Anti-nectin-4 antibody and use thereof

Abstract

The present invention relates to a novel antibody and antibody fragment specifically binding to Nectin-4, and a composition containing the antibody or the antibody fragment. In addition, the present invention relates to a nucleic acid encoding the antibody or the antibody fragment thereof, a host cell containing same, and a related use. Further, the present invention relates to therapeutic and diagnostic uses of the antibody and the antibody fragment.

Description

Anti-Nectin-4 antibodies and their uses

[0001] Cross-reference to related applications

[0002] This application is based on and claims priority to Chinese Patent Application No. 202410814016.X, filed on June 21, 2024, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to novel antibodies and antibody fragments that specifically bind to Nectin-4, as well as compositions containing said antibodies or antibody fragments. Furthermore, this invention relates to nucleic acids encoding said antibodies or antibody fragments thereof, host cells containing them, and related uses. Additionally, this invention relates to the therapeutic and diagnostic uses of these antibodies and antibody fragments.

[0004] Background of the Invention

[0005] Nectin-4, also known as PVRL4, is a cell adhesion protein belonging to the Nectin family. In humans, the Nectin family includes Nectin-1, Nectin-2, Nectin-3, and Nectin-4. Nectin-1 through Nectin-3 are primarily distributed in normal tissues: Nectin-1 (immune system organs), Nectin-2 (blood cells and sperm cells), and Nectin-3 (testes and placenta). Nectin-4 (also known as PVRL4) is unique in that it is expressed at high levels in normal embryos and fetuses, but its expression is limited in healthy adult tissues.

[0006] Nectin-4 is a type I membrane protein. The extracellular region of the dimer Nectin-4 consists of three Ig domains: Ig V and Ig C. In the human body, Nectin-4 plays a crucial role in cell-cell adhesion, signal transduction, and cell-cell interactions. However, studies have shown that it is highly expressed in various tumor cells, such as cervical cancer, urothelial carcinoma, breast cancer, lung cancer, gastric cancer, and head and neck cancer. It primarily promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway. Therefore, antibody-drug conjugates (ADCs) or immunostimulating antibody conjugates (ISACs) targeting Nectin-4 can selectively and stably bind with high affinity to Nectin-4 expressed on tumor cells, and exhibit tumor-specific uncoupling, avoiding damage to normal cells and significantly reducing toxic side effects, thus becoming a highly effective strategy for cancer treatment.

[0007] Therefore, there is a need in the field for a superior Nectin-4 antibody with improved properties.

[0008] Invention Overview

[0009] This invention relates to an antibody or fragment, such as an antigen-binding fragment, that specifically binds to Nectin-4, such as human, cynomolgus monkey, or mouse Nectin-4.

[0010] In some embodiments, the antibody of the present invention or its antigen-binding fragment specifically binds to human Nectin-4 (e.g., wild-type Nectin-4). In some embodiments, the affinity of the antigen-antibody binding is detected by ForteBio.

[0011] In some embodiments, the antibody or antigen-binding fragment of the present invention binds to the K+ of human Nectin-4. D Less than approximately 200 nM, 150 nM, 140 nM, 130 nM, 120 nM, 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 5 nM, 4 nM, or 3 nM, or K+ of human Nectin-4. D Greater than approximately 1 nM, or within any numerical range. In some embodiments, the K... D Between approximately 1 nM and 100 nM. In some implementations, the affinity for antigen-antibody binding is detected by ForteBio.

[0012] In some embodiments, the antibody of the present invention or its antigen-binding fragment binds to the K+ of cynomolgus monkey Nectin-4. D Less than approximately 100 nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, or 7.5 nM, or K+ of human Nectin-4. D Greater than approximately 1 nM, 2 nM, 3 nM, 4 nM, or 5 nM, or within any numerical range. In some embodiments, K... D Between approximately 5 nM and 70 nM. In some implementations, the affinity for antigen-antibody binding is detected by ForteBio.

[0013] In some embodiments, the antibody or antigen-binding fragment of the present invention is capable of specifically binding to cells expressing Nectin-4, for example, having considerable affinity.

[0014] In some embodiments, the antibody or antigen-binding fragment of the present invention has good specificity and antibody-dependent cell-mediated phagocytosis (ADCP), for example, it has ADCP activity in cells that highly express Nectin-4 but no ADCP activity in cells that do not express Nectin-4.

[0015] In some embodiments, the antibodies or antigen-binding fragments of the present invention are suitable for use in the preparation of ISAC (Immune-Stimulating Antibody Conjugate).

[0016] In some embodiments, the antibody or antigen-binding fragment of the present invention can induce ADCP in immune effector cells such as macrophages, for example, M1 macrophages.

[0017] In some embodiments, the antibodies or antigen-binding fragments of the present invention are effective in treating Nectin-4 related diseases or conditions, such as Nectin-4 positive tumors. Attached image description:

[0018] Figure 1 shows the ADCP activity of the antibody of the present invention in cell lines that highly express Nectin-4 (Figure 1A) and cell lines that do not express Nectin-4 (Figure 1B).

[0019] Figure 2 shows the binding affinity of the antibody of the present invention to a cell line that highly expresses Nectin-4.

[0020] Figure 3 shows the activity of the antibody of the present invention in inducing ADCP in M1 macrophages in the presence of cells expressing Nectin-4.

[0021] Invention Details

[0022] Before describing the invention in detail below, it should be understood that the invention is not limited to the specific methodologies, schemes, and reagents described herein, as these can vary.

[0023] I. Definition

[0024] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not restrictive.

[0026] When used in conjunction with a numerical value, the term "about" or "approximately" means to cover a range of numerical values ​​having a lower limit of 10% less than the specified numerical value and an upper limit of 10% greater than the specified numerical value, preferably covering a range of numerical values ​​having a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) less than the specified numerical value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) greater than the specified numerical value.

[0027] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0028] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover combinations of the stated elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region composed of that specific sequence.

[0029] "Isolated" antibodies or molecules are those that have been separated from components of their natural environment. In some embodiments, the antibody or molecule is purified to a purity of more than 95% or 99%, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0030] As used herein, “Nectin-4” refers to any naturally occurring Nectin-4 polypeptide (e.g., human or cynomolgus monkey Nectin-4 polypeptide) or a variant thereof. The term “Nectin-4” encompasses both “full-length” untreated Nectin-4 polypeptides and any form of Nectin-4 polypeptide produced by intracellular treatment. The term also encompasses naturally occurring variants of Nectin-4, such as those encoded by splice variants and allelic variants. The Nectin-4 polypeptides described herein can be isolated from a variety of sources, such as from humans or from another source, such as cynomolgus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human Nectin-4 protein is available under accession number Uniport ID: Q96NY8-1. In one embodiment of the invention, cynomolgus monkey Nectin-4 is available under accession number Uniport ID: L0N6D9.

[0031] As used herein, “antibody that binds to Nectin-4” or “anti-Nectin-4 antibody” or “antibody that specifically binds to Nectin-4” refers to an antibody that can bind to Nectin-4 with appropriate affinity.

[0032] In some respects, the anti-Nectin-4 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to Nectin-4 and other target antigens, such as bispecific antibodies.

[0033] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0034] The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0035] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It comprises a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, bivalent antibodies, dimorphic antibodies or fragments thereof, or camelid antibodies, dimorphic antibodies (diabody), single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab', which is a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. The Fv fragment consists of the VL and VH domains of the antibody single arm. The two domains, VL and VH, of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain using a recombinant approach, linking these two domains with a synthetic linker peptide, where the VL and VH regions pair to form a single-chain Fv (scFv).

[0036] The term "single-chain antibody (scAb)" is used herein in the broadest sense and specifically covers antibodies that are originally generated as a single, continuous polypeptide chain and have monospecific or multispecific (e.g., bispecific) properties. Such single-chain antibodies include, but are not limited to, those having two conjoined VL and VH regions. In one embodiment, the single-chain antibody is scFv.

[0037] "Bimeric antibodies" are small, divalent antibodies constructed through gene fusion; for example, they are dimers composed of two polypeptide chains. The VL and VH domains of each polypeptide chain in a bimeric antibody bind via linkers, thereby forming a dimer that encodes VL and VH domains within the same polypeptide chain, each with a different single-chain variable region. Bimeric antibodies typically have two antigen-binding sites.

[0038] The complementarity-determining region (CDR) or CDR is a region within the antibody variable domain that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0039] The following are exemplary schemes for defining the area of ​​a CDR using the Kabat, Chothia, or Kabat & Chothia (combined Kabat and Chothia schemes) schemes.

[0040] Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing manner. A CDR may also be determined based on having the same Kabat number position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0041] Unless otherwise stated, in this invention, when referring to the position of a residue in the antibody variable region (including heavy chain variable region residues and light chain variable region residues), it means the position numbered according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0042] In some embodiments, the CDR1 of the heavy chain variable region of the antibody in this invention is determined according to the Kabat & Chothia scheme, and CDR2 and CDR3 are determined according to the Kabat scheme. In some embodiments, the CDR of the light chain variable region of the antibody in this invention is determined according to the Kabat scheme.

[0043] In some embodiments, in the antibody of the present invention,

[0044] HCDR1 was determined according to the Kabat & Chothia scheme, while HCDR2 and HCDR3 were determined according to the Kabat scheme respectively.

[0045] LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0046] "Antibody that binds to the same or overlapping epitopes as the reference antibody" means an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay; conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay.

[0047] An antibody that competes with a reference antibody for binding to its antigen is one that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competitive assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competitive assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another; these assays include, for example, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), and sandwich competitive assays.

[0048] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0049] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that is capable of having at least 50%, 60%, 70%, 80%, 90%, or 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for measuring binding affinity and / or specificity.

[0050] The term "chimeric antibody" refers to an antibody molecule in which (a) a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered class, effector function, and / or species of constant region, or to a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug), which confers new properties to the chimeric antibody; or (b) a variable region or a portion thereof is altered, replaced, or exchanged with a variable region having different or altered antigen specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original mouse antibody.

[0051] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (such as a mouse monoclonal antibody) while exhibiting lower immunogenicity, for example, when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portions of the constant region and variable region with the corresponding portions of the human antibody).

[0052] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0053] As described herein, "conservative alteration" includes substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, a conservative alteration is a conservative substitution. A conservative substitution refers to the replacement of one amino acid with another amino acid of the same class, such as the replacement of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. For example, a conservative substitution often results in the replacement of a certain amino acid with a chemically similar amino acid. Tables of conservative substitutions of functionally similar amino acids are well known in the art. The following are eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conservative alteration," when applied to the amino acid sequence of the antibody molecule, refers to an amino acid modification that does not significantly affect or alter the target antigen binding characteristics of the antibody molecule of the present invention containing the amino acid sequence. For example, conserved alteration variants maintain at least 80%, 85%, 90%, 95%, 98%, 99%, or higher, such as 100-110%, binding affinity to the target antigen relative to the parent antibody.

[0054] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0055] An "immunoconjugate" is an antibody conjugated to one or more other substances (including, but not limited to, labels). In some embodiments, immunoconjugates encompass antibody-drug conjugates (ADCs) or immunostimulating antibody conjugates.

[0056] Antibody-drug conjugates (ADCs) typically consist of three main parts: an antibody that specifically recognizes antigens on the surface of cancer cells, a cytotoxic drug molecule, and a linker connecting the antibody and the drug. The antibody portion of the ADC specifically recognizes specific antigens on the surface of cancer cells. Once the antibody binds to the tumor cell surface antigen, the ADC is carried into the tumor cell via endocytosis, where it releases the cytotoxic drug, thereby killing the tumor cells.

[0057] Immunostimulating antibody conjugates (ICDs) typically consist of an antibody that specifically recognizes antigens on the surface of cancer cells, a molecule that activates the immune system, and a linker connecting the antibody and the molecule. When this conjugate binds to cancer cells, it can trigger the activity of immune cells, thereby enhancing the body's immune response against the tumor. Therefore, it combines the properties of both antibodies and immunostimulatory molecules. The antibody portion of the ICD is highly specific, recognizing and binding to specific antigens on the surface of cancer cells, while the immunostimulatory molecule linked to the antibody is designed to activate the immune system, for example, by promoting the proliferation, differentiation, or activation of immune cells. In some implementations, the immunostimulatory molecule may also have direct cytotoxicity, capable of killing or inhibiting the growth of cancer cells. Therefore, by activating the immune system, ICDs help establish immune memory, enabling the immune system to more effectively recognize and attack the same cancer cells in the future.

[0058] The term “therapeutic agent” as used herein encompasses any substance that is effective in preventing or treating diseases related to Nectin-4, including cytokines, other antibodies, small molecule drugs, or immunomodulators (such as immune activators).

[0059] The term "small molecule drug" refers to low-molecular-weight organic compounds capable of modulating biological processes. "Small molecule" is defined as a molecule with a molecular weight less than 10 kDa, typically less than 2 kDa, and preferably less than 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimics, and antibody mimics. As therapeutic agents, small molecules can penetrate cells more readily, are less susceptible to degradation, and are less likely to elicit an immune response than large molecules.

[0060] As used in this article, the term "immunomodulator" refers to natural or synthetic active agents or drugs that modulate immune responses. Immune responses can be humoral or cellular. Immunomodulators include immune activators or immunosuppressants.

[0061] The term "effective amount" refers to such an amount or dose of the antibody, fragment, conjugate, composition, or combination of the present invention, which, when administered to a patient in a single or multiple doses, produces the intended effect in a patient requiring treatment or prevention.

[0062] "Therapeutic effective amount" refers to the amount that, at the required dose and for the required duration, effectively achieves the desired therapeutic outcome. A therapeutic effective amount is also a amount in which any toxic or harmful effects of an antibody or antibody fragment or its conjugate or combination thereof are less than the beneficial therapeutic effect. Relative to an untreated subject, a "therapeutic effective amount" preferably inhibits measurable parameters (e.g., activity of the Nectin-4 signaling pathway, such as its activated PI2K / Akt) by at least about 20%, more preferably at least about 40%, and even more preferably at least about 50%, 60%, or 70%. In some embodiments, the term "therapeutic effective amount" as used herein is intended to define the amount of treatment necessary for the therapeutic condition (e.g., tumor) within a treatment regimen.

[0063] "Prophylactic effective dose" refers to the amount of medication administered at the required dose for the required duration to effectively achieve the desired preventive outcome. Typically, because prophylactic doses are administered to individuals before or at an early stage of the disease, the prophylactic effective dose will be less than the therapeutic effective dose.

[0064] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including the progeny of such cells. Host cells include “transformers” and “transformed cells,” which include the primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be identical to the parent cells in their nucleic acid content and may contain mutations. This document includes mutant progeny with the same function or biological activity screened or selected from the initially transformed cells.

[0065] As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the conjugated or fused reagent. The label itself may be detectable (e.g., radioisotope labeling or fluorescent labeling) or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. The term is intended to cover both direct labeling of probes or antibodies by conjugation (i.e., physical linking) to a detectable substance and indirect labeling of probes or antibodies by reaction with another directly labeled reagent.

[0066] The terms “individual,” “subject,” or “subject” as used herein are used interchangeably and include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0067] As used herein, the term "subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; or cells from any stage of pregnancy or development in the subject. Tissue samples may contain compounds that are naturally occurring and do not mix with tissues, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0068] "Nucleic acid encoding anti-Nectin-4 antibody or fragment thereof" refers to one or more nucleic acid molecules that encode the antibody heavy or light chain (or fragment thereof, such as the heavy chain variable region or light chain variable region), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0069] The "percentage of identity (%)" for an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to those in the specific amino acid sequence shown in this specification, after comparing the candidate sequence with the specific amino acid sequence shown herein and, if necessary, introducing vacancies to achieve the maximum percentage of sequence identity, and without considering any conserved substitutions as part of sequence identity. In some embodiments, the invention contemplates variants of the antibody molecules of the invention that have a considerable degree of identity with respect to the antibody molecules and their sequences specifically disclosed herein, for example, an identity of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher. These variants may contain conserved changes.

[0070] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0071] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject or individual administering the composition.

[0072] As used herein, the terms "drug combination" or "combination product" refer to non-fixed or fixed combinations, including but not limited to kits / reagents and pharmaceutical compositions. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the components produces an effect greater than that achieved by using any one ingredient alone in treating a disease or condition. A drug combination is not limited to fixing the individual components in the same pharmaceutical composition, kit, or formulation, but may be in separate formulations or pharmaceutical compositions. The components may each be in a separate formulation, and their formulations may be the same or different.

[0073] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiation therapy or surgery) to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0074] When used in this article, "treatment" means to slow down, interrupt, block, alleviate, stop, reduce, or reverse the onset of symptoms, complications, or biochemical indicators of a disease, relieve symptoms, or prevent or inhibit the further development of the disease, condition, or symptom.

[0075] When used in this article, "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0076] II antibody

[0077] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region, namely HCDR1, HCDR2 and HCDR3.

[0078] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises three complementary determinant regions (LCDRs) from the variable region of the light chain: LCDR1, LCDR2, and LCDR3.

[0079] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region and three complementarity-determining regions (LCDRs) from the light chain variable region.

[0080] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH). In some aspects, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a light chain variable region (VH). In some aspects, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0081] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region and a light chain constant region.

[0082] In some implementations, the heavy chain variable region described herein:

[0083] (i) An amino acid sequence comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 27-34; or

[0084] (ii) Contains or consists of an amino acid sequence selected from any one of SEQ ID NO:27-34; or

[0085] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any of SEQ ID NO:27-34, preferably, the amino acid alterations do not occur in the CDR region.

[0086] In some implementations, the light chain variable region described herein

[0087] (i) An amino acid sequence comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 35-42; or

[0088] (ii) Contains or consists of an amino acid sequence selected from any one of SEQ ID NO:35-42; or

[0089] (iii) An amino acid sequence comprising or consisting of the amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any of SEQ ID NO:35-42, preferably, the amino acid changes do not occur in the CDR region.

[0090] In some implementations, the three complementarity-determining regions (HCDRs) from the heavy chain variable region described herein, HCDR1, HCDR2, and HCDR3, are selected from...

[0091] (i) The three complementary determinant regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in any of SEQ ID NO:27-34;

[0092] (ii) A sequence comprising, in relation to any one of (i), at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conserved substitution) in the three HCDR regions.

[0093] The HCDR can be determined according to any scheme for determining the CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof.

[0094] For example, HCDR1 is determined according to the Kabat & Chothia scheme, and HCDR2 and HCDR3 are determined according to the Kabat scheme.

[0095] In some implementations, the three complementary determinant regions (LCDRs) from the light chain variable region described herein, LCDR1, LCDR2, and LCDR3, are selected from...

[0096] (i) The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in any of SEQ ID NO:35-42, or

[0097] (ii) A sequence comprising, in relation to any one of (i), at least one and no more than 5, 4, 3, 2, or 1 amino acid alteration (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions.

[0098] The LCDR can be determined according to any scheme for determining the CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof.

[0099] For example, LCDR1, 2 and 3 are determined according to the Kabat scheme.

[0100] In some embodiments, the HCDR1 described herein comprises or is composed of any of the amino acid sequences shown in SEQ ID NO:1-4, or the HCDR1 comprises an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to any of the amino acid sequences shown in SEQ ID NO:1-4.

[0101] In some embodiments, the HCDR2 described herein comprises or is composed of any of the amino acid sequences shown in SEQ ID NO:5-9, or the HCDR2 comprises an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to any of the amino acid sequences shown in SEQ ID NO:5-9.

[0102] In some embodiments, the HCDR3 described herein comprises or is composed of the amino acid sequence shown in any of SEQ ID NO: 10-13, or the HCDR3 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in any of SEQ ID NO: 10-13.

[0103] In some embodiments, the LCDR1 described herein comprises or is composed of the amino acid sequence shown in any of SEQ ID NO:14-18, or the LCDR1 comprises an amino acid sequence having one, two or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:14-18.

[0104] In some embodiments, the LCDR2 described herein comprises or is composed of the amino acid sequence shown in any of SEQ ID NO:19-22, or the LCDR2 comprises an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:19-22.

[0105] In some embodiments, the LCDR3 described herein comprises or is composed of any of the amino acid sequences shown in SEQ ID NO:23-26, or comprises an amino acid sequence having one, two, or three amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) compared to any of the amino acid sequences shown in SEQ ID NO:23-26.

[0106] In some specific embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises...

[0107] (i) HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:27 or 28, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:35 or 36;

[0108] (ii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:27, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:35;

[0109] (iii) HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:28, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:36;

[0110] (iv). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:29 or 30, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:37 or 38;

[0111] (v). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:29, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:37;

[0112] (vi) HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:30, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:38;

[0113] (vii). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:31 or 32, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39 or 40;

[0114] (viii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:31, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:39;

[0115] (ix). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:32, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:40;

[0116] (x). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:33 or 34, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:41 or 42;

[0117] (xi). HCDR1, HCDR2, and HCDR3 included in VH as shown in SEQ ID NO:33, and LCDR1, LCDR2, and LCDR3 included in VL as shown in SEQ ID NO:41; or

[0118] (xii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:34, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:42.

[0119] The HCDR and LCDR can be determined according to any scheme for determining the CDR, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof.

[0120] For example, HCDR1 is determined according to the Kabat & Chothia scheme, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are determined according to the Kabat scheme.

[0121] In some specific embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein...

[0122] (i) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23; or

[0123] (ii). The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24 or are composed of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24; or

[0124] (iii) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25 or are composed of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25; or

[0125] (iv) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 or are composed of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26.

[0126] In some implementations, the VH described herein includes HCDR1, HCDR2, and HCDR3, and the VL described herein includes LCDR1, LCDR2, and LCDR3, wherein...

[0127] (i) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23; or

[0128] (ii). The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24 or are composed of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24; or

[0129] (iii) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25 or are composed of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25; or

[0130] (iv) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 or are composed of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26.

[0131] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises:

[0132] (i) A VH comprising the amino acid sequence shown in SEQ ID NO:27 or 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or 36 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or 36;

[0133] (ii) A VH comprising the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35;

[0134] (iii) A VH comprising the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence;

[0135] (iv) A VH comprising the amino acid sequence shown in SEQ ID NO:29 or 30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:37 or 38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence;

[0136] (v) A VH comprising the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence;

[0137] (vi) A VH comprising the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence;

[0138] (vii) A VH comprising the amino acid sequence shown in SEQ ID NO:31 or 32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:39 or 40 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence;

[0139] (viii) A VH comprising the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence;

[0140] (ix). A VH comprising the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:40 or a VL comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:40;

[0141] (x) A VH comprising the amino acid sequence shown in SEQ ID NO:33 or 34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:41 or 42 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence;

[0142] (xi) A VH comprising the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VH composed of said amino acid sequence, and / or a VL comprising the amino acid sequence shown in SEQ ID NO:41 and an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL composed of said amino acid sequence; or (xii) A VH comprising the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VH composed of said amino acid sequence, and / or a VL comprising SEQ ID NO:33. The amino acid sequence shown in NO:42 is identical to an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence or to a VL composed of said amino acid sequence.

[0143] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0144] (i). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:27 or 28, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35 or 36;

[0145] (ii). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:35;

[0146] (iii) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:36;

[0147] (iv) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:29 or 30, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:37 or 38;

[0148] (v) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:37;

[0149] (vi) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:38;

[0150] (vii) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31 or 32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39 or 40;

[0151] (viii). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39;

[0152] (ix). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:40;

[0153] (x). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33 or 34, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41 or 42;

[0154] (xi). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:41; or

[0155] (xii). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:42.

[0156] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NO as follows:

[0157] SEQ ID NO:27 and SEQ ID NO:35;

[0158] SEQ ID NO:28 and SEQ ID NO:36;

[0159] SEQ ID NO:29 and SEQ ID NO:37;

[0160] SEQ ID NO:30 and SEQ ID NO:38;

[0161] SEQ ID NO:31 and SEQ ID NO:39;

[0162] SEQ ID NO:32 and SEQ ID NO:40;

[0163] SEQ ID NO:33 and SEQ ID NO:41; or

[0164] SEQ ID NO:34 and SEQ ID NO:42.

[0165] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain. In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain. In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention further comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain of the present invention comprises, or is composed of, the heavy chain variable region and the heavy chain constant region described herein. In some embodiments, the antibody light chain of the present invention comprises, or is composed of, the light chain variable region and the light chain constant region described herein. In some embodiments, the antibody of the present invention comprises two heavy chains and two light chains, or is composed of, two heavy chains and two light chains.

[0166] In some embodiments, the antibody heavy chain constant region described herein is or is derived from the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, such as the constant region of human IgG1, IgG2, IgG3, or IgG4. In some embodiments, the constant region is modified in the Fc region.

[0167] In some implementations, the heavy chain constant region

[0168] (i) An amino acid sequence comprising or consisting of said amino acid sequence; or

[0169] (ii) An amino acid sequence comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:53.

[0170] In some implementations, the heavy chain constant region described herein lacks a terminal lysine residue.

[0171] In some embodiments, the antibody light chain constant region described herein is or is derived from the lambda or Kappa light chain constant region, preferably the Kappa light chain constant region, such as the human lambda or Kappa light chain constant region. In some embodiments, the light chain constant region...

[0172] (i) An amino acid sequence comprising or consisting of, having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 54; or

[0173] (ii) An amino acid sequence containing or consisting of SEQ ID NO:54.

[0174] In some implementations, the heavy chain described herein includes

[0175] (i) An amino acid sequence comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:43-46;

[0176] (ii) Containing or consisting of an amino acid sequence selected from any one of SEQ ID NO:43-46; or

[0177] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in any of SEQ ID NO:43-46.

[0178] In some implementations, the light chain described herein includes

[0179] (i) An amino acid sequence comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:47-50;

[0180] (ii) Contains or consists of an amino acid sequence selected from any one of SEQ ID NO:47-50; or

[0181] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from any of SEQ ID NO:47-50.

[0182] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain, wherein

[0183] (i) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43, and / or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47;

[0184] (ii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44, and / or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:48;

[0185] (iii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:45, and / or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49; or

[0186] (iv) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:46, and / or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:50.

[0187] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises a heavy chain and a light chain, wherein

[0188] (i) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:43, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:47;

[0189] (ii). The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:44, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:48;

[0190] (iii) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:45, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:49; or

[0191] (iv) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:46, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:50.

[0192] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain, for example, two heavy chains and two light chains, wherein the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NO as follows:

[0193] SEQ ID NO:43 and SEQ ID NO:47;

[0194] SEQ ID NO:44 and SEQ ID NO:48;

[0195] SEQ ID NO:45 and SEQ ID NO:49; or

[0196] SEQ ID NO:46 and SEQ ID NO:50.

[0197] In one embodiment of the invention, the amino acid alterations described herein include substitutions, insertions, or deletions of amino acids. In some embodiments, the amino acid alterations described herein are conserved amino acid alterations. Preferably, the amino acid alterations described herein are amino acid substitutions, preferably conserved substitutions. In a preferred embodiment, the amino acid alterations described herein occur in regions outside the CDR (e.g., in the FR). More preferably, the amino acid alterations described herein occur in regions outside the heavy chain variable region and / or outside the light chain variable region.

[0198] In some embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation. The addition or deletion of glycosylation sites on the antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites. When the antibody contains an Fc region, the sugars attached thereto can be altered. In some applications, modification to remove unwanted glycosylation sites can be useful, for example, removing the fucosylation motif to enhance antibody-dependent cytotoxicity (ADCC) function. In other applications, galactoside modification can be performed to modify complement-dependent cytotoxicity (CDC). In some embodiments, it may be necessary to produce cysteine-engineered antibodies, such as “Thio MAb”, wherein one or more residues of the antibody are replaced with cysteine ​​residues. In some embodiments, the antibodies provided herein may be further modified to contain other non-protein motifs known in the art and readily available. Motifs suitable for antibody-derived activities include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-diane, poly-1,3,6-triane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0199] In some embodiments, the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention has one or more of the following characteristics:

[0200] (i) Demonstrates the same or similar binding affinity and / or specificity to Nectin-4 as defined herein by the anti-Nectin-4 antibodies (e.g., antibodies verified in the examples, such as chN4-P3-52, hzN4-P3-52, chN4-P3-66, hzN4-P3-66, chN4-P3-57, hzN4-P3-57, chN4-P3-48, or hzN4-P3-48);

[0201] (ii) Inhibition (e.g., competitive inhibition) of the binding of the present invention’s anti-Nectin-4 antibody (e.g., the antibody verified in the examples, such as chN4-P3-52, hzN4-P3-52, chN4-P3-66, hzN4-P3-66, chN4-P3-57, hzN4-P3-57, chN4-P3-48 or hzN4-P3-48) to Nectin-4;

[0202] (iii) Epitopes that bind to the same or overlapping epitopes as defined herein by the present invention (e.g., antibodies verified in the examples, such as chN4-P3-52, hzN4-P3-52, chN4-P3-66, hzN4-P3-66, chN4-P3-57, hzN4-P3-57, chN4-P3-48, or hzN4-P3-48);

[0203] (iv) Competing with the anti-Nectin-4 antibodies of the present invention as defined herein (e.g., antibodies verified in the examples, such as chN4-P3-52, hzN4-P3-52, chN4-P3-66, hzN4-P3-66, chN4-P3-57, hzN4-P3-57, chN4-P3-48, or hzN4-P3-48) to bind to Nectin-4;

[0204] (v) Having one or more of the biological characteristics of an anti-Nectin-4 antibody as defined herein (e.g., antibodies verified in the examples, such as chN4-P3-52, hzN4-P3-52, chN4-P3-66, hzN4-P3-66, chN4-P3-57, hzN4-P3-57, chN4-P3-48, or hzN4-P3-48).

[0205] In some embodiments, the anti-Nectin-4 antibody of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, preferably an antibody in the form of IgG1.

[0206] In some implementations, the anti-Nectin-4 antibody is a monoclonal antibody.

[0207] In some implementations, the anti-Nectin-4 antibody is humanized.

[0208] In some implementations, the anti-Nectin-4 antibody is a chimeric antibody.

[0209] In some embodiments, the anti-Nectin-4 antibody of the present invention is a full-length antibody.

[0210] In some embodiments, the anti-Nectin-4 antibody of the present invention also encompasses multispecific or bispecific antibodies that specifically bind to Nectin-4.

[0211] In one embodiment, the anti-Nectin-4 antibody of the present invention further encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antibody fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), bivalent antibodies, bispecific antibodies, or linear antibodies.

[0212] III. The nucleic acid of the present invention and the host cell containing it.

[0213] In one aspect, the present invention provides nucleic acids encoding any one strand or any monomer or domain of the antibody or antigen-binding fragment thereof of the present invention. Methods well known in the art can be used to generate polynucleotide sequences encoding each strand. For example, when expressed from a suitable expression vector, the polypeptide encoded by said nucleic acid can exhibit binding ability to human or cynomolgus monkey Nectin-4. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operatively linked in a reading frame to a nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby generating nucleic acids encoding said antibody heavy chain and / or light chain when expressed from a suitable expression vector.

[0214] In one aspect, the present invention provides a nucleic acid encoding any anti-Nectin-4 antibody or fragment thereof described herein. The nucleic acid may comprise an amino acid sequence encoding an amino acid sequence encoding a light chain variable region and / or a heavy chain variable region of the antibody, or a nucleic acid comprising an amino acid sequence encoding an amino acid sequence encoding an antibody's light chain and / or heavy chain.

[0215] For example, the nucleic acid of the present invention comprises a nucleic acid encoding an amino acid sequence selected from any one of SEQ ID NO:27-50, or a nucleic acid encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO:27-50. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequence encoding the molecule of the present invention can be generated using methods well known in the art, such as by de novo solid-phase DNA synthesis or by PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide conducive to purification can be fused to the N-terminus of the heavy chain and / or light chain of the antibody.

[0216] This invention also provides vectors containing the nucleic acids of this invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector of this invention is a pcDNA vector, such as pcDNA3.1 and / or pcDNA3.4 expression vectors.

[0217] In one embodiment, a host cell comprising the vector is provided. The present invention also provides a host cell comprising the nucleic acid or the vector. Suitable host cells for replicating and supporting the expression of the antibodies of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculation of large-scale fermenters to obtain sufficient quantities of antibodies for clinical applications. Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

[0218] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria, e.g., Escherichia coli.

[0219] For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. For instance, fungal and yeast strains whose glycosylation pathways have been “humanized” result in antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified for suspension growth can be used. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F, or 293T cells); and others. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NSO, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art.

[0220] IV. Production and purification of the antibody molecules of the present invention

[0221] In another aspect, the present invention provides a method for producing the anti-Nectin-4 antibody or an antigen-binding fragment thereof, the method comprising: culturing a host cell containing a polypeptide chain encoding the polypeptide chain under conditions suitable for expressing the polypeptide chain of the molecule; optionally further comprising assembling the polypeptide chain to produce the anti-Nectin-4 antibody or an antigen-binding fragment thereof under conditions suitable for assembling the polypeptide chain into the molecule.

[0222] To facilitate production and purification, anti-Nectin-4 antibodies or their antigen-binding fragments may be fused to a secretory signal peptide at the N-terminus or C-terminus (e.g., C-terminus), and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label. In some embodiments, the signal peptide comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:55.

[0223] For recombinant production, a polynucleotide encoding a polypeptide chain of the present invention's anti-Nectin-4 antibody or its antigen-binding fragment can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art.

[0224] In one embodiment, each polynucleotide encoding the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment can be transfected into a different vector, optionally the vector containing a heavy chain constant region or a light chain constant region (e.g., nucleotides encoding a heavy chain variable region are transfected into a vector containing a heavy chain constant region, or nucleotides containing a light chain variable region are transfected into a vector containing a light chain constant region, so that the vector can express the full-length heavy chain or the full-length light chain; or nucleotides encoding a heavy chain are transfected into a vector to express the full-length heavy chain, and nucleotides encoding a light chain are transfected into a vector to express the full-length light chain, so that the vector can express the full-length heavy chain or the full-length light chain).

[0225] Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. In one embodiment, one or more vectors encoding multiple chains of an antibody are transfected into the same host cell, such that the antibody chains assemble into a complete antibody within the host cell.

[0226] A variety of techniques can be used to achieve this goal, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0227] The molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0228] The purity of the anti-Nectin-4 antibody or its antigen-binding fragment of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high-performance liquid chromatography, etc. The physical / chemical properties and / or biological activity of the antibody molecules provided herein can be identified, screened, or characterized by a variety of assays known in the art.

[0229] V. Determination Method

[0230] The anti-Nectin-4 antibodies provided herein can be identified, screened, or characterized by their physical / chemical properties and / or biological activity using a variety of assays known in the art.

[0231] This invention also provides an assay for identifying biologically active anti-Nectin-4 antibodies. Biological activity may include, for example, binding to Nectin-4 (e.g., binding to human Nectin-4), antibody-dependent phagocytosis (ADCP), binding to cell surface Nectin-4 (binding to cells expressing Nectin-4), induction of macrophage (e.g., human M1 macrophage) endocytosis, and / or prevention or treatment of Nectin-4-related diseases or conditions. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0232] For the determination of the above-mentioned biological activities, please refer to the exemplary determination methods given in the embodiments.

[0233] It is understood that the immunoconjugate of the present invention can be used to replace or supplement the anti-Nectin-4 antibody for any of the above-described assays.

[0234] Understandably, any of the above assays can be performed using a combination of anti-Nectin-4 antibodies and other therapeutic agents.

[0235] VI. Immunoconjugates

[0236] In some embodiments, the present invention provides immunoconjugates comprising any anti-Nectin-4 antibody provided herein and other substances, such as any active agent or label suitable for forming immunoconjugates with Nectin-4 antibodies.

[0237] In some embodiments, the active agent suitable for forming an immunoconjugate with the Nectin-4 antibody may be, for example, a chemotherapeutic agent, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, or an immunomodulator, such as an immune activator.

[0238] In some implementations, the immunoconjugate is an antibody-drug conjugate, such as an ADC.

[0239] In some implementations, the immune conjugate is an immunostimulatory antibody conjugate (ISAC).

[0240] Therefore, the present invention also relates to a method for preparing immunoconjugates such as ADCs or ISACs using the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment thereof. The present invention also relates to the use of the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment thereof in preparing immunoconjugates such as ADCs or ISACs.

[0241] VII. Pharmaceutical compositions and pharmaceutical preparations

[0242] In some embodiments, the present invention provides compositions comprising any anti-Nectin-4 antibody described herein, or a fragment thereof (preferably an antigen-binding fragment thereof), or an immunoconjugate thereof, preferably pharmaceutical compositions. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, for example a pharmaceutical composition, comprises an anti-Nectin-4 antibody of the present invention, or a fragment thereof, or an immunoconjugate thereof, and a combination of one or more other therapeutic agents.

[0243] The present invention also includes compositions comprising anti-Nectin-4 antibodies or their immunoconjugates (including pharmaceutical compositions or pharmaceutical formulations), or compositions comprising polynucleotides encoding anti-Nectin-4 antibodies (including pharmaceutical compositions or pharmaceutical formulations). In some embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to Nectin-4, or one or more polynucleotides encoding one or more antibodies or fragments thereof against Nectin-4. These compositions may also comprise suitable pharmaceutical excipients, such as pharmaceutical carriers, pharmaceutical excipients, including buffers, known in the art.

[0244] As used in this article, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersion media, isotonic agents, and absorption delay agents.

[0245] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0246] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders or suspensions, liposomes, and suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0247] Pharmaceutical formulations containing the antibodies described herein can be prepared by mixing the antibodies of the present invention, having the desired purity, with one or more optional pharmaceutical excipients, preferably in the form of lyophilized formulations or aqueous solutions.

[0248] The pharmaceutical compositions or formulations of the present invention may also comprise more than one active ingredient, said active ingredient being required for a specific indication to be treated, preferably those active ingredients having complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (e.g., immunosuppressants or anti-inflammatory agents), or other antibodies. The active ingredients are suitably combined in amounts effective for the intended use.

[0249] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include a semi-permeable matrix of a solid hydrophobic polymer containing an antibody, said matrix being a shaped article, such as a film or microcapsule.

[0250] VIII. Drug Combinations and Medicine Boxes

[0251] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising the anti-Nectin-4 antibody of the present invention or a fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, and one or more other therapeutic agents (e.g., cytokines, chemotherapeutic agents, small molecule drugs, immunomodulators (e.g., immune activators) or other antibodies, etc.).

[0252] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0253] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0254] - A first container containing a pharmaceutical composition comprising an anti-Nectin-4 antibody or a fragment thereof or an immunoconjugate thereof;

[0255] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0256] The selection of other therapeutic agents that can be combined with the molecules of the present invention (e.g., anti-Nectin-4 antibody or fragments thereof or their immunoconjugates) depends on the intended use of the molecules of the present invention. For example, when the molecules of the present invention are used to treat tumors, the other therapeutic agents are any active agents suitable for treating tumors, such as antitumor agents or anticancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies, or immunomodulators (e.g., immune activators).

[0257] IX. Uses and Methods

[0258] In some embodiments, the present invention provides a method for inducing ADCP, such as ADCP in immune effector cells (e.g., macrophages, such as M1 macrophages), comprising administering to an individual a molecule of the present invention (e.g., the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment or its immunoconjugate), a pharmaceutical composition, a combination of drugs, or a kit. In some embodiments, the molecules of the present invention induce ADCP as a single agent or in combination.

[0259] In some embodiments, the present invention provides methods for preventing or treating diseases or conditions in an individual, including administering to the individual a molecule of the present invention (e.g., the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment or its immunoconjugate), a pharmaceutical composition, a pharmaceutical combination, or a kit. In some embodiments, the molecules of the present invention are used as monotherapy or in combination therapy to prevent or treat diseases or conditions in an individual.

[0260] In some embodiments, the present invention relates to molecules of the invention (e.g., the anti-Nectin-4 antibody of the invention or its antigen-binding fragment or its immunoconjugate), pharmaceutical compositions, pharmaceutical combinations or kits, for use in therapies, such as for treating the diseases or conditions mentioned herein.

[0261] In some embodiments, the present invention relates to methods of treating diseases or conditions using molecules of the present invention (e.g., the anti-Nectin-4 antibody of the present invention or its antigen-binding fragment or its immunoconjugate), pharmaceutical compositions, pharmaceutical combinations or kits, or uses for said treatment, or uses for preparing medicaments for said treatment.

[0262] In some embodiments, the disease or condition is a Nectin-4 related disease and / or condition. The term "Nectin-4 related disease and / or condition" involves aberrant activation of Nectin-4 or its mediated signaling pathways (e.g., the PI3K / Akt pathway), or involves Nectin-4 overexpression and / or increased levels of Nectin-4 in cells or organs or tissues, and / or aberrant Nectin-4 expression and / or aberrant expression of Nectin-4 variants in cells or organs or tissues. Such diseases and conditions include, for example, tumors.

[0263] In some embodiments, Nectin-4-mediated signaling pathways (e.g., the PI3K / Akt pathway) are aberrantly activated in patients with the disease or condition compared to healthy individuals. In some embodiments, Nectin-4 is aberrantly expressed (e.g., overexpressed, or expressed at increased levels, or expressed in aberrant variants) or has increased nucleic acid levels in patients with the disease or condition compared to healthy individuals. In some embodiments, Nectin-4 is expressed in cells, tissues, or organs in patients with the disease or condition, for example, at moderate or high levels. In some embodiments, Nectin-4 is present in the patient (e.g., at elevated levels, such as nucleic acid or protein levels or activity) compared to healthy individuals. In some embodiments, Nectin-4 is present in the patient's biological sample (e.g., cells or tissues) (e.g., at elevated levels, such as nucleic acid or protein levels or activity) compared to biological samples of healthy individuals (e.g., corresponding tissues or cells in healthy individuals), or compared to Nectin-4 in adjacent healthy tissues or cells of the patient.

[0264] In some embodiments, the antibodies of the present invention can prevent and / or treat diseases or conditions related to Nectin-4 by blocking the activation of downstream signaling pathways (e.g., the PI3K / Akt pathway) by Nectin-4.

[0265] In some embodiments, the disease or condition suitable for prevention and / or treatment according to the present invention is a tumor, such as cancer. In some embodiments, the tumor is a solid tumor or a hematologic malignancy (including lymphoma) and a metastatic lesion. In one embodiment, examples of solid tumors include malignant tumors. The cancer can be in an early, intermediate, or late stage, or metastatic. In some embodiments, the tumor is a tumor immune evader.

[0266] In some implementations, Nectin-4 or its mediated signaling pathways (e.g., the PI3K / Akt pathway) are abnormally activated in the tumor or tumor cells, for example, compared to healthy individuals or healthy cells adjacent to the tumor cells.

[0267] In some embodiments, the tumor is a Nectin-4 positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or aberrant activity of Nectin-4. In some embodiments, a Nectin-4 positive tumor or cancer refers to tumor cells expressing Nectin-4 in an individual with all types of tumor cancer. In some embodiments, the tumor cells of the individual express Nectin-4 or a variant thereof, such as moderate or high expression of Nectin-4, or expression of a Nectin-4 variant. In some embodiments, the individual has (e.g., elevated levels, such as nucleic acid or protein levels or activity) Nectin-4 (e.g., compared to a healthy individual) or has aberrant expression of Nectin-4. In some embodiments, the individual's biological sample (e.g., tumor cells or tumor tissue) has (e.g., elevated levels, such as nucleic acid or protein levels or activity) Nectin-4 (e.g., compared to a biological sample of a healthy individual (e.g., corresponding tissue or cells in a healthy individual), or compared to Nectin-4 in adjacent healthy tissue or cells of the individual) or has aberrant expression of Nectin-4.

[0268] In some implementations, the tumor is selected from, for example, breast cancer (e.g., triple-negative breast cancer), cervical cancer, ovarian cancer, urothelial carcinoma, colorectal cancer, esophageal cancer, liver cancer, thyroid cancer, bladder cancer, prostate cancer, lung cancer, stomach cancer, head and neck cancer, or pancreatic cancer.

[0269] In some embodiments, the antibodies or antibody fragments or immune conjugates or compositions or products of the present invention may delay the onset of the disease and / or disease-related symptoms.

[0270] In some embodiments, the present invention provides the use of the anti-Nectin-4 antibody of the present invention or a fragment thereof or an immunoconjugate or composition comprising the present invention in the production or preparation of a medicament for the purposes described herein, such as for the prevention or treatment of the related diseases or conditions mentioned herein.

[0271] In some embodiments, the preventive or therapeutic methods described herein also include administration to the subject or individual in combination with the antibody molecules or pharmaceutical compositions or immunoconjugates disclosed herein, and one or more other therapies, such as modes of treatment and / or other therapeutic agents. In some embodiments, anti-Nectin-4 antibodies or fragments thereof (and immunoconjugates, compositions, pharmaceutical compositions, formulations, etc., containing them) may also be administered in combination with one or more other therapies, such as modes of treatment and / or other therapeutic agents, for the purposes described herein, such as for the prevention and / or treatment of the related diseases or conditions mentioned herein.

[0272] The choice of treatment modality or other therapeutic agent depends on the intended use of the molecule of the invention. For example, when the molecule of the invention is used to treat tumors, the other therapeutic agent is any active agent suitable for treating tumors, such as antitumor agents or anticancer agents, including but not limited to cytokines, chemotherapeutic agents, small molecule drugs, other antibodies or immunomodulators; and / or the other treatment modality is selected, for example, from surgery or radiotherapy.

[0273] In some embodiments, the antibodies or fragments thereof described herein may be combined with other antibodies for separate administration, for example, as separate antibodies, or in combination (e.g., as bispecific or multispecific antibody molecules).

[0274] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations) and separate administration, in which case the antibody of the present invention may be administered before, simultaneously with, and / or after the administration of other therapeutic agents and / or therapies.

[0275] The antibodies or fragments thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, drug combinations, etc. comprising them) may be administered by any suitable method, including parenteral administration, and, if necessary, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous injection or infusion.

[0276] X. Methods and compositions for diagnosis and detection

[0277] In one aspect, the present invention also relates to methods for diagnosing and detecting antibodies of the present invention or antigen-binding fragments thereof, and compositions comprising such antibodies for diagnosing and detecting.

[0278] In some implementations, any anti-Nectin-4 antibody or fragment thereof (preferably an antigen-binding fragment) provided herein can be used to detect the presence of Nectin-4 in biological samples.

[0279] When used herein, the term "detection" includes both quantitative and qualitative detection. Exemplary detection methods may involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In some embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In some embodiments, the biological sample comprises cells or tissues.

[0280] In one implementation, an anti-Nectin-4 antibody or a fragment thereof is provided for use in diagnostic or detection methods.

[0281] In another aspect, a method for detecting the presence of Nectin-4 in a biological sample is provided. In some embodiments, the method comprises detecting the presence of the Nectin-4 protein in the biological sample. In some embodiments, Nectin-4 is human Nectin-4. In some embodiments, the method comprises contacting the biological sample with an anti-Nectin-4 antibody or a fragment thereof as described herein under conditions that allow the anti-Nectin-4 antibody or a fragment thereof to bind to Nectin-4, and detecting whether an antigen-antibody complex is formed between the anti-Nectin-4 antibody or a fragment thereof and Nectin-4. The formation of an antigen-antibody complex indicates the presence of Nectin-4. This method may be an in vitro or in vivo method. In one embodiment, the anti-Nectin-4 antibody or a fragment thereof is used to select subjects suitable for treatment utilizing the anti-Nectin-4 antibody or a fragment thereof, for example, where Nectin-4 is a biomarker for selecting said subjects.

[0282] In some embodiments, a labeled anti-Nectin-4 antibody or a fragment thereof is provided. Labeling includes, but is not limited to, labels or portions that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and portions that are indirectly detected, such as enzymes or ligands, for example, through enzymatic reactions or molecular interactions.

[0283] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-Nectin-4 antibody or a fragment thereof. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during or after treatment with other therapies.

[0284] In some implementations, Nectin-4 is detected before treatment, for example, before the start of treatment or before a treatment after a treatment interval.

[0285] In some embodiments, a method for treating the disease or condition of the present invention is provided, the method comprising: testing a subject (e.g., a sample) for the presence of Nectin-4, thereby determining a Nectin-4 value; comparing the Nectin-4 value with a control value; and if the Nectin-4 value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-Nectin-4 antibody or a fragment thereof (e.g., the anti-Nectin-4 antibody or a fragment thereof described herein) optionally in combination with one or more other therapies, thereby treating the disease.

[0286] These and other aspects and embodiments of the invention are described in the accompanying drawings (briefly described below) and the following detailed description of the invention, and are exemplified in the following embodiments. Any or all features discussed above and throughout this application may be combined in various embodiments of the invention. The following embodiments further illustrate the invention; however, it should be understood that the embodiments are described in an illustrative rather than limiting manner, and various modifications can be made by those skilled in the art. Example

[0287] Example 1: Development of anti-Nectin 4 antibody using the Single B technology platform

[0288] immunity

[0289] Balb / c mice (purchased from Vital River) were immunized with a mixture of human Nectin 4 extracellular fragment protein (Human Nectin-4 Acro NE4-H52H3) and adjuvant. The mice were immunized three times, with each immunization occurring two weeks apart via subcutaneous injection.

[0290] Single B cell fluorescence staining and sorting

[0291] Once the serum titer met the requirements, the spleen and leg bones of mice were harvested to obtain suspended lymphocytes. Next, B cells in the cell suspension were enriched using the EasySep Mouse Pan-B Cell Isolation Kit (Stemcell, 19844A). Cells were washed and resuspended with EasySep Buffer, and PerCP-Cy5.5 CD3 (BD, 100218), PerCP-Cy5.5CD8a (BD, 551162), PerCP-Cy5.5 CD4 (BD, 550954), PerCP-Cy5.5 Ly-6G / C (BD, 552093), PE-Cy 7Rat Anti-Mouse CD45R / B220 (BD, 552772), FITC (Invitrogen, A20181), and APC (Invitrogen, A20186)-labeled hu-Nectin 4 were added, followed by incubation in the dark for 1 hour. After washing with EasySep Buffer, APC-Cyanine 7 anti-mouse IgM Antibody (Biolegend, 406516) and Brilliant Violet 510 anti-mouse IgD Antibody (Biolegend, 405723) were added, and the cells were incubated in the dark for 20 min for staining. The washed B cell suspension was adjusted to an appropriate density and sorted using an Aria III flow cytometer. Single B cells meeting the following characteristics were identified: PerCP-Cy5.5 neg / PE-Cy7 pos / FITC pos / APC pos / APC-Cyanine 7 neg / Brilliant Violet 510 neg The samples were sorted into 96-well PCR plates containing lysis buffer and stored at -80°C.

[0292] Antibody variable region amplification, expression, and detection

[0293] After thawing 96-well PCR plates containing sorted B cells, amplification products of the antibody heavy and light chain variable region genes were obtained via RT-PCR and nested PCR. Paired amplification products were subjected to Sanger sequencing, and the sequencing results were analyzed and compared using MEGA7 software. Simultaneously, 48 clones were selected and constructed into eukaryotic expression vectors containing the constant region genes, transfected into Expi293 cells for expression, and purified. The purified antibodies were used to determine affinity.

[0294] The successfully expressed and purified antibodies were subjected to affinity assays using Fortebio. 27 clones bound to Human Nectin 4, 25 clones bound to Cyno Nectin 4, and 9 clones bound to Mouse Nectin 4. Based on the assay results and sequence diversity, four clones were selected as candidate molecules for further evaluation. The affinity results are shown in Table 1.

[0295] Table 1: Affinity results of four Nectin 4 clones

[0296] Example 2. Antibody Humanization

[0297] Using Discovery Studio software, chimeric antibodies obtained from hybridoma screening were humanized through the following steps: The CDR region of the chimeric antibody was determined; homologous sequences closest to the V / J regions of the heavy and light chains of the chimeric antibody were screened from human germline sequence databases; the CDR region of the chimeric antibody was constructed onto a human Germline backbone; based on sequence and structural characteristics, the amino acid positions in the backbone region that maintain the CDR function were determined, and reversion mutations (returning to the input amino acid type) were performed at these key sequence positions; finally, the sequence was synthesized to prepare the antibody. The amino acid sequences of the heavy and light chain variable regions of the humanized antibody are provided in the sequence information.

[0298] After obtaining the protein, the ability of the chimeric antibody and humanized antibody in this application to bind Nectin-4 was determined using biofilm thin-layer interferometry (BLI) (see Table 2).

[0299] Table 2. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0300] Based on the affinity data in Table 2, it can be concluded that the humanized molecules all maintained an affinity comparable to that of the chimeric antibody.

[0301] Example 3: Antibody-dependent phagocytosis (ADCP) function assay

[0302] This invention uses FcγRIIa-H reporter cells (G9871, Promega) to detect ADCP (Antibody-dependent cell-mediated phagocytosis) activity of Nectin-4 chimeric antibody. These cells are engineered Jurkat T cells. When FcγRIIa on the surface of these cells is bound and activated, it releases luciferase into the experimental system through the downstream signal NFAT (Nuclear Factor of Activated T), thereby allowing the detection of the degree of cell activation.

[0303] The specific method is as follows: Use a 96-well white flat-bottomed cell culture plate, add 6 × 10⁶ target cells of GS-CHO-Nectin-4 (Lonza) or GS-CHO-WT (Lonza) to each well. 4 3 × 10 effector cells FcγRIIa-H cells 4 Each plate was then incubated with the corresponding concentration of Nectin-4 monoclonal antibody at 37°C for 6 hours. The plate was then removed, and Bio-Glo (G7940, Promega) was added. Detection was performed using a microplate reader (Spectra, Molecular Devices). The antibody fraction of Nectin-4 ADC TADCEV, Enfortumab, was used as a control antibody (sequence and source can be found in patent US8637642B2). The results are shown in Figures 1A (GS-CHO-Nectin-4) and 1B (GS-CHO-WT). In the Nectin-4 high-expressing cell line GS-CHO-Nectin-4, the ADCP activities of clones N4-P3-52, N4-P3-66, N4-P3-57, and N4-P3-48 were all superior to those of Enfortumab. In the Nectin-4 non-expressing cell line GS-CHO-WT, clones N4-P3-52, N4-P3-66, N4-P3-57, and N4-P3-48 showed no ADCP activity. This indicates that the above four clones have good specificity and ADCP activity, and are suitable for the preparation of ISAC (Immune-Stimulating Antibody Conjugate).

[0304] Validation of the cellular binding capacity of four humanized antibodies.

[0305] Human GS-CHO-Nectin-4 cells were seeded into U-shaped 96-well plates at a density of 2 × 10⁶ cells per well. 5For each cell, the antibody to be detected was added to the corresponding cell well according to a series of concentration gradients (15, 5, 2, 1, 0.1852, 0.0617, 0.0206, 0.0069, 0.0023, 0.0008, 0.0003, 0.0001 μg / mL), and incubated at 4°C for 30 minutes. Then, the unbound portions were washed with PBS, and PE fluorescent secondary antibody against goat anti-human IgG (SouthernBiotech, 2040-09) was added. After incubation at 4°C for 30 minutes, the cells were washed twice with PBS, and the cells were detected using a flow cytometer (FACS CELESTA, BD). As shown in Figure 2, the humanized antibodies hzN4-P3-52, hzN4-P3-48, hzN4-P3-57, and hzN4-P3-66 exhibited considerable affinity for their corresponding chimeric antibodies N4-P3-52, N4-P3-48, N4-P3-57, and N4-P3-66 at the cellular level.

[0306] Example 5: Verification of macrophage endocytosis function of humanized antibodies

[0307] The ADCP activity of the humanized clone of Nectin-4 antibody was verified using induced differentiated human M1 macrophages. Macrophages and target cells were fluorescently labeled, and when both cells underwent ADCP with the aid of the antibody, a double-positive cell population could be observed.

[0308] M1 macrophage differentiation induction: Monocytes were isolated from fresh human peripheral blood mononuclear cells (PBMCs, TPCS) using a CD16-free human monocyte enrichment kit (19058, STEMCELL), and differentiated at a rate of 1×10⁻⁶. 6 Density resuspended in AIM Medium CTS (A3021002, Gibco) medium was placed in cell culture flasks and cultured at 37°C for 4 hours. The medium was then replaced with AIM medium. Medium CTS + 10% inactivated fetal bovine serum (FBS, SH30406.05, Hyclone) + 25 ng / ml human MCSF1 (216-MC-500, R&D) were cultured. For the following seven days, culture was performed every three days using AIM medium containing 10% inactivated fetal bovine serum. Medium CTS should be partially replaced once. Seven days later, use AIM medium. Medium CTS + 10% inactivated fetal bovine serum + 100 ng / ml IFNg (285-IF-100 / CF, R&D) was used for half-medium medium replacement, and macrophage endocytosis experiments were performed the next day.

[0309] Macrophage endocytosis assay: Macrophages induced to differentiate were digested with Accutase (A6964-500ML, Sigma). Cell density was adjusted using RPMI-1640 complete medium (22400-071, Gibco) containing 10% fetal bovine serum. 1×10⁶ cells were added to each well of a 96-well hypoabsorbent plate. 5 Target cells MDA-MB-468 (TCHu136, CAS Cell Bank) were labeled with CFSE (C34554, INVITROGEN) at 37℃ for 10 min, washed twice with RPMI-1640 complete medium containing 10% fetal bovine serum, and 2.5 × 10⁶ cells were added to each well of a 96-well low-absorption plate. 4 Target cells. Target cells and macrophages were mixed with serially diluted Nectin-4 antibody (10, 3.3, 1.11, 0.37, 0.12, 0.041, 0.014, 0.005, 0.002, 0.001 μg / mL) by pipetting and incubating at 37°C for 4 hours.

[0310] Flow cytometry staining: Macrophages were labeled with CD14 PE / Cy7 (557742, BD) and then flow cytometry was performed. Cells in the double positive areas of PE / Cy7 and CFSE were identified as the target cell population.

[0311] As shown in Figure 3, the humanized antibodies hzN4-P3-52, hzN4-P3-48, hzN4-P3-57, and hzN4-P3-66 can induce ADCP in M1 macrophages in the presence of human breast cancer cells MDA-MB-468, and their activity is comparable to that of the corresponding chimeric antibodies N4-P3-52, N4-P3-48, N4-P3-57, and N4-P3-66.

Claims

1. An anti-Nectin-4 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising: (i) HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:27 or 28, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:35 or 36; (ii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:27, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:35; (iii) HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:28, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:36; (iv). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:29 or 30, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:37 or 38; (v). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:29, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:37; (vi) HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:30, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:38; (vii). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:31 or 32, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:39 or 40; (viii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:31, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:39; (ix). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:32, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:40; (x). HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:33 or 34, and LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:41 or 42; (xi). HCDR1, HCDR2, and HCDR3 included in VH as shown in SEQ ID NO:33, and LCDR1, LCDR2, and LCDR3 included in VL as shown in SEQ ID NO:41; or (xii). HCDR1, HCDR2 and HCDR3 included in VH as shown in SEQ ID NO:34, and LCDR1, LCDR2 and LCDR3 included in VL as shown in SEQ ID NO:

42.

2. An anti-Nectin-4 antibody or its antigen-binding fragment, said antibody or its antigen-binding fragment comprising a first heavy chain complementarity-determining region (HCDR1), a second heavy chain complementarity-determining region (HCDR2), a third heavy chain complementarity-determining region (HCDR3), and a first light chain complementarity-determining region (LCDR1), a second light chain complementarity-determining region (LCDR2), and a third light chain complementarity-determining region (LCDR3), wherein... (i) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23 or are composed of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:5 or 6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:19 and SEQ ID NO:23; or (ii). The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24 or are composed of the amino acid sequences shown in SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15 or 16, SEQ ID NO:20, SEQ ID NO:24; or (iii) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25 or are composed of the amino acid sequences shown in SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:25; or (iv) The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:26 or are composed of the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:

26.

3. The anti-Nectin-4 antibody or its antigen-binding fragment according to claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 27-34, or is composed of or comprises an amino acid sequence selected from any one of SEQ ID NO: 27-34, or is composed of said amino acid sequence.

4. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-3, comprising a light chain variable region (VL), wherein the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 35-42, or is composed of or comprises an amino acid sequence selected from any one of SEQ ID NO: 35-42, or is composed of said amino acid sequence.

5. An anti-Nectin-4 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof (i) A VH comprising the amino acid sequence shown in SEQ ID NO:27 or 28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or 36 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or 36; (ii) A VH comprising the amino acid sequence shown in SEQ ID NO:27 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:35; (iii) A VH comprising the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence; (iv) A VH comprising the amino acid sequence shown in SEQ ID NO:29 or 30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:37 or 38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence; (v) A VH comprising the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence; (vi) A VH comprising the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence; (vii) A VH comprising the amino acid sequence shown in SEQ ID NO:31 or 32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:39 or 40 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence; (viii) A VH comprising the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence; (ix). A VH comprising the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:40 or a VL comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:40; (x) A VH comprising the amino acid sequence shown in SEQ ID NO:33 or 34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:41 or 42 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising said amino acid sequence; (xi) A VH comprising the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:41 or a VL comprising the amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:41; or (xii) A VH comprising the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or a VL comprising said amino acid sequence.

6. The anti-Nectin-4 antibody or its antigen-binding fragment according to claim 1 or 2, comprising a heavy chain variable region VH and a light chain variable region VL, wherein (i). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:27 or 28, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:35 or 36; (ii). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:27, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:35; (iii) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:28, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:36; (iv) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:29 or 30, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:37 or 38. (v) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:29, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:37; (vi) The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:30, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:38; (vii) The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31 or 32, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39 or 40; (viii). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:31, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:39; (ix). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:32, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:40; (x). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:33 or 34, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:41 or 42; (xi). The heavy chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:33, and the light chain variable region comprises or is composed of the amino acid sequence shown in SEQ ID NO:41; or (xii). The heavy chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:34, and the light chain variable region contains or is composed of the amino acid sequence shown in SEQ ID NO:

42.

7. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1-6, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region, said heavy chain constant region being or derived from a constant region of IgG1, IgG2, IgG3, or IgG4, such as the constant region of human IgG1, IgG2, IgG3, or IgG4, such as the IgG1 heavy chain constant region. (i) An amino acid sequence comprising or consisting of said amino acid sequence; or (ii) An amino acid sequence comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO:

53.

8. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1-7, wherein the antibody or antigen-binding fragment comprises a light chain constant region, said light chain constant region being or derived from a lambda or Kappa light chain constant region, such as a human lambda or Kappa light chain constant region, preferably, said light chain constant region (i) An amino acid sequence comprising or consisting of, having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 54; or (ii) An amino acid sequence containing or consisting of SEQ ID NO:

54.

9. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-8, comprising a heavy chain HC and / or a light chain LC, wherein the heavy chain comprises, or is composed of, the VH and a heavy chain constant region according to any one of claims 1-8; and / or the light chain comprises, or is composed of, the VL and a light chain constant region according to any one of claims 1-8.

10. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain The amino acid sequence comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO:43-46, or It contains an amino acid sequence selected from any one of SEQ ID NO:43-46, or is composed of an amino acid sequence selected from any one of SEQ ID NO:43-46.

11. The anti-Nectin-4 antibody or antigen-binding fragment thereof according to any one of claims 1-10, wherein the antibody or antigen-binding fragment thereof comprises a light chain, wherein the light chain The amino acid sequence comprising an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NO:47-50, or It contains an amino acid sequence selected from any one of SEQ ID NO:47-50, or is composed of an amino acid sequence selected from any one of SEQ ID NO:47-50.

12. An anti-Nectin-4 antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain and a light chain, wherein... (i) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

43. The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47; (ii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44; and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

48. (iii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:45; or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49; or (iv) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:46, and the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

50.

13. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-11, wherein the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein (i) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

43. The light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:47; (ii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44; and the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

48. (iii) The heavy chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:45; or the light chain comprises or is composed of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:49; or (iv) The heavy chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:46, and the light chain comprises or consists of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:

50.

14. An anti-Nectin-4 antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment comprises a heavy chain and a light chain, wherein (i) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:43, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:47; (ii) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:44, wherein... The light chain contains or is composed of the amino acid sequence of SEQ ID NO:48; (iii) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:45, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:49; or (iv) The heavy chain comprises or is composed of the amino acid sequence of SEQ ID NO:46, and the light chain comprises or is composed of the amino acid sequence of SEQ ID NO:

50.

15. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-14, wherein the antibody is a monoclonal antibody.

16. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-15, wherein the antibody is a humanized antibody or a chimeric antibody.

17. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-16, wherein the antigen-binding fragment is an antibody fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, dAb (domain antibody), bivalent antibody, or linear antibody.

18. The anti-Nectin-4 antibody or its antigen-binding fragment according to any one of claims 1-17, wherein the antibody or its antigen-binding fragment has one or more of the following properties: a) Binds to Nectin-4 in humans or cynomolgus monkeys with high affinity; b) It can specifically bind to cells expressing Nectin-4, for example, it has a considerable affinity; c) It has good specificity and antibody-dependent cell-mediated phagocytosis (ADCP), for example, it has ADCP activity in cells that highly express Nectin-4 but no ADCP activity in cells that do not express Nectin-4. d) Suitable for the preparation of ISAC (Immune-Stimulating Antibody Conjugate); e) Inducing ADCP in immune effector cells, such as macrophages, especially M1 macrophages; or f) It can effectively treat Nectin-4 related diseases or conditions, such as Nectin-4 positive tumors.

19. An isolated nucleic acid encoding any one strand of the anti-Nectin-4 antibody or its antigen-binding fragment as described in any one of claims 1-18.

20. A vector comprising the nucleic acid of claim 19, preferably an expression vector.

21. A host cell comprising the nucleic acid of claim 19 or the vector of claim 20, preferably, the host cell being prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

22. A method for preparing an anti-Nectin-4 antibody or an antigen-binding fragment thereof, the method comprising: The host cells of claim 21 are cultured under conditions suitable for expressing nucleic acids encoding the anti-Nectin-4 antibody or its antigen-binding fragment as described in any one of claims 1-18. Optionally, the antibody or its antigen-binding fragment can be separated. Optionally, the method further includes recovering the anti-Nectin-4 antibody or its antigen-binding fragment from the host cell; optionally, the antibody is purified, for example, by Protein A.

23. An immunoconjugate comprising an antibody against Nectin-4 as described in any one of claims 1-18 or an antigen-binding fragment thereof and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptosis-inducing agents, chelating agents, and immunomodulators, such as immune activators; for example, the immunoconjugate is an ADC or an ISAC.

24. A pharmaceutical composition comprising the anti-Nectin-4 antibody of any one of claims 1-18 or an antigen-binding fragment thereof or the immunoconjugate of claim 23, and optionally a pharmaceutical excipient.

25. A pharmaceutical combination comprising the anti-Nectin-4 antibody of any one of claims 1-18 or an antigen-binding fragment thereof or the immunoconjugate of claim 23, and one or more other therapeutic agents, such as those selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immune activators).

26. A method for preventing or treating Nectin-4-related diseases and / or conditions in an individual, the method comprising administering to the subject an effective amount of any one of claims 1-18 of an anti-Nectin-4 antibody or an antigen-binding fragment thereof, or the immunoconjugate of claim 23, or the pharmaceutical composition of claim 24, or the pharmaceutical combination of claim 25.

27. The method of claim 26, wherein the subject, compared to a healthy individual, has a disease or condition associated with abnormal expression of Nectin-4 or abnormal activation of its mediated signaling pathways (e.g., the PI3K / Akt pathway).

28. The method of claim 26 or 27, wherein the disease or condition is selected from tumors such as cancer, such as breast cancer (e.g., triple-negative breast cancer), cervical cancer, ovarian cancer, urothelial carcinoma, colorectal cancer, esophageal cancer, liver cancer, thyroid cancer, bladder cancer, prostate cancer, lung cancer, stomach cancer, head and neck cancer, or pancreatic cancer.

29. The method of any one of claims 26-28, wherein the method further comprises administering one or more other therapies, such as treatment modalities and / or other therapeutic agents, for example, said therapeutic agents are selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immune activators).

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