectin-4.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a graph showing results of a binding assay of 91 types of candidate antibodies to nectin-4.

[0022] FIG. 2 is a graph showing FACS binding full curves results for enfortumab to MDA- MB-468.

[0023] FIG. 3 is a graph showing FACS binding full curves results for ANX-ED133, ANX- ED141, ANX-ED143, and ANX-ED148 to normal keratinocytes.

[0024] FIG. 4 is a graph showing results of levels of relative maximal binding of ANX-ED133, ANX-ED141, ANX-ED143, and ANX-ED148 to normal keratinocytes.

[0025] FIG. 5 is a graph showing results of cytotoxicity of antibody-drug conjugates including ANX-ED133, ANX-ED141, ANX-ED143, and ANX-ED148 against NCI-H292 cell lines.

[0026] FIG. 6 is a graph showing results of cytotoxicity of antibody-drug conjugates including ANX-ED133, ANX-ED141 , ANX-ED143, and ANX-ED148 against normal keratinocytes.

[0027] FIG. 7 is a graph showing results of internalization activity of enfortumab and enfortumab-LALA, and ANX-ED143-LALA.DETAILED DESCRIPTION

[0028] It is to be noted that the term “a” or ‘"an” entity refers to one or more of that entity; for example, “an antibody,” is understood to represent one or more antibodies. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

[0029] As used herein, the term "antibody" refers to a general term for proteins involved in biological immunity by optionally acting on antigens, and types thereof are not particularly limited. A heavy chain and a light chain of an antibody include an antigen-binding site that includes a variable region and recognizes epitopes, and antigen specificity is revealed depending on variation in a sequence of the variable region. The variable region of the antigenbinding site is divided into a framework region (FR) that is less variable and a complementarity determining region (CDR) that is more variable, and both the heavy chain and the light chain include 3 CDR regions divided into CDR 1, 2, and 3 and 4 FR regions. The CDRs of each chain are typically named CDR1, CDR2, and CDR3 sequentially starting from the N-terminus and identified by the chain where a particular CDR is located.

[0030] As used herein, the term "complementarity' determining region" refers to a region of a variable region of an antibody imparting binding specificity for an antibody.

[0031] As used herein, the term "epitope" refers to a specific stereomolecular structure in antigen molecules to which an antibody is specifically bound.

[0032] As used herein, the term “polypeptide” or “peptide” is intended to encompass a singular “polypeptide / peptide” as well as plural “polypeptides / peptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide.” and the term “polypeptide” may be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acety lation, phosphory lation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or modification by non- naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis.

[0033] “Homology” or “identity'” or “similarity7” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology7can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology' between sequences is a function of the number of matching or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity7, though preferably less than 25% identity, with one of the sequences of the present disclosure.

[0034] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 98 % or 99 %) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences.

[0035] The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence having a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or complement thereof. A homolog of a double stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence whichhas a certain degree of homology7with or with the complement thereof. In one aspect, homologs of nucleic acids are capable of hybridizing to the nucleic acid or complement thereof. Likewise, "an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects, the equivalent polypeptide or polynucleotide has one, two, three, four or five additions, deletion, substitution and their combinations thereof as compared to the reference polypeptide or polynucleotide. In some aspects, the equivalent sequence retains the activity (e.g., epitope-binding) or structure (e.g., salt-bridge) of the reference sequence.

[0036] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility7, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity7of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, e.g., in US patent 5,892,019.

[0037] The term antibody encompasses various broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, 8, s) yvith some subclasses among them (e.g. , y 1- y4). It is the nature of this chain that determines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE, respectively. The immunoglobulin subclasses (isotypes) e.g., IgGi, IgGz, IgGs, IgG4, IgGs, etc. are yvell characterized and are knoyvn to confer functional specialization. Modified versions of each of these classes and isotypes are readily discernable to the skilled artisan in view of the instant disclosure and, accordingly, are within the scope of the instant disclosure. All immunoglobulin classes are clearly yvithin the scope of the present disclosure, the follo ving discussion will generally be directed to the IgG class of immunoglobulin molecules. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53.000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the lightchains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region.

[0038] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the disclosure include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs. single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti- idiotypic (anti -Id) antibodies (including, e.g., anti-Id antibodies to LIGHT antibodies disclosed herein). Immunoglobulin or antibody molecules of the disclosure can be of any type e.g.. IgG, IgE, IgM. IgD, IgA, and IgY), class (e.g., IgGL IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0039] Light chains are classified as either kappa or lambda (K, ). Each heavy chain class may be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tai 1” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.

[0040] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CK) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino- terminus of the antibody. The N-terminal portion is a variable region and at the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0041] The antibody may be a monoclonal antibody, a multispecific antibody (e.g., bispecific antibody), a human antibody, a humanized antibody, and / or a chimeric antibody (e.g.,humanized murine antibody). Also, the antibody may be a diabody, atriabody, and / or a tetrabody.

[0042] The antibody may be of any suitable class. As used herein, the term "class" refers to type of constant domain or constant region included in the heavy chain. As used herein, the term "constant domain" or "constant region" refers to a sum of domains of an antibody other than the variable region. The constant region is not directly involved in binding of antigens and exhibits various effector functions. The antibody may be an antibody of 5 major classes: IgA, IgD, IgE, IgG, IgM, or subclasses thereof (iso ty pe), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, or IgA3. Heavy chain constant domains corresponding to other classes of immunoglobulins are referred to as a, 5, e, y. and p, respectively. According to the present disclosure, the antibody may be an antibody of class IgG, IgA, or IgM, or any fragment thereof.

[0043] Throughout the specification, the antibody includes an "antigen-binding fragment" or "antibody fragment" of an antibody having antigen-binding ability. The antigen-binding fragment may be selected from the group consisting of antibody fragments including one or more complementarity determining regions, such as scFv, (scFvty, scFv-Fc, Fab, Fab' and F(ab')2. Among the antibody fragments, the Fab has a structure including variable regions of the light chain and the heavy chain, a constant region of the light chain, and a first constant region CHI of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that Fab' has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain CHI. The F(ab')2 antibody is generated as a cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv refers to a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In the two-chain Fv, a heavy chain variable region is bound to the light chain variable region by a non-covalent bond. In the single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally bound to each other by a covalent bond via a peptide linker or directly bound to each other at the C-terminus forming a structure similar to a dimer such as the two-chain Fv.

[0044] A position of CDR in the VH or VL region may be defined by Kabat, et al., [Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)] or IMGT numbering system, and both are known in the art. The IMGT numbering is defined to compare variable domains regardless of antigen receptor, chain type, specifies (See ["Unique database numbering system for immunogenetic analysis"Immunology7Today, 18, 509 (1997)] by Lefranc M.P.; and ["The IMGT unique numbering for Immunoglobulins, T cell receptors and Ig-like domains" The Immunologist, 7, 132-136 (1999)] by Lefranc M.P.).

[0045] Antibodies disclosed herein may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be condricthoid in origin (e.g, from sharks).

[0046] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CHI domain, a hinge (e.g, upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the disclosure may comprise a polypeptide chain comprising a CHI domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CHI domain and a CH3 domain; a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CHI domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the disclosure comprises a polypeptide chain comprising a CH3 domain.Further, an antibody for use in the disclosure may lack at least a portion of a CH2 domain (e.g, all or part of a CH2 domain). As set forth above, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that they vary in amino acid sequence from the naturally occurring immunoglobulin molecule.

[0047] The heavy chain constant region of an antibody disclosed herein may be derived from different immunoglobulin molecules. For example, a heavy chain constant region of a polypeptide may comprise a CHI domain derived from an IgGi molecule and a hinge region derived from an IgGa molecule. In another example, a heavy chain constant region can comprise a hinge region derived, in part, from an IgGi molecule and, in part, from an IgGs molecule. In another example, a heavy chain portion can comprise a chimeric hinge derived, in part, from an IgGi molecule and. in part, from an IgG4 molecule.

[0048] As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or constant lambda domain.

[0049] A “light chain-heavy chain pair” refers to the collection of a light chain and heavy chain that can form a dimer through a disulfide bond between the CL domain of the light chain and the CHI domain of the heavy chain.

[0050] As previously indicated, the subunit structures and three-dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino terminal variable domain of an immunoglobulin heavy chain and the term “CHI domain” includes the first (most amino terminal) constant region domain of an immunoglobulin heavy chain. The CHI domain is adjacent to the VH domain and is amino terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0051] By “specifically binds” or “has specificity to,” it is generally meant that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity7between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity ” is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody “A” may be deemed to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be said to bind to epitope “C” with a higher specificity than it has for related epitope “D.”Anti-Nectin-4 Antibody

[0052] The anti-nectin-4 antibodies and antigen-binding fragments thereof provided herein may show significantly reduced binding affinity to the normal cells, such as keratinocytes, as compared to enfortumab, but the relative maximal binding level to the cancer cells was comparable (see the examples) to enfortumab. Also as demonstrated in the examples, antibodydrug conjugates (ADC) including antibodies and antigen-binding fragments of the present disclosure exhibited significantly reduced toxicity to normal cells such as keratinocytes, while maintaining comparable cytotoxicity against cancer cells, as compared to enfortumab. Thus, the anti-nectin-4 antibodies and antigen-binding fragments thereof provided herein provide safeoptions than enfortumab, and the ADCs thereof have comparable therapeutic effect while with reducing side effects.

[0053] In an embodiment, the antibody or antigen-binding fragment thereof for nectin-4, as provided herein, has at least one amino acid substitution relative to enfortumab, and said antibody or antigen-binding fragment thereof may have adjusted binding affinity to cells. Surprisingly, the antibody or antigen-binding fragment thereof for nectin-4, as provided herein, exhibited significantly reduced affinity to normal cells (e.g., keratinocytes), while showing affinity to cancer cells that were reduced less significantly. As a result, the antibody or antigenbinding fragment of the present disclosure can be more selectively bound to cancer cells rather than normal cells. Further, ADCs including the antibody or antigen-binding fragment thereof as provided herein, showed significantly reduced cytotoxicity against normal cells (e.g., keratinocytes), while showing similar cytotoxicity' against cancer cells.

[0054] In an embodiment, the anti-nectin-4 antibody of the present disclosure may have greater affinity (binding affinity) for cancer cells as compared to affinity for normal cells (cancer cell affinity / normal cell affinity), specifically, greater affinity for cancer cells expressing nectin-4 compared to affinity' for normal keratinocytes. In an embodiment, anti-nectin-4 antibodies of the present disclosure, may have improved affinity for cancer cells as compared to affinity for normal keratinocytes in comparison with a reference antibody comprising a heavy chain variable region (VH) including an amino acid sequence of SEQ ID NO: 11 and a light chain variable region (VL) including an amino acid sequence of SEQ ID NO: 12. In some embodiments, the reference antibody is enfortumab.

[0055] ‘‘Maximal binding level” is a quantitative measure used to express the binding activity of an antibody to cells in an assay, such as flow cytometry or cell-based ELISA, or similar assays. It is the maximal binding signal level (such as fluorescence intensity' or absorbance) that can be obtained from the sample under saturating antibody conditions (i.e., the maximal binding signal level or upper asymptote of the 4-parameter curve, at saturating concentrations). “Relative maximal binding level” may be used to express the relative binding level of an antibody to cells in an assay, calculated by dividing the maximal binding level of the antibody of interest with the maximal binding level of a reference antibody, then multiplying by 100 to convert the ratio into a percentage. This normalization allows for direct comparison of antibodies binding across different samples or experimental conditions.

[0056] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with relative maximal binding of about 90%- 120% about 95%-120%. about 97%-120%, about 99%-120%, about 100%-120%, about 90%- 1 15%, about 95%- 115%, about 97%-l 15%, about 99%-l 15%, about 90%-l 10% , about 95%- 110% , about 97%-l 10%, about 99%-l 10%, about 100%- 110%, about 90%-105% , about 95%- 105%, about 97%-105%, about 99%-105%, about 100%-105%. about 90%-100%, about 95%- 100%, about 97%-100%, or about 99%-100% relative to a reference antibody having a VH including the amino acid sequence of SEQ ID NO: 11 and a VL including the amino acid sequence of SEQ ID NO: 12. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with relative maximal binding of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%. 106%, 107%, 108%. 109%, 110%, 115%. or 120% relative to the reference antibody. In some embodiments, the reference antibody is enfortumab. In certain embodiments, the cancer cell is a solid cancer cell. For example, the cancer may be selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer. In some embodiments, the cancer is selected from a group consisting of TNBC, NSCLC, and bladder cancer. In some embodiments, the cancer cell is MDA-MB-468 cell, NCI-H292 cell, NCI-H322 cell, or HT-1376 cell.

[0057] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a non-cancer cell with relative maximal binding of about 95% or less, about 93% or less, about 91% or less, about 90% or less, about 89% or less, about 88% or less, about 87% or less, about 86% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or about 50% or less relative to the reference antibody having a VH including the amino acid sequence of SEQ ID NO: 11 and a VL including the amino acid sequence of SEQ ID NO: 12. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with relative maximal binding of about 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 77%, 75%, 73%, 71%, 70%, 65%, 60%, 55%, 50%, 40%, 30%, 20% or 10% relative to the reference antibody. In some embodiments, the reference antibody is enfortumab.

[0058] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with relative maximal binding level of about 95%-120% relative to a reference antibody a reference antibody, while the antibody or antigenbinding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 95% or less relative to the reference antibody, wherein the reference antibody comprises a heavy chain variable region (VH) including an amino acid sequence of SEQ ID NO: 11 and a light chain variable region (VL) including an amino acid sequence of SEQ ID NO: 12. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with relative maximal binding level of about 95%-l 15% relative to a reference antibody a reference antibody, while the antibody or antigen-binding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 95% or less relative to the reference antibody. The anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with relative maximal binding level of about 95%-l 10% relative to a reference antibody a reference antibody, while the antibody or antigen-binding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 90% or less relative to the reference antibody. The anti-nectin-4 antibody or antigenbinding fragment thereof of the present disclosure may bind to a cancer cell with relative maximal binding level of about 97%-l 10% relative to a reference antibody a reference antibody, while the antibody or antigen-binding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 90% or less relative to the reference antibody. In some embodiments, the reference antibody is enfortumab.

[0059] In an embodiment, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to cancer cells expressing intermediate or high levels of nectin-4 more specifically than to normal keratinocytes expressing low levels of nectin-4. Therefore, because the anti-nectin-4 antibody of the present disclosure exhibits lower binding affinity for, lower internalization of, and / or lower cytotoxicity against normal keratinocytes than cancer cells, compared to enfortumab, side effects of skin toxicity caused by enfortumab may be effectively reduced. Especially, safety of the anti-nectin-4 antibody of the present disclosure is greater, as it has significantly reduced binding affinity’, internalization, and cytotoxicity' against normal keratinocytes than cancer cells.

[0060] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with an ECso value of about 0. 12 nM or greater. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may to a cancer cell with an ECso value of about 0.15nM or greater, about 0.18 nM or greater, about 0.2 nM or greater, about 0.25 nM or greater, about 0.29 nM or greater, about 0.3 nM or greater, about 0.4 nM or greater, about 0.5 nM or greater, about 0.6 nM or greater, about 0.7 nM or greater, about 0.8 nM or greater, about 1.0 nM or greater, about 1.5 nM or greater, about 2.0 nM or greater, about 2.2 nM or greater, about 2.5 nM or greater, about 3.0 nM or greater, about 3.5 nM or greater, about 4.0 nM, about 4.5 nM, about 5.0 nM, about 5.5 nM, about 6.0 nM, about 6.5 nM, about 7.0 nM, about 7.5 nM, about 8.0 nM, about 8.5 nM, about 9.0 nM, about 9.5 nM. about 10 nM or greater.

[0061] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with an ECso value of about 3 nM or lower. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with an ECso value of about 2.9 nM or less, about 2.8 nM or less, about 2.7 nM or less, about 2.6 nM or less, about 2.5nM or less, about 2.4 nM or less, about 2.3 nM or less, about 2.2 nM or less, about 2 nM or less, about 1.5 nM or less, about 1.2 nM or less, about 1.0 nM or less, about 0.8 nM or less, about 0.5 nM or less, about 0.3 nM or less, about 0.2 nM or less, about 0. 1 nM or less.

[0062] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with an ECso value in the range of about 0. 15-2.5 nM. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a cancer cell with an ECso value of about 0.15-2.5 nM, about 0.15-2.4 nM, about 0.15-2.3 nM, about 0.15-2.2 nM, about 0.15-2.0 nM, about 0.15-E7 nM, about 0.15- 1.5 nM, about 0.15-E0 nM, about 0.20-2.5 nM, about 0.20-2.4 nM, about 0.20-2.3 nM, about 0.20-2.2 nM, about 0.20-2.0 nM, about 0.20-1.7 nM, about 0.20-1.5 nM, about 0.20-1.0 nM, about 0.25-2.5 nM, about 0.25-2.4 nM, about 0.25-2.3 nM, about 0.25-2.2 nM. about 0.25-2.0 nM, about 0.25-1 .7 nM, about 0.25-1 .5 nM, or about 0.25-1 .0 nM. In some embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a cancer cell with an ECso value of about 0. 15 nM, about 0.20 nM, about 0.25 nM, about 0.29 nM, about 0.30 nM, about 0.40nM, about 0.50 nM, about 0.60 nM, about 0.70 nM. about 0.80nM, about 0.90 nM, about 1.00 nM, about 1.50 nM, about 2.00 nM, about 2.10 nM, about 2.20 nM, about 2.30 nM, about 2.40 nM, or about 2.50 nM.

[0063] In certain embodiments, the cancer cell is a solid cancer cell. For example, the cancer may be selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer. In some embodiments, the cancer is selected from a group consisting of TNBC, NSCLC, and bladder cancer. In some embodiments, the cancer cell is MDA-MB-468 cell, NCI-H292 cell, NCI-H322 cell, or HT- 1376 cell.

[0064] In certain embodiments, the cancer cell is blood cancer cell. For example, the cancer may be selected from the group consisting of leukemia (including acute leukemias (<?.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g.. chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy7chain disease.

[0065] In certain embodiments, the anti-nectin-4 antibody7or antigen-binding fragment thereof of the present disclosure binds to a non-cancer cell with ECso of 0.3 nM or greater. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a non-cancer cell with ECso of about 0.3 nM or greater, about 0.5 nM or greater, about 0.7 nM or greater, about 0.9 nM or greater, about 1.0 nM or greater, about 1.1 nM or greater, about 1.2 nM or greater, about 1.3 nM or greater, about 1.4 nM or greater, about 1.5 nM or greater, about 1.6 nM or greater, about 1.7 nM or greater, about 1.8 nM or greater, about 1.9 nM or greater, about 2.0 nM or greater, about 2.5 nM or greater, about 3.0 nM or greater, about 3.5 nM or greater, about 3.6 nM or greater, about 3.7 nM or greater, about 4.0 nM or greater, about 4.5 nM or greater, about 5.0 nM or greater, about 5.5 nM or greater, about 6.0 nM or greater, about 6.5 nM or greater, about 7.0 nM or greater, about 7.5 nM or greater, about 8.0 nM or greater, about 8.5 nM or greater, about 9.0 nM or greater, about 9.5 nM or greater, or about 10 nM or greater.

[0066] In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a non-cancer cell with an ECso value in the range of about 0.3- 6.0 nM. For example, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure may bind to a non-cancer cell with an ECso value in the range of about 0.3-5.5 nM, about 0.3-5.0 nM, about 0.3-4.5 nM, about 0.3-4.0 nM, about 0.3-3.7 nM, 0.5-5.5 nM, about 0.5-5.0 nM, about 0.5-4.5 nM, about 0.5-4.0 nM, about 0.5-3.7 nM, 0.7-5.5 nM, about 0.7-5.0 nM, about 0.7-4.5 nM, about 0.7-4.0 nM, about 0.7-3.7 nM, 0.9-5.5 nM, about 0.9-5.0 nM, about 0.9-4.5 nM, about 0.9-4.0 nM, about 0.9-3.7 nM, about 1.0-5.5 nM, about 1.0-5.0 nM, about 1.0-4.5 nM, about 1.0-4.0 nM, about 1.0-3.7 nM, about 1.2-5.5 nM, about 1.2-5.0 nM, about 1.2-4.5 nM, about 1.2-4.0 nM, about 1.2-3.7 nM, about 1.5-5.5 nM, about 1.5-5.0 nM, about 1.5-4.5 nM, about 1.5-4.0 nM, about 1.5-3.7 nM, about 1.7-5.5 nM, about 1.7-5.0 nM, about 1.7-4.5 nM, about 1.7-4.0 nM, or about 1.7-3.7 nM. In certain embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof of the present disclosure binds to a non-cancer cell with an ECso value of about 0.3 nM, 0.5 nM, 0.7 nM, 0.9 nM, 1.0 nM, 1.1 nM, 1.2 nM, 1.3 nM, 1.4 nM, 1.5 nM, 1.6 nM, 1.7 nM, 1.8 nM, 1.9 nM, 2.0 nM, 2.2 nM, 2.3 nM,2.4 nM, 2.5 nM, 2.6 nM. 2.7 nM, 2.8 nM, 2.9 nM, 3.0 nM, 3.1 nM. 3.2 nM, 3.3 nM, 3.4 nM.3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, 4.0 nM, 4. 1 nM, 4.2 nM, 4.3 nM, 4.4 nM, 4.5 nM,4.6 nM, 4.7 nM, 4.8 nM, 4.9 nM, or 5.0 nM.

[0067] Also provided herein is an antibody or antigen-binding fragment thereof having a binding specificity to human nectin-4, wherein the antibody or the antigen-binding fragment thereof binds to a cancer cell with a greater affinity compared to a non-cancer cell, wherein ECso for the non-cancer cell is at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8- fold, at least 1.9-fold, at least 2.0-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.7-fold, at least 3.0-fold, at least 3.4-fold, or at least 3.5- fold than EC so for the cancer cell.

[0068] The non-cancer cell may be any normal cell that is not a tumor cell. In certain embodiments, the non-cancer cell is keratinocyte.

[0069] In certain embodiments, the ECso is measured by FACS.

[0070] In certain embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD of 1.5 x 10‘8M or greater. For example, the antibody or antigen-bindingfragment thereof may bind to nectin-4 with a KD of 1.5 x 10xM or greater, 1.7 x 10xM or greater. 1.9 x |()xM or greater, 2.1 x 10xM or greater, 2.3 x | ()xM or greater, 2.5 x | ()xM or greater, 2.7 x | ()xM or greater. 2.9 x | ()xM or greater, 3. 1 x | ()xM, 3.3 x 10xM or greater, 3.5 x |()xM or greater, 3.7 x 1 ()xM or greater, 3.9 x 10xM or greater, 4.1 x 10xM or greater, 4.3 x 10xM or greater, 4.5 x 10xM or greater, 4.7 x 10sM or greater, 4.9 x | ()xM or greater, 5.1 x 10xM or greater, 5.3 x 10xM or greater, 5.5 x 10xM or greater, 5.7 x 108M or greater, 5.9 x 108M or greater, 6. 1 x 108M or greater, 6.3 x 108M or greater. 6.5 x 10sM or greater, 6.7 x 10xM or greater, 6.9 x 1 ()sM or greater, or 7.0 x I ()8M or greater. In certain embodiments, the KD is measured by binding assay using Biolayer Interferometry (BLI).

[0071] In certain embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD of two folds or greater (e.g. 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 80-fold, 100-fold, or higher) compared to a reference antibody having a VH including the amino acid sequence of SEQ ID NO: 11 and a VL including the amino acid sequence of SEQ ID NO: 12. In some embodiments, the reference antibody is enfortumab.

[0072] In certain embodiments, the antibody or antigen-binding fragment thereof binds to nectin-4 on a cancer cell with binding affinity of about 50% to about 90% (or about 50% to about 80%, about 50% to about 70%, about 50% to about 60%, about 60% to about 90%, about 60% to about 80%, about 60% to about 70%, about 70% to about 90%, about 70% to about 80%, or about 80% to about 90%) compared to a reference antibody having a VH including the amino acid sequence of SEQ ID NO: 11 and a VL including the amino acid sequence of SEQ ID NO: 12. In some embodiments, the reference antibody is enfortumab.

[0073] In certain embodiments, the cancer cell is a solid cancer cell. For example, the cancer may be selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer. In some embodiments, the cancer is selected from a group consisting of TNBC, NSCLC, and bladder cancer. In some embodiments, the cancer cell is MDA-MB-468 cell. NCI-H292 cell, NCI-H322 cell, or HT- 1376 cell.

[0074] In certain embodiments, the cancer cell is blood cancer cell. For example, the cancer may be selected from the group consisting of leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic. promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g, Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.

[0075] In certain embodiments, the antibody or antigen-binding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 80% to about 90% (or about 85% to about 90%, or about 80% to about 85%) relative to the reference antibody having a VH including the amino acid sequence of SEQ ID NO: 11 and a VL including the amino acid sequence of SEQ ID NO: 12. In some embodiments, the reference antibody is enfortumab.

[0076] In certain embodiments, the antibody or antigen-binding fragment thereof exhibits B- value (slope) between about 0.70 or greater (or about 0.8. 0.9, 1.0, 1.1, 1.2. 1.3, 1.4, 1.5, 1.7. 2.0, 2.5 or greater) for dose dependent 4-parameter curve for binding to MDA-MB-468 under full curve binding FACS assay.

[0077] In some embodiments, the antibodies and antigen-binding fragments thereof are ANX- ED133, ANX-ED141, ANX-ED143 and ANX-ED148 or have CDRs and vanable regions including amino acid sequences shown in Tables 1-2. Also included are those that include the same CDRs as illustrated herein. In some embodiments, the disclosed antibodies and fragments include those that bind to the same epitope as those illustrated here, and those that compete with the instantly disclosed in binding to nectin-4.

[0078] In accordance with one embodiment of the present disclosure, provided is an antibody or antigen-binding fragments thereof that includes the heavy chain and light chain variable domains with the CDR regions disclosed herein, as well as their biological equivalents.Table 1. Sequences of CDRs of the anti-nectin-4 antibodiesTable 2. Sequences of variable regions

[0079] It is appreciated that CDRs can be modified to include those having one, two or three amino acid addition, deletion and / or substitutions. In some embodiments, the substitutions can be conservative substitutions.

[0080] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g, aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g, threonine, valine, isoleucine) and aromatic side chains (e.g, tyrosine, phenylalanine, tty pt ophan. histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and / or composition of side chain family members.

[0081] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids.Table 3. Amino Acid Similarity MatrixTable 4. Conservative Amino Acid Substitutions

[0082] It will also be understood by one of ordinary' skill in the art that antibodies as disclosed herein may be modified such that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a designated protein may be similar, e.g.. have a certain percent identity to the starting sequence, e.g., it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[0083] Provided herein is an anti-nectin-4 antibody or antigen-binding fragment thereof including: a heavy chain variable region including a heavy chain complementarity7determining region 1 (HCDR1) including an amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) including an amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (HCDR3) including an amino acid sequence of SEQ ID NO: 3; and a light chain variable region including a light chain complementarity determining region 1 (LCDR1) including an amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) including an amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (LCDR3) including an amino acid sequence of SEQ ID NO: 6, wherein the anti-nectin-4 antibody or antigen-binding fragment thereof includes at least one, two or three amino acid substitution at the heavy chain variable region and the light chain variable region.

[0084] Also provided herein is an anti-nectin-4 antibody or antigen-binding fragment thereof including: a heavy chain variable region including a heavy chain complementarity7determining region 1 (HCDR1) including an amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) including an amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (HCDR3) including an amino acid sequence of SEQ ID NO: 3; and a light chain variable region including a light chain complementarity determining region 1 (LCDR1) including an amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) including an amino acidsequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (LCDR3) including an amino acid sequence of SEQ ID NO: 6, wherein the anti-nectin-4 antibody or antigen-binding fragment thereof includes at least one, two or three amino acid substitution at the heavy chain CDRs and the light chain CDRs.

[0085] In an embodiment, the antibody or antigen-binding fragment thereof may include HCDR1, HCDR2, HCDR3, LCDR1, LCDR2. and LCDR3 below: 1) HCDR1 including an amino acid sequence of SEQ ID NO: 1 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 1; 2) HCDR2 including an amino acid sequence of SEQ ID NO: 2 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 2; 3) HCDR3 including an amino acid sequence of SEQ ID NO: 3 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 3; 4) LCDR1 including an amino acid sequence of SEQ ID NO: 4 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 4; 5) LCDR2 including an amino acid sequence of SEQ ID NO: 5 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 5; and 6) LCDR3 including an amino acid sequence of SEQ ID NO: 6 or including an amino acid sequence having at least one amino acid substitution in SEQ ID NO: 6.

[0086] In certain embodiments, the amino acid substitution is alanine substitution (i.e.. substitution to alanine).

[0087] In an embodiment, the anti-nectin-4 antibody or antigen-binding fragment thereof may include at least one amino acid substitution, specifically, about 1 to 5, about 1 to 4. about 1 to 3, or about 1 to 2 amino acid substitutions. In some embodiments, the anti-nectin-4 antibody or antigen-binding fragment thereof may include one amino acid substitution.

[0088] In an embodiment, the at least one amino acid substitution may include an amino acid substitution at one or more positions selected from the group consisting of a 6thposition of SEQ ID NO: 4, a 6thposition of SEQ ID NO: 5, a 1stposition of SEQ ID NO: 6, and a 7thposition of SEQ ID NO: 6.

[0089] In an embodiment, the anti-nectin-4 antibody or antigen-binding fragment thereof may have adjusted affinity for nectin-4 because the amino acid at the 1stposition of SEQ ID NO: 6 corresponding to the first amino acid of LCDR3 is substituted with an amino acid other than Q.Specifically, by substituting the amino acid at the above-described position, affinity for cancer cells expressing nectin-4 may be maintained or slightly reduced while significantly reducing affinity for normal keratinocytes.

[0090] In an embodiment, the at least one amino acid substitution may include at least substitution selected from the group consisting of the following amino acid substitutions: 1) substitution of isoleucine, the 6thamino acid of SEQ ID NO: 4, with alanine; 2) substitution of glutamine, the 6thamino acid of SEQ ID NO: 5, with alanine; 3) substitution of glutamine, the 1stamino acid of SEQ ID NO: 6, with alanine; and 4) substitution of proline, the 7thamino acid of SEQ ID NO: 6, with alanine.

[0091] In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and 2) a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 10. LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 6.

[0092] In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and 2) a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 7, and LCDR3 including an amino acid sequence of SEQ ID NO: 6.

[0093] In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and 2) a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 8.

[0094] In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including anamino acid sequence of SEQ ID NO: 3; and 2) a light chain variable region including LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 9.

[0095] In certain embodiments, the antibodies and antigen-binding fragment thereof include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a peptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 13. In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and 2) a light chain variable region including an amino acid sequence of SEQ ID NO: 13.

[0096] In certain embodiments, the antibodies and antigen-binding fragment thereof include a light chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a peptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 14. In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and 2) a light chain variable region including an amino acid sequence of SEQ ID NO: 14.

[0097] In certain embodiments, the antibodies and antigen-binding fragment thereof include a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a peptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 15. In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and 2) a light chain variable region including an amino acid sequence of SEQ ID NO: 15.

[0098] In certain embodiments, the antibodies and antigen-binding fragment thereof include a light chain variable region comprising an ammo acid sequence of SEQ ID NO: 11. or a peptide having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 16. In an embodiment, the antibody or antigen-binding fragment thereof may include: 1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11 ; and 2) a light chain variable region including an amino acid sequence of SEQ ID NO: 16.

[0099] In consideration of variation having biologically equivalent activity, the antibody according to an aspect or nucleic acid molecules encoding the same may be interpreted to include a sequence exhibiting substantial identity to a sequence set forth in the SEQ ID NOs.

[0100] In certain embodiments, the antigen-binding fragment thereof provided herein includes is a F(ab')2, a F(ab)2, a Fab', a Fab, a Fv, or a scFv.

[0101] In certain embodiments, the antibody or antigen-binding fragment thereof provided herein further comprises a heavy chain constant region, a light chain constant region, an Fc region, or the combination thereof.

[0102] In certain embodiments, the antibody or antigen-binding fragment thereof is of an isotype of IgGl, IgG2, IgG3 or IgG4. In certain embodiments, the antibody or antigen-binding fragment thereof is of an isotype of IgG.

[0103] The Fc region can be engineered to enhance or attenuate / eliminate effector function. IgG antibodies can induce direct anti-tumor effects by way of indirect anti-tumor effects via the Fc-mediated effector functions that engage other immune cells or killer mechanisms. “Effector functions” or “antibody effector functions” as used herein refer to biological activities attributable to the binding of Fc region of an antibody to its effectors such as Cl complex and Fc receptor (FcyRIIa or FcyRIIIa). Exemplary effector functions include: complement dependent cytotoxicity (CDC) induced by interaction of antibodies and Clq on the Cl complex; antibody -dependent cell-mediated cytotoxicity (ADCC) induced by binding of Fc region of an antibody to Fc receptor on an effector cell; and antibody dependent cell mediated phagocytosis (ADCP) , where nonspecific cytotoxic cells that express FcyRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell. Among the four IgG subclasses, IgGl and IgG3 induce the strongest Fc-effector functions. However, since IgGl has the longest half-life and is more stable than IgG3, most therapeutic antibodies with Fc-mediated functions are of IgGl isotype.

[0104] In certain embodiments, the Fc region of the antibodies provided herein is engineered to enhance the effector function, such as ADCC or CDC. Various methods (mainly Fc mutations) can be found to enhance the Fc-mediated effector function, such as those described in the PCT publications W02007024249A2 and WO2011044368A1, which are incorporated herein by reference in their entireties. In certain embodiments, the Fc region is engineered to include S298A / E333A / K334A mutations in human IgGl, according to EU numbering. Such Fc mutation combination is reported to enhance binding to FcyRIIIa. thus enhancing ADCC (See Shields R.L. et al, J. Biol. Chem 276:6591-6604 (2001)).

[0105] In certain embodiments, the Fc region of the antibodies provided herein is engineered to reduce or eliminate the effector function. In certain embodiments, the Fc region of the antibodies provided herein includes S228P, L234A, L235A, L235E, N297Q, S298N, T299A, Y300T and / or Y300S mutation. In certain embodiments, the Fc region of the antibodies provided herein includes S228P mutation. In certain embodiments, the Fc region of the antibodies provided herein includes L235E mutation. In certain embodiments, the Fc region of the antibodies provided herein includes S298P mutation. In certain embodiments, the Fc region of the antibodies provided herein includes L234A mutation and L235A mutation. In certain embodiments, the Fc region of the antibodies provided herein includes S228P, F234A, and / or L235A mutation. In certain embodiments, the Fc region of the antibodies provided herein includes S228P and T299A mutation. In certain embodiments, the Fc region of the antibodies provided herein includes S298N and Y300S mutations. In certain embodiments, the Fc region of the antibodies provided herein includes S298N and Y300T mutations. In certain embodiments, the Fc region of the antibodies provided herein includes N297Q, S298N and Y300S mutations. In certain embodiments, the Fc region of the antibodies provided herein includes S298N, T299A and Y300S mutations. In certain embodiments, the Fc region is derived from human IgGl or IgG4. All the mutations are numbered according to EU numbering.

[0106] In certain embodiments, the Fc region of the antibodies provided herein includes H268Q / V309L / A330S / P331S mutation. In certain embodiments, the Fc region of the antibodies provided herein includes V234A / G237A / P238S / H268A / V309L / A330S / P33 IS mutation. In certain embodiments, the Fc region is derived from human IgG2. All the mutations are numbered according to EU numbering.

[0107] In certain embodiments, substitutions can be made in the constant regions of antibodies of the present disclosure to prolong half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004). Exemplary substitutions include a Gin at position 250 and / or a Leu at position 428 (Eu numbering) for increasing the half-life of an antibody.

[0108] In certain embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in more detail below. For example, an antibody of the disclosure may comprise a flexible linker sequence, or may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[0109] Antibodies, variants, or derivatives thereof of the disclosure include derivatives that are modified, z.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to the epitope. For example, but not by way of limitation, the antibodies can be modified, e.g, by glycosylation, acetylation, pegylation. phosphorylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the antibodies may contain one or more non- classical amino acids.

[0110] The antibodies can be delectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-tagged antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.[OHl] Provided is a composition for diagnosing cancer including the anti-nectin-4 antibody or antigen-binding fragment thereof and a labeling group.

[0112] The labeling group may be, for example, a dye detectable by imaging, or a paramagnetic, radioactive, or fluorescent group.

[0113] The labeling group may include, as a detection label, an enzyme, a fluorescent substance, a ligand, a luminescent substance, a microparticle, an enzy matic label, a magneticsubstance, a dye detectable by imaging, a paramagnetic group, a fluorescent group, or a radioisotope. Examples of the enzy me used as detection labels include acetylcholinesterase, alkaline phosphatase, P-D-galactosidase, horseradish peroxidase and P-latamase. examples of the fluorescent substance include fluorescein, Eu3+, Eu3+chelate, cryptate, FITC, and RITC, examples of the ligand include biotin derivatives, examples of the luminescent substance include acridinium esters and isoluminol derivatives, examples of the microparticle include colloidal gold and colored latex, and examples of the radioisotope include57Co,3H, and125I. The presence of an analyte may be quantitatively or qualitatively identified by visual inspection or using a sensor based on the degree of color development of the detection label. According to an embodiment, the presence of the analyte may be identified by preparing a conjugate of the fluorescent substance, among the detection labels, and a ligand of an antigen, an antibody, or the like, and detecting the degree of color development detected by irradiating light to a composite in which the conjugate is bound to a binder fixed to a porous membrane pad via the analyte or competitively by using a sensor.Bi-functional Molecules and Combination Therapies

[0114] Nectin-4 is overexpressed on tumor cells. As a tumor antigen targeting molecule, an antibody or antigen-binding fragment specific to nectin-4 can further include a second portion specific to a second antigen to generate a combination therapy or a bispecific antibody. The second portion can be an antibody or antigen-binding fragment specific to an immune cell, or an antibody or antigen-binding fragment specific to an immune checkpoint.

[0115] In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a monocyte, a macrophage, a neutrophil, a dendritic cell, a phagocyte, a natural killer cell, an eosinophil, a basophil, and a mast cell. Molecules on the immune cell which can be targeted include, for example, CCL1, CD3, CD16, CD19, CD28, and CD64. Other examples include PD-1, PD-L1, CTLA-4, LAG-3 (also known as CD223), CD28, CD122, 4-1BB (also known as CD137). TIM3, OX-40 or OX40L, CD40 or CD40L. LIGHT, ICOS / ICOSL.GITR / GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272), killer-cell immunoglobulin-like receptors (KIRs), and CD47.

[0116] Different format of bispecific antibodies are also provided. In some embodiments, each of the anti-nectin-4 fragment and the second fragment each is independently selected from aFab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific antibody further includes a Fc fragment.

[0117] Multifunctional (e.g., bifunctional) molecules that include not just antibody or antigen binding fragment are also provided. As a tumor antigen targeting molecule, an antibody or antigen-binding fragment specific to nectin-4, such as those described here, can be combined with an immune cytokine or ligand optionally through a peptide linker. The linked immune cytokines or ligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 10, IL- 12, IL-13, IL-15, GM-CSF, TNF-a, CD40L, OX40L, CD27L, CD30L, 4-1BBL, LIGHT and GITRL. Such bi-functional molecules can combine the immune checkpoint blocking effect with tumor site local immune modulation.Chimeric Antigen Receptors (CAR)

[0118] The present disclosure provides a chimeric antigen receptor (CAR) or an engineered T cell receptors (TCRs) comprising the anti-nectin-4 antigen-binding fragment thereof provided herein.

[0119] CARs and TCRs are genetically engineered receptors. These engineered receptors can be readily inserted into and expressed by immune cells, such as T cells. A CAR can recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen. When these antigens exist on tumor cells, an immune cell that expresses the CAR can target and kill the tumor cell.

[0120] CARs can be engineered to bind to an antigen (such as a cell-surface antigen) by incorporating an antigen binding molecule that interacts with that targeted antigen. Preferably, the antigen binding molecule is an antibody fragment thereof, and more preferably one or more single chain antibody fragment C‘scFv”). An scFv is a single chain antibody fragment having the variable regions of the heavy and light chains of an antibody linked together. It will be appreciated that the antigen binding molecule is typically contained w ithin the extracellular portion of the CAR such that it is capable of recognizing and binding to the antigen of interest. Bispecific and multispecific CARs are contemplated within the scope of the invention, w ith specificity to more than one target of interest.

[0121] An example CAR includes, in addition to the antigen binding molecule, a transmembrane domain and an activation domain. In some embodiments, the CAR can further include an extracellular domain between the antigen binding molecule and the transmembrane domain. The extracellular domain may comprise a hinge domain. In some embodiments, the CAR can further include a co-stimulatory domain.

[0122] The antigen binding portion of the CAR of the instant disclosure can be an antibody or antigen binding fragment (in particular a scFv) targeting human nectin-4, as disclosed herein.

[0123] Chimeric antigen receptors may comprise an extracellular domain. Extracellular domains of particular use in this invention may be derived from (z.e., comprise) all or some of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD- 1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein. IgGl, lgG2, IgG3. IgG4. IgA, IgD, IgE. IgM, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19. CD4. CD8alpha. CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244. 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGLL CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMFL CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or any combination thereof. The extracellular domain may be derived either from a natural or from a synthetic source.

[0124] Extracellular domains often comprise the hinge portion, sometimes referred to as the “spacer” region. A variety of hinges can be employed in accordance with the invention, including portions or derivatives of the molecules as listed above. In certain embodiments, the hinge portion is a hinge region of IgGl, IgG2. IgG3, IgG4, IgA, IgD. IgE, IgM, CD28. or CD8 alpha, any truncation thereof, or any combination thereof.

[0125] The CAR can be designed with a transmembrane domain that is fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular domain of the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0126] Non-limiting examples of such proteins include CD28, CD8alpha, CD8beta, 4-1BB, B7- H3. BAFFR, BLAME. BTLA. CD100, CD103, CDl la. CDl lb, CDl lc, CDl ld, CD160, CD 18, CD 19, CD 19a, CD2, CD247, CD27, CD276, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD79a, CD84, CD96, CDS, CEACAM1, CRT AM, DAP-10, DNAM1, Fc gamma receptor, GADS, GITR, HVEM, IA4, ICAM-1, ICOS, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4. ITGA6, ITGAD. ITGAE, ITGAL, ITGAM, ITGAX. ITGB1, ITGB2, ITGB7. KIRDS2. LAT, LFA-1, LIGHT, LTBR, Ly9, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80, OX-40, PAG, PD-1, PSGL1, SELPLG, SLAM, SLAMF4, SLAMF6, SLAMF7, SLP-76, TNFR2, TNFSF14, TRANCE, VLA1, VLA-6, a cytokine receptor, a MHC class 1 molecule, a SLAM protein, a TNF receptor protein, a Toll ligand receptor, an activating NK cell receptor, an immunoglobulin protein, and an integrin.

[0127] The intracellular (cytoplasmic) domain of the engineered T cells of the invention can provide activation of at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may refer to cytolytic activity or helper activity, including the secretion of cytokines. The intracellular domain may include at least an activation domain. The intracellular domain can also include one or more costimulatory domains.

[0128] A “costimulatory domain” as used herein refers to a molecule that provides a signal which mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. Costimulatory domain can provide a signal in addition to the primary signal provided by an activating molecule as described herein.

[0129] Chimeric antigen receptors may incorporate costimulatory (signaling) domains to increase their potency. See U.S. Patent Nos. 7,741,465. and 6,319,494. as well as Krause et al. and Finney et al. (supra), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). For example, CD28 is a costimulatory protein found naturally on T-cells. A variety of costimulatory molecules are set forth herein, but it will be appreciated that additional costimulatory molecules are also included within the scope of this invention.

[0130] It will be appreciated that suitable costimulatory domains within the scope of the invention include the signaling domain (or other suitable portion) of CD28, OX-40, 4- 1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CDl-la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins. Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19. CD4, CD8alpha. CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD1 Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1. CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150. IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, or fragments or any combination thereof. It will be appreciated that additional costimulatory molecules, or fragments thereof, not listed above are within the scope of the invention.

[0131] An “activation domain” refers to a molecule on a T cell, e.g., the TCR / CD3 complex that specifically binds with a cognate stimulatory ligand present on an antigen present cell. Suitable activating molecules are described herein. The activation domain is part of theintracellular (cytoplasmic) portion of a CAR. The intracellular domain of a CAR can provide activation of at least one of the normal effector functions of the immune cell. Effector function of a T cell, for example, may refer to cytolytic activity or helper activity, including the secretion of cytokines.

[0132] CD3 is an element of the T cell receptor on native T cells, and has been shown to be an important intracellular activating element in CARs. In a preferred embodiment, the CD3 is CD3 zeta, CD3 epsilon, CD3 delta, or CD3 gamma.

[0133] Immune cells enclosing a CAR of the present disclosure or one or more polynucleotides encoding the CAR are also provided.

[0134] As described, an engineered receptors can be inserted into and expressed by an immune cell, such as T cell, which can recognize a specific antigen and, when bound to that antigen, activate the immune cell to attack and destroy the cell bearing that antigen.

[0135] Various types of immune cells have been tested for expressing CARs. Non-limiting examples include T cells, NK cells, macrophages and monocytes. A T cell, in some embodiment, may be an alpha beta T cell. In some embodiments, the T cell is a gamma delta T cell.Polynucleotides Encoding the Antibodies and Methods of Preparing the Antibodies

[0136] The present disclosure also provides isolated polynucleotides or nucleic acid molecules encoding the antibodies, CARs, variants or derivatives thereof of the disclosure. The polynucleotides of the present disclosure may encode the entire heavy and light chain variable regions of the antigen-binding polypeptides. CARs, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules. Additionally, the polynucleotides of the present disclosure may encode portions of the heavy and light chain variable regions of the antigen-binding polypeptides, CARs, variants or derivatives thereof on the same polynucleotide molecule or on separate polynucleotide molecules.

[0137] Provided is an isolated nucleic acid encoding the anti-nectin-4 antibody or antigenbinding fragment thereof, or CARs as provided herein.

[0138] Provided is a vector including the isolated nucleic acid.

[0139] Provided is a host cell including the vector or the isolated nucleic acid.

[0140] Provided is a method of manufacturing an anti-nectin-4 antibody or antigen-binding fragment thereof, the method including expressing the antibody or antigen-binding fragment thereof by culturing the host cell.

[0141] According to another aspect of the present disclosure, provided is an isolated nucleic acid encoding the antibody or antigen-binding fragment thereof or CARs. The same parts described above are also applied to the nucleic acid.

[0142] As used herein, the term "nucleic acid" refers to a comprehensive term including DNA and RNA molecules, and nucleotide, as a basic structural unit thereof, includes not only natural nucleotides but also analogues in which a sugar or base is modified. The sequence of the nucleic acid encoding the heavy chain and light chain variable region according to an embodiment may be modified. The modification includes addition, deletion, or nonconservative or conservative substitution of nucleotides.

[0143] The nucleic acid is interpreted to include a nucleotide sequence exhibiting substantial identity to the nucleotide sequence of the nucleic acid as well. Substantial identity refers to a nucleotide sequence having a homology of 80 % or more, more specifically, a homology of 90 % or more, even more specifically, 95 % or more, and most specifically, 99 % or more in the case of aligning the nucleotide sequence of an aspect to correspond to another nucleotide sequence as much as possible, and analyzing the aligned sequence by using algorithm commonly used in the art.

[0144] According to another aspect of the present disclosure, provided is a vector including the isolated nucleic acid. The same parts described above are also applied to the vector.

[0145] For expression of an antibody or fragments thereof in an appropriate host cell, the vector may be obtained by constructing a DNA encoding partial or full-length light chains and heavy chains by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma that expresses a target antibody), and may include essential regulatory elements operably linked for expression of the inserted DNA (gene). The "operably linked" refers to functional linkage of a nucleic acid expression control sequence for performing generalfunctions and a nucleic acid sequence encoding a target protein or RNA, indicating that the gene is expressed by the expression control sequence.

[0146] The "expression control sequence" refers to a DNA sequence to regulate expression of an operably linked DNA sequence in a certain host cell. Such a control sequence includes a promotor for initiating transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence for terminating transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer. Those skilled in the art may recognize that a design of an expression vector may vary by selecting a different control sequence depending on factors such as choice of host cell to be transformed, and the level of protein expression.

[0147] Types of the vector are not particularly limited, as long as the vector is commonly used in the fields of cloning and antibody production, and examples thereof may include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and virus vectors. The plasmids may be plasmids derived from E. coll (pBR322. pBR325. pUCl 18. pUCl 19. and pET-21b(+)), plasmids derived from Bacillus subtilis (pUBl 10 and pTP5), and plasmids derived from yeasts (YEpl3, YEp24, and YCp50), and the virus may be retrovirus, animal virus such as adenovirus or vaccinia virus, and insect virus such as baculovirus. A pComb3 vector commonly used for phage display may be used, and vectors commonly used to express proteins in mammalian cells to express antibodies in mammalian cells, such as pcDNA or pVITRO may be used.

[0148] According to another aspect of the present disclosure, provided is an isolated host cell transformed with the vector. The same parts described above are also applied to the host cell.

[0149] As used herein, the term "transformation" refers to a process in molecular biology by which a DNA chain fragment or plasmid including a foreign gene different from that originally contained in a cell is introduced into the cell and binds to DNA present in the original cell, thereby changing genetic characteristic of the cell. The vector is transfected into a host cell. Various techniques commonly used in introduction of exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic cells for transfection, such as electrophoresis, calcium phosphate precipitation. DEAE-dextran transfection, or lipofection may be used.

[0150] The antibody or antigen-binding fragment thereof according to an aspect may be expressed in microorganisms such as bacteria (E. coli) or yeast, or eukary otic cells, particularly, mammalian host cells in consideration of applicability to mammalian cells. The mammalian host cell may be, for example, one selected from the group consisting of Chinese Hamster Ovary (CHO) cell, NSO myeloma cell, COS cell, SP2 cell, F2N cell, HEK293 cells, and antibody -producing hybridoma cell, but is not limited thereto.

[0151] According to another aspect of the present disclosure, provided is a method of preparing an anti-nectin-4 antibody or antigen-binding fragment thereof, the method including expressing the antibody or antigen-binding fragment thereof by culturing the isolated host cell. The same parts described above are also applied to the method.

[0152] The method may include transforming a host cell used to produce the antibody or antigen-binding fragment thereof according to an aspect with a vector operably linked to a DNA encoding the antibody or antigen-binding fragment thereof. Types of the selected host cell and the expression vector are as described above, and this process may be performed by selecting an appropriate transformation method. In the case where a recombinant expression vector encoding the antibody is introduced into a mammalian host cell, the antibody may be produced by culturing the host cell for a sufficient period of time for expression of the antibodyin the host cell, or more specifically, for a sufficient period of time for secreting the antibody into a culture medium in which the host cells is cultured.

[0153] In addition, the method may include producing a polypeptide of the antibody or an antigen-binding fragment according to an aspect from a recombinant expression vector introduced into the host cell by further culturing the transformed isolated host cell. A composition of the culture medium for culturing the selected host cell, and a culture time may be appropriately selected, and the antibody molecules produced by the host cell may be accumulated in cytoplasm of the cell, secreted out of the cell or into the culture medium by an appropriate signaling sequence, or targeted into periplasm, and the like. In addition, the antibody according to an aspect may be refolded to have a functional structure such that binding specificity to nectin-4 is maintained by using a method known in the art. Also, in the case of producing antibodies in the form of IgG, the heavy chain and the light chain may be expressed in separate cells and brought into contact with each other to construct a complete antibody, orthe heavy chain and the light chain may be expressed in the same cell to construct a complete antibody in the cell.

[0154] Also, the method may further include recovering the antibody or antigen-binding fragment thereof produced in the isolated host cell. A recovery method may be appropriately selected and controlled in consideration on polypeptide characteristics of the antibody or antigen-binding fragment thereof produced in the host cells, characteristics of the host cells, expression method, or targeting of the polypeptide. For example, the antibody or antigenbinding fragment thereof secreted into the culture medium may be recovered by a method including collecting the culture medium used to culture the host cell, and removing impurities by centrifugation. If required, the cell may be lysed within a range not affecting the functional structure of the antibody or antigen-binding fragment to release the antibody present in specific organelles or cytoplasm and recover the antibody.

[0155] The recovered antibody may be subjected to additional processes for impurity removal and concentration by chromatography, filtration using a filter, dialysis, and the like. Separation or purification of the recovered antibody may be performed by a separation and purification method commonly used in proteins, such as chromatography. The chromatography may include, for example, affinity chromatography including protein A column, protein G column, and protein L column, ion exchange chromatography, or hydrophobic chromatography. In addition to the above-described chromatography, the antibody may be isolated and purified by combining filtration, ultrafiltration, salting out, dialysis, and the like.Antibody-Drug Conjugates

[0156] According to another aspect of the present disclosure, provided is an antibody-drug conjugate including the anti-nectin-4 antibody or antigen-binding fragment thereof and a drug. The same parts described above are also applied to the conjugate.

[0157] As used herein, the term "antibody-drug conjugate (ADC)" refers to a conjugate in which the antibody (or antigen-binding fragment thereof) of the present disclosure is linked to a drug, and may also be referred to as an immunoconjugate. The antibody-drug conjugate may be prepared by various methods known in the art. In the antibody-drug conjugate, an anticancer drug should be stably bound to the antibody until the anticancer drug is delivered to a target cancer cell. The drug delivered to the target should be released from the antibody and inducethe death of the target cell. To this end, the drug needs to be stably bound to the antibody while also having cytotoxicity sufficient for inducing the death of the target cell at the time of being released from the target cell.

[0158] The antibody -drug conjugate may be one in which the drug is directly bound to the antibody, or one in which the drug is indirectly bound to the antibody or antigen-binding fragment thereof via any means such as a linker. The linker may include a non-cleavable linker or a cleavable linker. In general, the antibody-drug conjugate may be designed such that the drug is indirectly bound to the antibody via a means such as a linker and then released as the linker is cleaved in a target cell.

[0159] In general, it is know n that the ADC is ideally designed such that the drug is indirectly bound to the antibody via a means such as a linker and cleaved in a target cell. The linker may be cleaved in an intracellular environment, e.g., by a cleaving agent present in lysosomes or endosomes, for example, may be a peptide linker cleaved by a peptidase or protease in a cell, such as lysosome or endosome protease. In general, the peptide linker is at least two amino acids long. For example, the cleavable linker may be pH sensitive, which means that it may be susceptible to hydrolysis at a certain pH value. In general, the pH sensitive linkers may be hydrolyzed under acidic conditions. For example, an acid labile linker that may be hydrolyzed in lysosomes such as hydrazone, semi carbozone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, and the like. As another example, the linker may be cleaved under reducing conditions and may be, for example, a disulfide linker. Various disulfide bonds may be formed by using N-succinimidyl-S-acetylthioacetate (SATA), N-succinimidyl-3-(2- pyridyldithiojpropionate (SPDP), N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB). and N- succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene (SMPT).

[0160] The drug and / or drug-linker may be conjugated randomly via lysine of the antibody, or conjugated via cysteine exposed when the disulfide bond chain is reduced. In some cases, the linker-drug may be bound via cysteine present in a genetically engineered tag, such as a peptide or protein. The peptide or protein may have deletion at the carboxy terminal of the peptide or protein, or addition by a covalent bond of a spacer unit at the carboxy (C) terminal of the peptide or protein.

[0161] As used herein, the term "drug" may include, without limitation, any substances to be used to treat disease such as a substance by binding to the antibody of the present disclosure to increase the efficacy of the therapeutic antibody itself, increasing the half-life of the antibody in the blood, or arriving at a target location to eliminate a cause of disease such as cancer in the target location. Examples thereof include, but are not limited to, cytotoxic drugs, toxins, cytokines, chemokines, antibiotics, enzymes such as nucleases, radionuclides, photosensitizers, photothermal nanomaterials, nanoparticles, and micelles.

[0162] In the present disclosure, the drug may be directly linked (bound) to the antibody, or indirectly linked thereto via a known means. In addition, the drug bound to the antibody may be applied in any specific carrier form (e.g., a form supported on micelles, nanoparticles, liposomes, or dendrimers) of the drug.

[0163] The cytotoxic drug refers to a drug that may be used to treat a disease, and examples of drugs having anticancer activity (anticancer drugs) include microtubulin structure formation inhibitors, meiosis inhibitors, topoisomerase inhibitors, or DNA intercalators. The cytotoxic drug may include camptothecin, maytansinoid, auristatin, dolastatin, trichothecene, CC-1065 drug (NSC 298223), calicheamicin, enediynes, taxane, anthracycline, methotrexate, adriamycin, vindesine, vinca alkaloid, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, daunomycin. etoposide, teniposide, carminomycin, aminopterin, dactinomycin, bleomycin, esperamicin, 5-fluorouracil, melphalan, nitrogen mustar (mechlorethamine HCL), cis-platinum and homologues thereof, cisplati, CPT-11, docetaxel, monomethyl auristatin E, monomethyl auristatin F, emtansine (DM1), or derivatives thereof, but is not limited thereto.

[0164] The antibody -drug conjugate may be one having a molar drug-antibody / antibody fragment ratio (DAR) of 1 or more, i.e., one in which at least one drug molecule is attached to the antibody / antibody fragment. The antibody-drug conjugate may have a DAR of about 1 to about 20. specifically, an average DAR of about 1 to about 20, about 1 to about 16, about 1 to about 12. about 1 to about 10, about 1 to about 8, about 1 to about 7, about 1 to about 6, about 1 to about 5, about 2 to about 20, about 2 to about 1 , about 2 to about 12, about 2 to about 10, about 2 to about 8, about 2 to about 7, about 2 to about 6, about 2 to about 5, about 4 to about 20, about 4 to about 16, about 4 to about 12, about 4 to about 10, about 4 to about 8, about 4 to about 7, about 4 to about 6, about 4 to about 5, about 5 to about 20. about 5 to about 16, about 5 to about 12, about 5 to about 10, about 5 to about 8, or about 5 to about 6. In someembodiments, the average DAR may be about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 15, or 20.

[0165] In an embodiment, the antibody-drug conjugate including the anti-nectin-4 antibody of the present disclosure may be one with reduced toxicity against normal cells, specifically, one with reduced cytotoxicity against normal keratinocytes.

[0166] In an embodiment, the at least one amino acid substitution of the anti-nectin-4 antibody of the present disclosure may be intended to reduce toxicity' of the antibody-drug conjugate including the antibody or antigen-binding fragment thereof against normal keratinocytes, specifically, the at least one amino acid substitution may be intended to increase toxicity of the antibody-drug conjugate including the antibody or antigen-binding fragment thereof against cancer cells expressing nectin-4 compared to toxicity thereof to normal keratinocytes.

[0167] In an embodiment, the antibody-drug conjugate including ANX-ED133, ANX-ED141. ANX-ED143, or ANX-ED148, which is the anti-nectin-4 antibody of the present disclosure, may have reduced toxicity against normal cells compared to an antibody-drug conjugate including enfortumab. In the case where the toxicity of the antibody-drug conjugate including enfortumab against normal cells is set to 100 %. the antibody-drug conjugate including the anti- nectin-4 antibody of the present disclosure may have a toxicity of about 5% to about 20%. about 5% to about 22%, about 5% to about 24%, about 5% to about 26%, about 5% to about 28%, about 5% to about 30%, about 5% to about 32%, about 5% to about 36%, about 5% to about 38%. about 5% to about 40%, about 5% to about 50%, about 10% to about 20%, about 10% to about 22%. about 10% to about 24%, about 10% to about 26%, about 10% to about 28%, about 10% to about 30%, about 10% to about 32%, about 10% to about 36%, about 10% to about 38%, about 10% to about 40%, about 10% to about 50%, about 15% to about 20%, about 15% to about 22%, about 15% to about 24%, about 15% to about 26%, about 15% to about 28%. about 15% to about 30%, about 15% to about 32%, about 15% to about 36%, about 15% to about 38%. about 15% to about 40%, about 15% to about 50%. about 20% to about 22%, about 20% to about 24%, about 20% to about 26%, about 20% to about 28%, about 20% to about 30%, about 20% to about 32%, about 20% to about 36%, about 20% to about 38%, about 20% to about 40%, about 20% to about 50%, about 22% to about 24%. about 22% to about 26%. about 22% to about 28%, about 22% to about 30%. about 22% to about 32%, about 22% to about 36%, about 22% to about 38%, about 22% to about 40%, about 22% to about50%, about 24% to about 26%, about 24% to about 28%, about 24% to about 30%, about 24% to about 32%, about 24% to about 36%, about 24% to about 38%, about 24% to about 40%, about 24% to about 50%, about 26% to about 28%, about 26% to about 30%. about 26% to about 32%, about 26% to about 36%, about 26% to about 38%, about 26% to about 40%, about 26% to about 50%, about 28% to about 30%, about 28% to about 32%, about 28% to about 36%, about 28% to about 38%, about 28% to about 40%, about 28% to about 50%. about 30% to about 32%, about 30% to about 36%, about 30% to about 38%, about 30% to about 40%, about 30% to about 50%, about 32% to about 36%, about 32% to about 38%, about 32% to about 40%, about 32% to about 50%, about 36% to about 38%, about 36% to about 40%, about 36% to about 50%, about 38% to about 40%, about 38% to about 50%, or about 40% to about 50%.

[0168] In an embodiment, the antibody-drug conjugate including the anti-nectin-4 antibody of the present disclosure may be one having increased toxicity' against cancer cells compared to normal cells (cancer cell toxicity / normal cell toxicity), specifically, one in which cytotoxicity' against cancer cells increase compared to cytotoxicity against normal keratinocytes.

[0169] In an embodiment, the antibody-drug conjugate including ANX-ED133, ANX-ED141, ANX-ED143, or ANX-ED148, which are anti-nectin-4 antibody of the present disclosure, have increased toxicity against cancer cells compared to the antibody-drug conjugate including enfortumab. In the case where the cancer cell toxicity / normal cell toxicity ratio of the antibodydrug conjugate including enfortumab is set to 1, the antibody-drug conjugate including the anti- nectin-4 antibody of the present disclosure may exhibit a toxicity of about 1.5 to about 10, specifically, a cancer cell toxicity / normal cell toxicity ratio of about 1.5 to about 10, about 1.5 to about 8, about 1.5 to about 6, about 1.5 to about 5.5, about 1.5 to about 5.0, about 1.5 to about 4.5, about 1.5 to about 3.0, about 2.0 to about 10, about 2.0 to about 8, about 2.0 to about 6, about 2.0 to about 5.5, about 2.5 to about 10, about 2.5 to about 8, about 2.5 to about 6, about 2.5 to about 5.5, about 2.5 to about 5.0, about 2.5 to about 4.5, about 2.5 to about 4.0, about 3.0 to about 10, about 3.0 to about 8, about 3.0 to about 6, or about 3.0 to about 5.5.

[0170] In certain embodiments, the antibody-drug conjugate is conjugated to vedotin.Pharmaceutical Compositions

[0171] According to another aspect of the present disclosure, provided is a pharmaceutical composition for preventing or treating cancer including the anti-nectin-4 antibody or antigenbinding fragment thereof or an antibody-drug conjugate including the anti-nectin-4 antibody or antigen-binding fragment thereof and a drug. The same parts described above are also applied to the composition. In some embodiments, the composition further includes a second anticancer agent (e.g., an immune checkpoint inhibitor).

[0172] Provided is a pharmaceutical composition for preventing or treating cancer including the anti-nectin-4 antibody or antigen-binding fragment thereof, the multifunctional (e.g.. bifunctional) molecule, or the CAR, and a pharmaceutically acceptable carrier.

[0173] Provided is a pharmaceutical composition for preventing or treating cancer including the antibody-drug conjugate.

[0174] According to another aspect of the present disclosure, provided is a pharmaceutical composition for preventing or treating cancer including the anti-nectin-4 antibody or antigenbinding fragment thereof. The same parts described above are also applied to the composition.

[0175] The composition may further include a drug, and specifically the drug may be directly linked to the anti-nectin-4 antibody or antigen-binding fragment thereof or indirectly linked thereto via a linker.

[0176] The pharmaceutical composition may include a pharmaceutically acceptable carrier. The "pharmaceutically acceptable carrier" may refer to a carrier or diluent that does not stimulate living organism nor inhibit biological activity and properties of an administered compound. In this regard, the "pharmaceutically acceptable" means not inhibiting the activity of an active ingredient nor having toxicity beyond what an application (prescription) target may adapt. Types of carrier usable in the present disclosure may be any carrier as long as the carrier is generally used in the art and is pharmaceutically acceptable. Non-limiting examples of the carrier may be lactose, dextrose, maltodextrin, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, ethanol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, a saline solution, sterile water, Ringer's solution, buffered saline, an albumin injection solution,methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. They may be used alone or a mixture of at least two thereof may be used. The pharmaceutical composition including a pharmaceutically acceptable carrier in addition to an active ingredient may be prepared into formulations for oral or parenteral administration according to the route of administration by a common method know n in the art. The pharmaceutical composition may be prepared as formulations for oral administration such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, preparations for external use. suppositories, or sterile injectable solutions according to common methods, respectively.

[0177] The pharmaceutical composition may be formulated by using a diluent or excipient commonly used in the art such as a filler, an extender, a binder, a humectant, a disintegrant, and a surfactant, but may not be limited thereto.

[0178] In the case where the pharmaceutical composition is prepared into a formulation for oral administration, the pharmaceutical composition may be prepared into formulations such as powders, granules, tablets, pills, sugar coating tablets, capsules, liquids, gels, syrups. suspensions, and wafers together with a suitable carrier, by a method known in the art. In this regard, examples of suitable pharmaceutically acceptable carrier may include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol, starches such as com starch, potato starch, and wheat starch, celluloses such as cellulose, methylcellulose, ethylcellulose, sodium carboxymethyl cellulose, and hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, and the like. In a case of being made into preparations, the preparations may include a diluent and / or excipient such as a filler, an extender, a binder, a humectant, a disintegrant, and a surfactant.

[0179] In the case where the pharmaceutical composition of the present disclosure is prepared into a parenteral formulation, the pharmaceutical composition may be prepared into injections, transdermal drugs, nasal inhalers, and suppositories, together with a suitable carrier by a method known in the art. As a suitable carrier in the case of injections, sterilized w ater, ethanol, polyol such as glycerol and propylene glycol, or a mixture thereof may be used. Specifically, isotonic solutions such as Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, and 5% dextrose, and the like may be used. In the case of transdermal drugs, the pharmaceutical composition may be prepared into ointments,creams, lotions, gels, liquids for external use, pastes, liniments, aerosols, or the like. In the case of nasal inhalers, the pharmaceutical composition may be prepared in the form of aerosol spray by using an appropriate propellent such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide, and in the case of suppositories, a base therefor may include witepsol, tween 61, polyethylene glycol, cacao butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, glycerogelatin, or the like.

[0180] The pharmaceutical composition may be administered in a pharmaceutically acceptable amount. The term "pharmaceutically acceptable amount" refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and an effective dosage level may be determined by the severity of the disease, the activity of the drug, age, body weight, health status, gender, and drug sensitivity of the patient, administration time, route of administration, and excretion rate, and duration of treatment of the composition of the present disclosure used, drug used in combination or simultaneously with the composition used, and other factors well known in the medical field. The pharmaceutical composition of the present disclosure may be administered alone or in combination with other components know n to exhibit therapeutic effects on cancer diseases. It is important to administer a minimum amount for achieving a maximum effect without side effects in consideration of all of the above-described factors.

[0181] The dosage of the pharmaceutical composition may be determined by those skilled in the art in consideration of the purpose of use, the severity of a disease, age, body weight, gender, and anamnesis of a patient, types of substances used as active ingredients, and the like. For example, the pharmaceutical composition of the present disclosure may be administered in a dose of about 0. 1 ng to about 1,000 mg / kg, specifically, 1 ng to about 100 mg / kg per an adult, and administration frequency of the composition of the present disclosure is not particularly limited, but may be once a day or several times a day at divided doses. The dose or administration frequency does not limit the scope of the present disclosure in any way.

[0182] According to another aspect of the present disclosure, provided is a method of preventing or treating cancer, the method including administering the pharmaceutical composition to a subject. The same parts described above are also applied to the method.

[0183] As used herein, the term "subject" refers to a subject that require treatment of a disease, more specifically, a mammal such as a human or non-human primate, rodent (rat, mouse, guinea pig. etc.), dog, cat, horse, cow, sheep, pig, goat, camel, or antelope.

[0184] The pharmaceutical composition may be administered in a single dose or multiple doses in a pharmaceutically effective amount. In this case, the composition may be administered after being prepared in a formulation of liquid, powder, aerosol, injection, infusion (Ringer), capsule, pill, tablet, suppository, or patch. The pharmaceutical composition may be administered via any known route as long as the pharmaceutical composition arrives at a target tissue.

[0185] The pharmaceutical composition may be administered by. but not particularly limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, or rectal administration, depending on the purpose. However, in the case of oral administration, the pharmaceutical composition may be administered in an unformulated form. Because the active ingredient of the pharmaceutical composition may be denatured or decomposed by gastric acid, the composition for oral administration may be administered in a protected formulation in which an active ingredient is coated to be degraded in the stomach or as an oral patch. Also, the composition may be administered by any device capable of transporting an active ingredient to a target cell.

[0186] According to another aspect of the present disclosure, provided is a composition for diagnosing cancer including the anti-nectin-4 antibody or antigen-binding fragment thereof and a labeling group. The same parts described above are also applied to the composition.

[0187] The cancer may be cancer expressing nectin-4.

[0188] The labeling group may be linked to the anti-nectin-4 antibody or antigen-binding fragment thereof.Treatment Methods

[0189] As described herein, the antibodies or antigen-binding fragments, multifunctional (e.g., bifunctional) molecules, ADCs, CARs, variants or derivatives or compositions of the present disclosure may be used in certain treatment and diagnostic methods.

[0190] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of cancer (tumor) by administration of the pharmaceutical composition according to the present disclosure.

[0191] As used herein, the term "treatment" refers to any action that ameliorates or beneficially changes the symptoms of cancer (tumor) by administration of the pharmaceutical composition according to the present disclosure.

[0192] As used herein, the term "cancer" is a general term for disease caused by cells having an aggressive characteristic of dividing and growing beyond normal growth limits of cells, an invasive characteristic of invading surrounding tissues, and / or a metastatic characteristics of spreading to other parts of the body, and the cancer may be used in the same sense as malignant tumor.

[0193] By “subject” or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired. Mammalian subjects include humans, domestic animals, farm animals, and zoo, sport, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.

[0194] As used herein, phrases such as “to a patient in need of treatment” or “a subject in need of treatment” includes subjects, such as mammalian subjects, that would benefit from administration of an antibody or composition of the present disclosure used, e.g, for detection, for a diagnostic procedure and / or for treatment.

[0195] Provided is a method of preventing or treating cancer, the method including administering the antibody-drug conjugate or a composition including the same to an individual.

[0196] The present disclosure is further directed to antibody-based therapies which involve administering the antibodies or ADCs or compositions of the disclosure to a patient such as an animal, a mammal, and a human for treating one or more of the disorders or conditions described herein. Therapeutic compounds of the disclosure include, but are not limited to. antibodies or ADCs or compositions of the disclosure (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies of the disclosure (including variants and derivatives thereof as described herein).

[0197] The antibodies or ADCs or compositions of the disclosure can also be used to treat or inhibit cancer. In some embodiments, the cancer cells in the patient express or overexpress nectin-4. As provided above, nectin-4 can be overexpressed in tumor cells, in particular urothelial carcinoma, breast cancer, lung cancer, and ovarian cancer. Inhibition of nectin-4 has been shown to be useful for treating the tumors.

[0198] In an embodiment, the cancer may be a nectin-4-positive or nectin-4-overexpressing cancer. Specifically, the cancer may include at least one selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer.

[0199] Accordingly, in some embodiments, provided are methods for treating a cancer in a patient in need thereof. The method, in one embodiment, entails administering to the patient an effective amount of an antibody or an ADC or compositions of the present disclosure. In some embodiments, at least one of the cancer cells (e.g., stromal cells) in the patient over-express nectin-4.

[0200] Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies, are also provided in the present disclosure. A suitable cell can be used, that is put in contact with an anti-nectin-4 antibody of the present disclosure (or alternatively engineered to express an anti- nectin-4 antibody of the present disclosure). In some embodiments, the antibody is presented in a chimeric antigen receptor (CAR). Upon such contact or engineering, the cell can then be introduced to a cancer patient in need of a treatment. The cancer patient may have a cancer of any of the types as disclosed herein. The cell (e.g., T cell) can be, for instance, a tumorinfiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation.

[0201] In some embodiments, the cell is isolated from the cancer patient him- or her-self. In some embodiments, the cell is provided by a donor or from a cell bank. When the cell is isolated from the cancer patient, undesired immune reactions can be minimized. In some embodiments, the cell can be autologous. In some embodiments, the cell can be allogeneic.

[0202] In some embodiments, cancers may be a solid cancer. In some embodiments, the cancers may be liquid or blood cancer. Non-limiting examples of cancers include gastric cancer,colorectal cancer, hepatocarcinoma, bladder cancer, liver cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, neuroendocrine cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, oesophageal cancer, ovarian cancer, renal cancer, melanoma, prostate cancer and thyroid cancer.

[0203] Additional diseases or conditions associated with increased cell survival, that may be treated, prevented, diagnosed and / or prognosed with the antibodies or variants, or derivatives thereof of the disclosure include, but are not limited to, progression, and / or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic. promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillaiy carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

[0204] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative or compositions thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in theart. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0205] Methods of administration of the antibodies, ADCs, variants or derivatives, or compositions include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The antigen-binding polypeptides, ADCs or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides or ADCs of the disclosure may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.

[0206] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrastemal, subcutaneous and intra-articular injection and infusion.

[0207] Administration can be systemic or local. In addition, it may be desirable to introduce the antibodies or ADCs or compositions of the disclosure into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g, by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0208] It may be desirable to administer the antigen-binding polypeptides, ADCs or compositions of the disclosure locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction, with a w ound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous. or gelatinous material, including membranes, such as silastic membranes, or fibers.Preferably, when administering a protein, including an antibody or an ADC, of the disclosure, care must be taken to use materials to which the protein does not absorb.

[0209] The amount of the antibodies or ADCs or compositions of the disclosure which will be effective in the treatment, inhibition and prevention of an immune or malignant disease, disorder or condition can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease, disorder or condition, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0210] As a general proposition, the dosage administered to a patient of the antigen-binding polypeptides or ADCs of the present disclosure is ty pically 0.1 mg / kg to 100 mg / kg of the patient's body weight, between 0. 1 mg / kg and 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies or ADCs of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.

[0211] In an additional embodiment, the compositions of the disclosure are administered in combination with cytokines. Cytokines that may be administered with the compositions of the disclosure include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL- 15, anti-CD40, CD40L. and TNF-a.

[0212] In additional embodiments, the compositions of the disclosure are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0213] According to another aspect of the present disclosure, provided is a method of screening for an anti-nectin-4 antibody or antigen-binding fragment thereof capable of reducing toxicity of nectin-4 antibody-drug conjugate against normal cells, the method including 1) constructinga candidate antibody binding to nectin-4; 2) identifying affinity of the constructed candidate antibody for normal keratinocytes: 3) identity affinity of the constructed candidate antibody for nectin-4-expressing cancer cells: and 4) selecting an antibody with increased affinity for cancer cells compared to affinity for normal keratinocytes. The same parts described above are also applied to the method.

[0214] In the method, the identifying of affinity may include identifying binding strength of the candidate antibody to nectin-4 or nectin-4-expressing cells.

[0215] Also provided herein is a method of screening for an anti-nectin-4 antibody or antigenbinding fragment thereof capable of reducing toxicity of an anti-nectin-4 antibody-drug conjugate against normal cells, the method including 1) constructing a candidate antibody binding to nectin-4; 2) identify ing affinity of the constructed candidate antibody for normal keratinocytes; 3) identifying affinity of the constructed candidate antibody for cancer cells expressing nectin-4; and 4) selecting an antibody having increased affinity for cancer cells compared to affinity for normal keratinocytes.Diagnostic Methods

[0216] Over-expression of nectin-4 is observed in certain tumor samples, and patients having nectin-4-over-expressing cells are likely responsive to treatments with the anti-nectin-4 antibodies of the present disclosure. Accordingly, the antibodies of the present disclosure can also be used for diagnostic and prognostic purposes.

[0217] As used herein, the term "diagnosis" refers to identifying the presence or characteristics of a pathological condition. For the object of the present disclosure, the diagnosis may refer to determining whether a cancer expressing nectin-4 is developed.

[0218] A sample that preferably includes a cell can be obtained from a patient, which can be a cancer patient or a patient desiring diagnosis. The cell be a cell of a tumor tissue or a tumor block, a blood sample, a urine sample or any sample from the patient. Upon optional pretreatment of the sample, the sample can be incubated with an antibody of the present disclosure under conditions allowing the antibody to interact with a nectin-4 protein potentially present in the sample. Methods such as ELISA can be used, taking advantage of the anti-nectin-4 antibody, to detect the presence of the nectin-4 protein in the sample.

[0219] Presence of the nectin-4 protein in the sample (optionally with the amount or concentration) can be used for diagnosis of cancer, as an indication that the patient is suitable for a treatment with the antibody, or as an indication that the patient has (or has not) responded to a cancer treatment. For a prognostic method, the detection can be done at once, twice or more, at certain stages, upon initiation of a cancer treatment to indicate the progress of the treatment.Advantageous Effects of Invention

[0220] The novel anti-nectin-4 antibody of the present disclosure has appropriately adjusted affinity for nectin-4 and reduced cytotoxicity against normal cells compared to cytotoxicity against cancer cells, and thus the antibody and the antibody -drug conjugate including the antibody may exhibit excellent therapeutic effects while significantly reducing side effects.Mode for the Invention

[0221] Hereinafter, an aspect will be described in more detail with reference to the following examples. However, the following examples are merely presented to exemplify the aspect and the scope of the aspect is not limited thereto, and these examples are provided to fully convey the concept of the disclosure to those skilled in the art.Other embodiments

[0222] Embodiment 1. An anti-nectin-4 antibody or antigen-binding fragment thereof, comprising: a heavy chain variable region comprising a heavy chain complementarity determining region 1 (HCDR1) including an amino acid sequence of SEQ ID NO: 1, a heavy chain complementarity determining region 2 (HCDR2) including an amino acid sequence of SEQ ID NO: 2, and a heavy chain complementarity determining region 3 (HCDR3) including an amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1 ) including an amino acid sequence of SEQ ID NO: 4, a light chain complementarity determining region 2 (LCDR2) including an amino acid sequence of SEQ ID NO: 5, and a light chain complementarity determining region 3 (LCDR3) including an amino acid sequence of SEQ ID NO: 6, wherein the anti-Nectin-4 antibody or antigen-binding fragment thereof comprises at least one amino acid substitution in the heavy chain variable region and the light chain variable region.

[0223] Embodiment 2. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1 , wherein the at least one amino acid substitution comprises an amino acid substitution at one or more positions selected from the group consisting of a 6th position of SEQ ID NO: 4, a 6th position of SEQ ID NO: 5, a 1st position of SEQ ID NO: 6, and a 7th position of SEQ ID NO: 6.

[0224] Embodiment 3. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the at least one amino acid substitution comprises at least one substitution selected from the group consisting of the following amino acid substitutions:1) substitution of I. the 6th amino acid of SEQ ID NO: 4, with A;2) substitution of Q, the 6th amino acid of SEQ ID NO: 5, with A;3) substitution of Q, the 1st amino acid of SEQ ID NO: 6, with A; and4) substitution of P, the 7th amino acid of SEQ ID NO: 6, with A.

[0225] Embodiment 4. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region comprising HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and2) a light chain variable region comprising LCDR1 including an amino acid sequence of SEQ ID NO: 10, LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 6.

[0226] Embodiment 5. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region comprising HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and2) a light chain variable region comprising LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 7, and LCDR3 including an amino acid sequence of SEQ ID NO: 6.

[0227] Embodiment 6. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region comprising HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an amino acid sequence of SEQ ID NO: 3; and2) a light chain variable region comprising LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 8.

[0228] Embodiment 7. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen -binding fragment thereof comprises:1) a heavy chain variable region comprising HCDR1 including an amino acid sequence of SEQ ID NO: 1, HCDR2 including an amino acid sequence of SEQ ID NO: 2, and HCDR3 including an ammo acid sequence of SEQ ID NO: 3; and2) a light chain variable region comprising LCDR1 including an amino acid sequence of SEQ ID NO: 4, LCDR2 including an amino acid sequence of SEQ ID NO: 5, and LCDR3 including an amino acid sequence of SEQ ID NO: 9.

[0229] Embodiment 8. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen -binding fragment thereof comprises:1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and2) a light chain variable region including an amino acid sequence of SEQ ID NO: 13.

[0230] Embodiment 9. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and2) a light chain variable region including an amino acid sequence of SEQ ID NO: 14.

[0231] Embodiment 10. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 1 1; and 2) a light chain variable region including an amino acid sequence of SEQ ID NO: 15.

[0232] Embodiment 11. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody or antigen-binding fragment thereof comprises:1) a heavy chain variable region including an amino acid sequence of SEQ ID NO: 11; and2) a light chain variable region including an amino acid sequence of SEQ ID NO: 16.

[0233] Embodiment 12. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antibody is a chimeric antibody, a multispecific antibody, a human antibody, or a humanized antibody.

[0234] Embodiment 13. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or a two-chain Fv or single-chain Fv (scFv) fragment.

[0235] Embodiment 14. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein the at least one amino acid substitution is performed to adjust affinity of the antibody or antigen-binding fragment thereof for Nectin-4.

[0236] Embodiment 15. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1 , wherein the at least one amino acid substitution is performed to increase affinity of the antibody or antigen-binding fragment thereof for cancer cells expressing Nectin-4 as compared to affinity for normal keratinocytes.

[0237] Embodiment 16. The anti-nectin-4 antibody or antigen-binding fragment thereof of Embodiment 1, wherein a ratio of affinity for cancer cells expressing Nectin-4 to affinity for normal keratinocytes (Nectin-4-expressing cancer cell affinity / normal keratinocyte affinity) of the anti-Nectin-4 antibody or antigen-binding fragment thereof is improved as compared to that of enfortumab.

[0238] Embodiment 17. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of Embodiment s 1 to 16.

[0239] Embodiment 18. A vector comprising the isolated nucleic acid of Embodiment 17.

[0240] Embodiment 19. A host cell comprising the vector of Embodiment 18.

[0241] Embodiment 20. A method of preparing an anti-Nectin-4 antibody or antigen-binding fragment thereof, the method comprising expressing the antibody or antigen-binding fragment thereof by culturing the host cell of Embodiment 19.

[0242] Embodiment 21. A pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof of any one of Embodiment s 1 to 16.

[0243] Embodiment 22. The pharmaceutical composition of Embodiment 21, wherein the composition further comprises a drug.

[0244] Embodiment 23. The pharmaceutical composition of Embodiment 21 , wherein the cancer is a cancer overexpressing Nectin-4.

[0245] Embodiment 24. The pharmaceutical composition of Embodiment 21, wherein the cancer comprises at least one selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer.

[0246] Embodiment 25. A composition for diagnosing cancer, comprising: the antibody or antigen-binding fragment thereof of any one of Embodiment s 1 to 16; and a labeling group.

[0247] Embodiment 26. An antibody-drug conjugate comprising: the antibody or antigenbinding fragment thereof of any one of Embodiment s 1 to 16; and a drug.

[0248] Embodiment 27. The antibody-drug conjugate of Embodiment 26, wherein the drug is linked to the antibody or antigen-binding fragment thereof via a linker.

[0249] Embodiment 28. The antibody-drug conjugate of Embodiment 26, wherein the at least one amino acid substitution included in the antibody or antigen-binding fragment thereof reduces toxicity of the antibody -drug conjugate against normal keratinocytes.

[0250] Embodiment 29. The antibody-drug conjugate of Embodiment 26, wherein the at least one amino acid substitution is performed to increase toxicity of the antibody-drug conjugate including the antibody or antigen-binding fragment thereof against cancer cells expressing Nectin-4 as compared to toxicity to normal keratinocytes.

[0251] Embodiment 30. The antibody-drug conjugate of Embodiment 26, wherein toxicity of the antibody-drug conjugate against normal keratinocytes is reduced as compared to toxicity of an antibody -drug conjugate including enfortumab.

[0252] Embodiment 31. The antibody-drug conjugate of Embodiment 26, wherein toxicity' of the antibody-drug conjugate against Nectin-4-expressing cancer cells relative to toxicity of the antibody-drug conjugate against normal keratinocytes is increased as compared to that of an antibody-drug conjugate including enfortumab.

[0253] Embodiment 32. A pharmaceutical composition for preventing or treating cancer, comprising the antibody-drug conjugate of Embodiment 26.

[0254] Embodiment 33. The pharmaceutical composition of Embodiment 32, wherein the cancer is a cancer overexpressing Nectin-4.

[0255] Embodiment 34. The pharmaceutical composition of Embodiment 32, wherein the cancer comprises at least one selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer.

[0256] Embodiment 35. A method of screening for an anti -Nectin-4 antibody or antigenbinding fragment thereof capable of reducing toxicity of an anti-Nectin 4 antibody-drug conjugate against normal cells, the method comprising:1) constructing a candidate antibody that binds to Nectin-4;2) identifying affinity of the constructed candidate antibody for normal keratinocytes;3) identifying affinity of the constructed candidate antibody for Nectin-4-expressing cancer cells; and4) selecting an antibody with improved affinity for cancer cells as compared to affinity for normal keratinocytes.EXAMPLESEXAMPLE 1: Discovery of Novel Anti-Nectin-4 Antibody

[0257] An experiment was performed as described below to develop novel anti-nectin-4 antibodies with affinity for nectin-4.1.1: Designing and Constructing Novel Anti-Nectin-4 Antibody

[0258] Candidate antibodies were constructed by introducing mutations into enfortumab that is a known anti-nectin-4 antibody. First, a total of 91 types of candidate antibodies were derived by constructing 91 mutations in which a certain amino acid in the CDR region of enfortumab is substituted.1.2: First Screening - Analysis of Affinity for Nectin-4

[0259] In order to develop a novel anti-nectin-4 antibody having reduced cytotoxicity, a first screening was performed on the 91 types of candidate antibodies.

[0260] Specifically, the first screening was performed based on the results of affinity analysis of nectin-4 protein. In order to evaluate the affinity of the above candidate antibodies for nectin- 4 protein, a binding assay for nectin-4 was performed using an Octet device. In the above binding assay, a RED 384 device from Fortebio using biolayer interferometry technology was used to analyze the affinity- between nectin-4 protein and antibodies. In order to load the antibody onto the biosensor, a protein G biosensor capable of binding to the Fc portion of the antibody was used. Each antibody was diluted to 33.3 nM using assay buffer (PBS with 1% BSA) and then loaded onto the protein G biosensor. Nectin-4 protein was diluted to three concentrations (100 nM, 33.3 nM, 11.1 nM) using assay buffer (PBS with 1% BSA) and bound to the antibody loaded on the protein G biosensor, and the degree of binding w as evaluated. Binding was performed for 600 sec, and dissociation for 300 sec. Based on the sensorgram results, the binding strength between the antibody and nectin-4 was confirmed using the octet analysis program. Meanwhile, enfortumab, a known anti-nectin-4 antibody, was used as a control.

[0261] As a result of the above analysis, binding affinity of the total of 91 candidate antibodies for nectin-4 was identified (Table 5 and FIG. 1). In order to preform first screening to select antibodies with adjusted affinity for nectin-4 compared to enfortumab, as a control, antibodies with reduced KD (dissociation constant) by twice or more (21.0 nM) compared to that of enfortumab were selected (shaded portion of FIG. 1), and a total of 25 types of variant antibodies were obtained by the first screening.Table 51.3: Second Screening - Analysis on Level of Binding to Nectin-4-expressing Cell

[0262] In order to develop novel anti-nectin-4 antibodies, second screening was performed on the 25 ty pes of candidate antibodies selected by the first screening.

[0263] Specifically, in order to evaluate levels of binding to nectin-4 under in vitro conditions, FACS binding assay was performed on the 25 types of candidate antibodies for binding to MDA-MB-468, which is a breast cancer cell line known to express nectin-4 at intermediate levels. FACS Verse equipment from BD bioscience was used. MDA-MB-468 cells in passagewere detached from the flask using Tr psin-EDTA. neutralized with media, and only the cells were downed through centrifugation. The cells were lysed using assay buffer (PBS with 1% FBS) and the cell number was confirmed using a cell counter. Based on the counted cell number, 100,000 MDA-MB-468 cells were placed per well. For the above binding assay, each antibody was diluted 5-fold from 167 nM in assay buffer (PBS with 1% FBS). Binding was confirmed at a total of seven concentrations of the antibody. The diluted antibodies were added to the wells and incubated with the cells at 4 °C for 30 minutes. After incubation, the cells were washed and incubated with anti-hlgG-FITC detection antibody at 4 °C. After washing off the unbound detection antibody, the cells were analyzed by FACS. A 4-parameter curve was generated using the mean fluorescence intensity (MFI) values and the results were compared.

[0264] FACS binding assay was performed by selecting a certain concentration point (1.34 nM).

[0265] As a result of the analysis, FACS binding assay was performed on MDA-MB-468 at a concentration of 1.34 nM of the 25 types of candidate antibodies, and binding levels were identified (Table 6). The anti-nectin-4 antibodies to be discovered in the present disclosure are those with appropriately adjusted affinity to nectin-4, and clones with too high levels or too low levels of binding to MDA-MB-468 are not target clones finally desired to obtain, and thus 14 types of antibodies exhibiting the binding levels of 50.0 % to 90.0 % compared to that of enfortumab were selected by the second screening.Table 61.4: Third Screening - Analysis on Level of Binding to Nectin-4-expressing Cell

[0266] In order to develop novel anti-nectin-4 antibodies, third screening was performed on the 14 types of candidate antibodies selected by the second screening.

[0267] Specifically, based on the FACS binding assay method of Example 1.3, a full curve binding FACS assay was performed on the 14 types of candidate antibodies selected by the second screening and MDA-MB-468. The FACS binding of enfortumab antibody to MDA-MB- 468 in the full curve binding FACS assay was shown in FIG. 2.

[0268] As a result, it was confirmed that dose dependent 4-parameter curves were not properly formed in most clones. Because non-specific binding and defects in physical properties and the like may be caused in the case where the 4-parameter curve is not properly formed, and 4 types of antibodies (ANX-ED133, ANX-ED141, ANX-ED143, ANX-ED148) were selected as final candidate antibodies by the third screening.

[0269] As a result of identifying relative binding levels (% relative binding) based on ECso values of the selected 4 types of antibodies, binding activity of about 5 % to about 40 % w as confirmed compared to enfortumab although the relative binding levels of the 4 types of antibodies were different (Table 7).Table 7

[0270] Subsequently, based on the max binding of the selected 4 types of antibodies, a relative maximal binding level was calculated. The maximal binding level corresponds to a saturated value at an upper portion of the 4-parameter curve, indicating a level at which the antibody may maximally bind to nectin-4 at a certain concentration or more. As a result of analyzing relative maximal binding levels, it was confirmed that the selected 4 types of antibodies maintains the maximal binding levels similar to that of enfortumab (Table 8).Table 8

[0271] Specific amino acid sequences of CDR regions of the finally selected 4 types of antibodies (ANX-ED133, ANX-ED141 , ANX-ED143, and ANX-ED148) and enfortumab, as the control, are shown in Table 9, and specific amino acid sequences of variable heavy chain regions and variable light chains are shown in Table 10.Table 9Table 10EXAMPLE 2: Evaluation on Binding Level of Novel Anti-Nectin-4 Antibody to Normal Keratinocyte

[0272] An experiment was performed as described below to evaluate the level of binding of 4 types of the novel anti-nectin-4 antibodies selected in Example 1 to normal keratinocytes.

[0273] Specifically, based on the descriptions given above in Examples 1.3 and 1.4, FACS assay (full curve binding FACS) was performed on normal keratinocytes that are normal cells for the four antibodies. FACS Verse equipment from BD bioscience, was used as in the MDA- MB-468 FACS binding equipment. Since keratinocytes were known to express nectin-4 as their differentiation progresses, 2% FBS and 1.5 mM CaCh were added to the keratinocyte dermal cell basal media and then grown for 3-4 d to induce differentiation. The differentiation-induced keratinocytes were detached from the flask using Trypsin-EDTA, neutralized using media, and only the cells were downed by centrifugation. The cells were lysed using assay buffer (PBS with 1% FBS), and the cell number was confirmed using a cell counter device. Based on the counted cell number, keratinocytes were seeded at 100,000 cells per well. For the above binding assay, each antibody was diluted 5-fold from 167 nM in assay buffer (PBS with 1% FBS). Binding was confirmed at a total of 7 antibody concentrations. The diluted antibodies were added to the wells and incubated with the cells at 4 °C for 30 minutes. After incubation, thecells were washed and incubated with anti-hlgG-FITC detection antibody at 4 °C. After washing away the unbound antibody, the cells were analyzed by FACS. A 4-parameter curve was generated using the mean fluorescence intensity (MFI) value and the results were compared.

[0274] As a result, it w as confirmed that among the 4 types of antibodies, ANX-ED133, ANX- ED143 and ANX-ED148 antibodies had reduced binding level to the normal keratinocytes compared to enfortumab (FIG. 3, FIG. 4, and Table 11). Particularly, it was confirmed that the relative maximal binding level, to the cancer cells w as not reduced compared to enfortumab (Table 11), but the relative maximal binding level to the normal keratinocytes was reduced by 10 % or more compared to enfortumab.Table 11

[0275] The reduction in the maximal binding level to the cells indicates that less antibodies bind to the normal keratinocytes, compared to enfortumab, although an overdose of the antibodies was used. Thus, it was confirmed that affinity of the antibody for normal keratinocytes was reduced compared to enfortumab, and an antibody -drug conjugate prepared based on the antibody may have reduced toxicity7against normal keratinocytes that are normal skin cells.

[0276] Therefore, it may be expected that ANX-ED133, ANX-ED143. and ANX-ED148 having reduced relative maximal binding level value to normal keratinocytes finally have excellent safety' profiles.EXAMPLE 3: Evaluation on Cytotoxicity of Antibody-drug Conjugates Including Anti- Nectin-4 Antibodies

[0277] An experiment was performed as described below to evaluate cytotoxicity of antibodydrug conjugates including the novel anti-nectin-4 antibody prepared in Example 2 against normal skin cells and cancer cells.

[0278] Specifically, in order to compare and evaluate cytotoxicity with enfortumab vedotin that is an approved antibody-drug conjugate, antibody-drug conjugates (ADC), in which each of the 4 types of antibodies derived in Example 2 was conjugated with vedotin, were constructed, and cytotoxicity of the constructed ADCs against normal keratinocytes, as normal cells, and NCI- H292 cell lines, as lung cancer cell lines, were evaluated. For the evaluation of cytotoxicity, NCI-H292 cells or normal keratinocytes (respectively, 1,000 cell / well and 15,000 cell / well) were inoculated onto 96-well plate. For NCI-H292 cells, RPMI with 10% FBS was used, and for normal keratinocytes, 2% FBS and 1.5 mM CaC12 were used in dermal cell basal media for differentiation. The NCI-H292 cells were treated with 9 diluted antibody-vedotin conjugates in a final concentration range of 800 nM to 0.5 nM, respectively. The normal keratinocytes were treated with 9 diluted antibody-vedotin conjugates in a final concentration range of 625 nM to 0.00004 nM. respectively. The cells were incubated at 37 °C for 7 days. After incubation, celltiter blue was added to each well, followed by incubation for 4 hours more. Tn the case of cell titer blue, living cells show fluorescence when resazurin is reduced to resorufin, but dead cells do not show fluorescence because this reduction reaction does not occur. Therefore, it can be used to distinguish between living and dead cells. Fluorescence was read using a SpectraMax plate reader, and a 4-parameter curve was generated with the read values and the results were compared.

[0279] As a result, in the case of the ADCs in which the 4 types of antibodies developed in the present disclosure were conjugated with vedotin, it was confirmed that cytotoxicity against NCI-H292 cells, as cancer cells, slightly decreased (75.9 to 83.7 %) in comparison with the enfortumab-vedotin ADC (FIG. 5 and Table 12), representing slight decrease in efficacy for the ADCs from the 4 antibodies compared to enfortumab ADC. On the contrary, it was confirmed that cytotoxicity to normal keratinocytes, as normal cells, was about 16.0 to about 38.0 % in comparison with the enfortumab-vedotin ADC indicating a significant reduction (FIG. 6 and Table 12), representing significant improvement in safety for the ADCs from the 4 antibodies compared to enfortumab ADC. Particularly, in the case of ANX-ED143, it was confirmed that cytotoxicity against NCI-H292 cells was 83.7 % compared to that of enfortumab-vedotin ADC, but cytotoxicity against normal keratinocytes was 16.0 % compared to that of enfortumab- vedotin ADC, indicating that cytotoxicity against normal cells was significantly reduced compared to that against cancer cells.Table 12

[0280] Therefore, based on the results, the novel anti-nectin-4 antibodies of the present disclosure had significantly reduced cytotoxicity against normal cells compared to enfortumab, as a control, by appropriately adjusting affinity for nectin-4, and thus the antibody-drug conjugate including the antibody and the antibody may have excellent therapeutic effects while reducing side effects.EXAMPLE 4: Generation of Fc-engineered Antibodies

[0281] Fc-engineered antibodies were generated from enfortumab (“WT’?) and ANX-ED143 (‘’ED143’’). Specifically, antibodies having two point mutations at Fc region (L234A and L235A) were generated. (“WT-LALA” and “ED143-LALA”). Sequences of the Fc-engineered antibodies are shown in Table 13 below.Table 13

[0282] Binding of the Fc-engineered antibodies was measured using a FcyRIIIa binding assay, and their binding affinity to FcyRIIIa was compared with enfortumab (WT). Specifically, to measure the FcyRIIIa binding activity of the candidate substance, the surface plasmon resonance (SPR) method was used. FcyRIIIa ligand at a concentration of 3.0 ug / mL was immobilized on the Series S sensor chip CM5 (CM5 chip) at a level of 150 RU. The candidate substance was flowed through the CM5 chip immobilized with FcyRIIIa ligand at various concentrations (0, 32, 80, 200, 250, and 1,250 nM). The change in resonance angle according to the strength of binding to the FcyRIIIa ligand during the time the candidate substance passes through the immobilized CM5 chip is displayed in the form of sensorgrams, and the Resonance Unit (RU) value was confirmed through the two state reaction model analysis of the displayed sensorgram. The stronger the binding to the FcyRIIIa ligand, the larger the Resonance Unit (RU) value, so the FcyRIIIa binding activity is indicated through RU.

[0283] In Table 14 and FIG. 7, the result from the assay is shown in Resonance Unit (RU) and % relative RU compared to enfortumab (WT). As shown in Table 14 and FIG. 7, the antibodies with LALA mutations exhibited minimal binding to FcyRIIIa, thus these antibodies will have minimal Fc-mediated effector functions, compared to wild-type antibodies.Table 14aRelative to WT antibodyEXAMPLE 5 - Internalization of Anti-Nectin-4 Antibodies

[0284] Internalization activity of the Fc-engineered antibodies and enfortumab were measured. Specifically, the cells (HT-1376. NCI-H322, MDA-MB-468, NCI-H292) were plated at a density of 10,000 cells / 96 well. The next day, the antibody samples were prepared at 10 ug / mL and labeled using labeling reagent (Incucyte® Human Fabfluor-pH Orange Antibody Labeling Dyes, Cat # 4812). Specifically, the antibody samples and labeling reagent were prepared at the same concentration (each 10 ug / mL), mixed equal amounts in a microtube, and stood at 37 degrees for 15 minutes. The labeled antibodies were added to the cells plated yesterday and placed in a CO2 incubator at 37 degrees for 24 hours. After 24 hours, the fluorescence (RFP, red fluorescent protein) signal in the cells was measured using Cytation 7 imaging equipment.Since the labeled antibodies show fluorescence only when they are internalized in the cell and encounter the lysosome (fluorescence is shown in the low pH environment of the lysosome), the stronger the measured RFP signal, the greater the internalization into the cell. Accordingly, the intensity of the fluorescence value obtained using the Cytation 7 imaging equipment represents the internalization activity.

[0285] In Table 15. the result from the assay is shown in % relative intensity compared to normal human immunoglobulin G (IgG). As shown in Table 15, the ADX-ED143 antibodies exhibited greater internalization compared to enfortumab (“WT”).Table 15

[0286] The above description of the disclosure is for illustrative purposes, and those skilled in the art to which the disclosure belongs will be able to understand that the examples and embodiments can be easily modified without changing the technical idea or essential features of the disclosure. Thus, it is clear that the above-described embodiments of the present disclosure are illustrative in all aspects and do not limit the present disclosure.

Claims

CLAIMS1 . An antibody or antigen-binding fragment thereof having a binding specificity to human nectin-4, wherein the antibody or antigen-binding fragment thereof binds to a cancer cell with relative maximal binding level of about 97%-110% relative to a reference antibody, while the antibody or antigen-binding fragment thereof binds to a non-cancer cell with relative maximal binding level of about 90% or less relative to the reference antibody, wherein the reference antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO.: 11, and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO.: 12.

2. The antibody or the antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD value of 2. 1 x 1 O'8M or greater.

3. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD value of two folds or greater compared to the reference antibody.

4. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 on a cancer cell with binding affinity of about 50% to about 90% compared to the reference antibody.

5. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof binds to anon-cancer cell with binding affinity of about 80% to about 90% relative to the reference antibody.

6. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof exhibits B-value (slope) between about 0.70 or greater for dose dependent 4-parameter curve for binding to a cancer cell under full curve binding FACS assay.

7. An antibody or antigen-binding fragment thereof having a binding specificity to human nectin cell adhesion molecule 4 (nectin-4), wherein the antibody or the antigen-binding fragmentthereof binds to a cancer cell with ECso of 0.25 nM or greater, and binds to a non-cancer cell with ECso of 1.0 nM or greater.

8. The antibody or the antigen-binding fragment thereof of claim 7, wherein the antibody or antigen-binding fragment thereof binds to the cancer cell with ECso of about 0.25-2.5 nM.

9. The antibody or the antigen-binding fragment thereof of claim 7 or 8. wherein the antibody or antigen-binding fragment thereof binds to the non-cancer cell with ECso of about 1.0- 4.0 nM.

10. The antibody or antigen-binding fragment thereof of any one of claims 7-9, wherein the ECso is measured by FACS.

11. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the cancer cell is a solid cancer cell.

12. The antibody or antigen-binding fragment thereof of claim 11 , wherein the cancer cell is MDA-MB-468 cell.

13. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the non-cancer cell is keratinocyte.

14. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof shows greater internalization activity compared to the reference antibody.

15. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having one or more amino acid substitution relative to the amino acid sequence of SEQ ID NO: 11; or a light chain variable region having one or more amino acid substitution relative to the amino acid sequence of SEQ ID NO: 12.

16. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDR) HCDR1. HCDR2, and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and wherein one of the LCDR sequence is selected from the group consisting of SEQ ID NOs: 7, 8, 9, and 10.

17. An antibody or antigen-binding fragment thereof having a binding specificity to human nectin-4 antibody or antigen-binding fragment thereof, wherein the antibody or the antigenbinding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining regions (CDR) HCDR1, HCDR2, and HCDR3, and a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3, and wherein: the HCDR1 comprises an amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 1; the HCDR2 comprises an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 2; the HCDR3 comprises an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having one, two, or three amino acid substitution relative to SEQ ID NO: 6, wherein at least one of HCDR1, HCDR2, HCDR3. LCDR1, LCDR2, LCDR3 has one or more amino acid substitutions relative to SEQ ID Nos: 1, 2, 3, 4, 5, or 6, respectively.

18. The antibody or antigen-binding fragment thereof of claim 17, wherein: the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 2;the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having an alanine substitution relative to SEQ ID NO: 6.

19. The antibody or antigen-binding fragment thereof of claim 17 or 18, wherein: the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4 or 10; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5 or 7; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6, 8 or 9.

20. The antibody or antigen-binding fragment thereof of any one of claims 17-19, wherein:(a) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 10; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(b) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6;(c) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3;the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8; or (d) the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1: the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 9.

21. The antibody or antigen-binding fragment thereof of any one of claims 17-20, wherein(a) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 1 , and the VL comprises the amino acid sequence of SEQ ID NO: 13, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 13;(b) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 14, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 14;(c) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 15, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 15; or(d) the VH comprises the amino acid sequence of SEQ ID NO: 11, or a peptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 11 and the VL comprises the amino acid sequence of SEQ ID NO: 16, or a peptide having at least 90% sequence identity7to the amino acid sequence of SEQ ID NO: 16.

22. The antibody or antigen- binding fragment thereof of any one of claims 17-20, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 10; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acidsequence of SEQ ID NO: 6.

23. The antibody of claim 22, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 13.

24. The antibody or antigen-binding fragment thereof of any one of claims 17-20, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 7; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 6.

25. The antibody of claim 24, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 15.

26. The antibody or antigen- binding fragment thereof of any one of claims 17-20, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 8.

27. The antibody of claim 26, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 16.

28. The antibody or antigen-binding fragment thereof of any one of claims 17-20, wherein the HCDR1 comprises the amino acid sequence of SEQ ID NO: 1; the HCDR2 comprises the amino acid sequence of SEQ ID NO: 2; the HCDR3 comprises the amino acid sequence of SEQ ID NO: 3; the LCDR1 comprises the amino acid sequence of SEQ ID NO: 4; the LCDR2 comprises the amino acid sequence of SEQ ID NO: 5; and the LCDR3 comprises the amino acid sequence of SEQ ID NO: 9.

29. The antibody of claim 28, wherein the VH comprises the amino acid sequence of SEQ ID NO: 11, and the VL comprises the amino acid sequence of SEQ ID NO: 16.

30. The antibody or antigen-binding fragment thereof of any one of claims 17-29, which binds to a cancer cell with relative maximal binding of about 97%-l 10% relative to a reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, while binds to a non-cancer cell with relative maximal binding of about 90% or less relative to the reference antibody.

31. The antibody or the antigen-binding fragment thereof of any one of claims 16-29, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD of 2. 1 x 10‘8M or greater.

32. The antibody or the antigen-binding fragment thereof of any one of claims 17-31, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with a KD of two folds or greater compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

33. The antibody or the antigen-binding fragment thereof of any one of claims 17-32, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity with about 50% to about 90% compared to the reference antibody having a heavy7chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

12. under full curve binding FACS assay against a cancer cell.

34. The antibody or the antigen-binding fragment thereof of any one of claims 17-32, wherein the antibody or antigen-binding fragment thereof has binding affinity to non-cancer cell of about 80% to about 90% relative to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

35. The antibody or the antigen-binding fragment thereof of any one of claims 17-34, wherein the antibody or antigen-binding fragment thereof exhibits B-value (slope) between about 0.70 or greater for dose dependent 4-parameter curve for binding to MDA-MB-468 under full curve binding FACS assay.

36. The antibody or antigen-binding fragment thereof of any one of claims 17-35, which binds to a cancer cell with EC 50 of 0.25 nM or greater, and binds to a non-cancer cell with EC50 of 1.0 nM or greater.

37. The antibody or antigen-binding fragment thereof of any one of claims 17-36, which binds to a cancer cell with a greater affinity compared to a non-cancer cell, wherein EC50 for the noncancer cell is at least 1.5-fold greater than EC50 for the cancer cell.

38. The antibody or the antigen-binding fragment thereof of any one of claims 17-37, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity to a cancer cell with EC50 of about 0.25-2.5 nM.

39. The antibody or the antigen-binding fragment thereof of any one of claims 17-38, wherein the antibody or antigen-binding fragment thereof binds to nectin-4 with binding affinity to a non- cancer cell with EC50 of about 1.0-4.0 nM.

40. The antibody or antigen-binding fragment thereof of any one of claims 36-39, wherein the EC50 is measured by FACS.

41. The antibody or the antigen-binding fragment thereof of any one of claims 17-40, wherein the antibody or antigen-binding fragment thereof shows greater internalization activity compared to the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 11, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

42. The antibody or antigen-binding fragment thereof of any one of claims 30-41, wherein the cancer cell is a solid cancer cell.

43. The antibody or antigen-binding fragment thereof of claim 42, wherein the cancer cell is MDA-MB-468 cell.

44. The antibody or antigen-binding fragment thereof of any one of claims 30-43, wherein the non-cancer cell is keratinocyte.

45. The antibody or antigen-binding fragment thereof of any one of the preceding claims, which is a F(ab')2, a F(ab)2, a Fab', a Fab, a Fv, or a scFv.

46. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or the combination thereof.

47. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is of an isotype of IgGl, IgG2, IgG3 or IgG4.

48. The antibody or antigen-binding fragment thereof of claim 45, wherein the antibody or antigen-binding fragment thereof is of an isotype of human IgGl.

49. The antibody or antigen-binding fragment thereof of claim 45, wherein a constant region of the antibody or antigen-binding fragment has L234A and L235 A mutations.

50. The antibody or antigen-binding fragment thereof of any preceding claim, wherein the reference antibody having a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 1 1, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12 is enfortumab.

51. A nucleic acid encoding the antibody or antigen-binding fragment thereof of any one of claims 1-50.

52. A vector comprising the nucleic acid of claim 51.

53. A host cell comprising the nucleic acid of claim 51, or the vector of claim 52.

54. A method of preparing an anti-nectin-4 antibody or antigen-binding fragment thereof, the method comprising expressing the antibody or antigen-binding fragment thereof by culturing the host cell of claim 53.

55. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-50, and a pharmaceutically acceptable earner.

56. A pharmaceutical composition for treating cancer, comprising the antibody or antigenbinding fragment thereof of any one of claims 1-50.

57. A method of treating cancer in a patient in need thereof, comprising administering to the patient the antibody or antigen-binding fragment thereof of any one of claims 1-50 or the pharmaceutical composition of claim 55.

58. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-50 or the pharmaceutical composition of claim 55 for the preparation of a medicament for treating cancer.

59. The antibody or antigen-binding fragment thereof of any one of claims 1-50 or the pharmaceutical composition of claim 55 for use in the treatment of cancer in a patient in need thereof.

60. The method of claim 57 or the use of claim 58 or the antibody or antigen- binding fragment thereof or pharmaceutical composition for use of claim 59, wherein the cancer is solid cancer.

61. The method, the use or the antibody or antigen-binding fragment thereof or pharmaceutical composition for use of claim 60, wherein the solid cancer is selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and esophageal cancer.

62. The method, the use or the antibody or antigen-binding fragment thereof or pharmaceutical composition for use of claim 60, wherein the cancer is selected from a group consisting of TNBC. NSCLC, and bladder cancer.

63. The method, the use or the antibody or antigen-binding fragment thereof or pharmaceutical composition for use of claim 60, wherein the cancer cell expresses nectin-4.