Humanized anti-nectin-4 antibody

Abstract

The present invention relates to a humanized antibody having specificity for Nectin-4 and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to a humanized antibody having specificity for Nectin-4 and uses thereof. [Background technology]

[0002] Nectins are adhesion molecules that help organize epithelial and endothelial junctions and function as receptors for the entry of herpes simplex virus, measles virus, and poliovirus.

[0003] Nectins belong to the immunoglobulin superfamily and are homologs of the poliovirus receptor (PVR / CD155), also known as poliovirus receptor-bound proteins (PRRs). Five members have been described to date: PVR / CD155, nectin-1 / PRR1 / CD111, nectin-2 / PRR2 / CD112, nectin-3 / PRR3, and nectin-4 / PRR4. Their ectodomains consist of three immunoglobulin-like (Ig) V-, C-, and C-type domains that share 30–55% identity in their amino acid sequences.

[0004] Expression of nectin / PRR molecules is generally widespread in tissues, including hematopoietic, neural, endothelial, and epithelial cells, with the exception of nectin-3 and nectin-4, which show a more restricted expression profile.

[0005] Nectin-4 is a particularly interesting target. It is expressed during fetal development, but its expression is reduced and highly restricted in adult tissues compared to that of other members of the nectin family. Nectin-4 is a tumor-associated antigen in 83% of bladder cancers, 78% of breast cancers (mainly triple-negative and ERBB2+), 71% of pancreatic cancers, 55% of lung cancers, 57% of ovarian cancers, 59% of head and neck cancers, and 55% of esophageal cancers.

[0006] Nectin-4 expression in these pathologies is associated with poor prognosis, likely due to its ability to confer increased migration, proliferation, and metastasis formation capabilities on tumor cells in vitro.

[0007] In normal tissues, Nectin-4 is only detected in the skin, salivary glands, bladder, and esophagus. The recent approval by health authorities of enfortumumab vedotin for the second-line treatment of advanced urothelial carcinoma completes the validation of Nectin-4 as a target for cancer therapy. Summary of the Invention [Means for solving the problem]

[0008] The present invention relates to a humanized antibody having specificity for Nectin-4, an antigen-binding fragment thereof, and uses thereof.

[0009] In particular, the present invention provides humanized antibodies derived from the monoclonal anti-Nectin-4 antibody 5A12.2 mAb.

[0010] Humanized variants derived from 5A12.2, which can be provided in high yields, offer a new approach to improving the therapeutic index of Nectin-4-positive cancer treatment, combined with reduced skin toxicity and high antitumor selectivity and efficacy.

[0011] Surprisingly, the antibodies of the present invention can be provided in significantly higher yields than non-humanized antibodies. This has been demonstrated for small-scale and large-scale production approaches. Such high-yield, reproducible production greatly facilitates pharmaceutical use.

[0012] The first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that binds to a discontinuous epitope on Nectin-4 consisting of amino acids L81, H83, Y86, G87, H89, S91, P92, and E95 of SEQ ID NO: 1, as determined using deep mutational scanning (DMS). DMS is known to those skilled in the art. Essentially, all possible amino acid changes in a given protein are first synthesized. The activity of each of these protein variants is assayed in parallel using barcodes for each variant. The effect of each mutation is identified by comparing the activity to the wild-type protein. A detailed example of DMS is provided herein below.

[0013] Preferably, the antibodies of the present invention have better avidity for their dimeric epitope on Nectin-4 than the prior art antibody HA22. HA22 is a human monoclonal anti-Nectin-4 antibody described in WO 2012 / 047724, the contents of which are incorporated herein by reference. "Avidity," as used herein, describes the overall or cumulative strength of a protein-protein complex, i.e., a measure of the total strength of all noncovalent interactions between antibodies binding to their ligand(s). Avidity is determined by three parameters: the binding affinity of the complex, the valency of the proteins, and the structural arrangement of the proteins in the complex. The antibodies of the present invention are not, for example, the antibody mAb 5A12.2 obtainable by the hybridoma deposited at the CNCM under accession number CNCM I-5407, as described in WO 2021 / 069508.

[0014] The antibodies of the present invention are monoclonal antibodies (mAbs) or monoclonal antibody fragments characterized by a particular amino acid sequence. Unless otherwise indicated, the term "monoclonal" refers to a single species, i.e., a single amino acid composition, of the antibody or antibody fragment.

[0015] The antigen-binding site of an antibody of the present invention comprises a heavy chain variable domain / region (VH) and / or an antibody light chain variable domain / region (VL), or a VH / VL pair. The variable domain / region refers to each of the light and heavy chain pairs directly involved in binding the antibody to the antigen. The heavy chain variable domain is abbreviated as "VH" and the light chain variable domain is abbreviated as "VL".

[0016] The term "antigen-binding site" refers to the region(s) of an antibody molecule to which a ligand (e.g., an antigen, i.e., Nectin-4, or an antigenic fragment thereof) actually binds and which is derived from the antibody.

[0017] The antigen-binding site of an antibody according to the present invention can contain six complementarity-determining regions (CDRs), which contribute to varying degrees to the affinity of the binding site for the antigen. There are three heavy chain variable domain CDRs (CDR-H1, CDR-H2, and CDR-H3) and three light chain variable domain CDRs (CDR-L1, CDR-L2, and CDR-L3). Functional antigen-binding sites consisting of fewer CDRs (i.e., where binding specificity is determined by three, four, or five CDRs) are also within the scope of the present invention. For example, less than the complete set of six CDRs may be sufficient for binding. In some cases, one VH domain or one VL domain may be sufficient.

[0018] According to the present invention, only the VH region or its CDRs may constitute the complete antigen-binding site. In certain embodiments, the antibody comprises only the VH region or its CDRs as defined herein. In other embodiments, the antibody comprises a VH region or its CDRs as defined herein together with a VL region or its CDRs, particularly a VL region or its CDRs as defined herein.

[0019] The positions of CDRs within a VH or VL region may be defined according to Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) or the IMGT numbering system, both of which are known to those skilled in the art. The IMGT numbering system is defined for comparing variable domains regardless of antigen receptor, chain type, or species (Lefranc M.-P., "Unique database numbering system for immunogenetic analysis," Immunology Today, 18, 509 (1997); Lefranc M.-P., "The IMGT unique numbering for immunoglobulins, T cell receptors, and Ig-like domains," The Immunologist, 7, 132-136 (1999)).

[0020] A further aspect of the present invention is a humanized monoclonal antibody or an antigen-binding fragment thereof that binds to Nectin-4, comprising: (a) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; a variable heavy chain (VH) region comprising complementarity determining regions (CDRs) CDR-H1 set forth in SEQ ID NO: 10 or 11, CDR-H2 set forth in SEQ ID NO: 12 or 13, and CDR-H3 set forth in SEQ ID NO: 14; (b) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6 to 9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a variable light chain (VL) region comprising CDR-L1 set forth in SEQ ID NO: 15 or 16, CDR-L2 set forth in SEQ ID NO: 17 or 18, and CDR-L3 set forth in SEQ ID NO: 19.

[0021] The specific amino acid sequences represented by SEQ ID NOs used herein are provided in the attached sequence listing. SEQ ID NOs: 10-19 define the six CDR sequences of the humanized antibodies of the present invention according to the Kabat numbering system. Unless otherwise specified, the Kabat system is used herein.

[0022] According to the present invention, substitution of one or two amino acids in these CDR sequences is possible. In certain embodiments, conservative amino acid substitutions, i.e., substitution of an amino acid with another amino acid having similar biochemical properties, such as substitution of an aliphatic amino acid, e.g., Gly, Ala, Val, Leu, or Ile, for another aliphatic amino acid, substitution of a basic amino acid, e.g., His, Lys, or Arg, for another basic amino acid, substitution of an acidic amino acid or its amide, e.g., Asp, Glu, Asn, or Gln, for another acidic amino acid or its amide, substitution of an aromatic amino acid, e.g., Phe, Tyr, or Trp, for another aromatic amino acid, or substitution of a hydroxy- or sulfur-containing amino acid, e.g., Ser, Thr, Met, or Cys, for another hydroxy- or sulfur-containing amino acid, are preferred.

[0023] An "antigen-binding fragment" of an antibody refers to a molecule comprising a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. The term also encompasses fusion proteins, e.g., with non-immunoglobulin peptides or polypeptides, and conjugates with non-proteinaceous structures, e.g., labels or toxins. The terms "antigen-binding fragment of an antibody," "antigen-binding fragment thereof," "fragment of an antibody," or "fragment thereof" may be used interchangeably herein.

[0024] The antibodies or antigen-binding fragments thereof of the present invention may be monovalent or multivalent, i.e., they may contain a single antigen-binding site or multiple antigen-binding sites. For example, Fab fragments have a single antigen-binding site, IgG class antibodies or Fv or scFv fragments have two antigen-binding sites, and IgM class antibodies have five antigen-binding sites. The term "antibody" also encompasses heterospecific antibodies, e.g., heterobispecific antibodies, having different antigen-binding sites, particularly antibodies directed against two different epitopes on an antigen. As used herein, an "epitope" is the region of an antigen bound by an antibody. The term "epitope" includes any polypeptide determinant capable of specific binding to an antibody.

[0025] According to another preferred embodiment, the humanized antibody or antigen-binding fragment thereof comprises: (a) a VH region comprising an amino acid sequence set forth in SEQ ID NOs: 2 to 5; (b) a VL region comprising the amino acid sequence set forth in SEQ ID NOs: 6 to 9.

[0026] More specifically, the antibody or antigen-binding fragment thereof comprises: (i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; (ii) a VH region comprising an amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; (iii) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising an amino acid sequence according to SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% identity thereto; (iv) a VH region comprising an amino acid sequence according to SEQ ID NO: 5 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and It may comprise a VL region comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

[0027] "Percent (%) amino acid sequence identity" with respect to a peptide or polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in that particular peptide or polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST.

[0028] The antibody of the present invention may be a chimeric antibody, a multispecific antibody, in particular a bispecific antibody, a human antibody, a humanized antibody or an antigen-binding fragment thereof.

[0029] According to the present invention, a "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0030] A "multispecific antibody" binds to two or more different epitopes. The epitopes can be on the same antigen or on different antigens. A preferred example of a multispecific antibody is a "bispecific antibody" that binds to two different epitopes.

[0031] The term "humanized antibody" or "humanized form of antibody" refers to an antibody in which both the heavy and light chains have been humanized as a result of antibody engineering. Humanized chains are typically chains in which the V-region amino acid sequence has been analyzed as a whole and altered to be more homologous to human germline sequences than to the germline sequences of the species of origin. For example, a "humanized antibody" may be prepared by grafting murine CDRs into the framework regions of a human antibody. See, e.g., Riechmann, L. et al., Nature 332 (1988) 323-327 and Neuberger, MS et al., Nature 314 (1985) 268-270. Other forms of humanized antibodies encompassed by the present invention are those in which the constant regions have been further modified or altered from those of the original antibody to generate the properties described herein. Humanization assessment is based on the resulting amino acid sequence, not the methodology itself.

[0032] The antibodies of the present invention can be of any suitable class. The term "class" refers to the type of constant domain or constant region possessed by the heavy chain. As used herein, "constant domain" or "constant region" refers to the sum of the domains of an antibody other than the variable region. The constant region is not directly involved in antigen binding but exhibits various effector functions. The antibody can be any of the five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, or any subclass (isotype) thereof, such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. Antibodies or fragments thereof of class IgG, IgA, or IgM are particularly preferred according to the present invention.

[0033] According to a preferred embodiment, the antibody of the invention is selected from the class IgG, for example the subclasses IgG1, IgG2, IgG3 or IgG4, the class IgM, the class IgA or an antigen-binding fragment thereof.

[0034] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody of the present invention or a fragment thereof to an epitope of the Nectin-4 antigen. A measure of the binding strength of an antibody is called affinity. Methods for determining such binding and / or affinity using in vitro assays are known to those skilled in the art. According to the present invention, detection by flow cytometry, immunohistochemistry, and / or fluorescence are described and are particularly preferred herein.

[0035] The affinity of binding of an antibody to an antigen is determined by the Ka (rate constant for the association of the antibody from the antibody / antigen complex), KD (dissociation constant), and K dis (kD / ka), i.e., the term "Ka," as used herein, refers to the association rate of a particular antibody-antigen interaction, while the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. Such values for antibodies can also be determined using methods well established in the art and also described herein.

[0036] The antibodies and antigen-binding fragments thereof according to the invention preferably exhibit a dissociation constant KD of at least 3.5, preferably at least 4.0, more preferably at least 4.2, and most preferably at least 4.5 (nM).

[0037] Of course, specific binding to human nectin-4 expressed by tumors is preferred. Thus, the antibodies provided herein preferably exhibit specific binding to nectin-4 and do not exhibit substantial cross-reactivity or cross-reactivity with other proteins, particularly proteins of the human nectin family such as nectin-1.

[0038] Antibodies or antigen-binding fragments thereof can be produced in a suitable host cell comprising a nucleic acid molecule, e.g., a DNA molecule, encoding an antibody VH region or VL region, or encoding a complete antibody or antibody fragment, or a vector or vector system, i.e., a plurality of vectors comprising said nucleic acid molecule(s), preferably operably linked to an expression control sequence, particularly a heterologous expression control sequence. Methods for providing antibodies of the invention are described herein. The host cell may be any known host cell for producing antibodies or antibody fragments, for example, a prokaryotic cell such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, e.g., a CHO cell or a hybridoma cell.

[0039] Surprisingly, the humanized antibodies of the present invention, particularly those characterized by a VH region comprising the amino acid sequences set forth in SEQ ID NOs: 2 to 5 and a VL region comprising the amino acid sequences set forth in SEQ ID NOs: 6 to 9, can be provided in higher yields than the corresponding parent chimeric 5A12.2 antibodies (see Tables 2 and 3).

[0040] According to a further embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a labeling group and / or an effector group attached to the antibody or antigen-binding fragment. The labeling group may be, for example, a dye, a paramagnetic group, a radioactive group, or a fluorogenic group that can be detected by imaging. Preferred effector groups are therapeutic groups, in particular cytotoxic agents such as chemotherapeutic agents, drugs, anti-inflammatory agents, radioisotopes, toxins such as topoisomerase poisons, enzymes such as nucleases and their fragments, growth inhibitors, antibiotics, and all suitable anticancer and antitumor agents known to those skilled in the art. Particularly preferred is the topoisomerase poison camptothecin, and its derivatives and / or structural analogs such as exatecan and its derivatives such as deruxtecan.

[0041] A further aspect of the present invention relates to an antibody-drug conjugate (ADC) comprising a humanized antibody or antigen-binding fragment thereof described herein and a drug conjugated to a reactive amino acid residue on the antibody or antigen-binding fragment, e.g., an amino acid residue having a side chain comprising an amino group, a hydroxy group, or a thiol group, or to a reactive group in the antibody glycan structure.

[0042] Preferably, the drug is conjugated to a reactive thiol group in the side chain of a cysteine residue on the antibody or antigen-binding fragment thereof.

[0043] The drug of the antibody conjugate of the present invention is preferably a topoisomerase I inhibitor, an auristatin, or a maytansinoid.

[0044] A topoisomerase I inhibitor is a compound that can form a ternary complex with topoisomerase I and DNA, thereby preventing DNA religation and introducing DNA strand breaks into the cellular genome. The topoisomerase I inhibitor may be selected from, for example, camptothecin or its analogs, indenoisoquinolines, and indolocarbazoles. In certain embodiments, the topoisomerase I inhibitor is camptothecin or its analogs, i.e., a compound comprising the pentacyclic basic structure of camptothecin and, optionally, modified substituents that result in the presence of an additional ring. Specific examples are camptothecin, topotecan, irinotecan, SN-38, belotecan, and exatecan, including derivatives thereof such as deruxtecan, lurtotecan, or atiratecan. In a particularly preferred embodiment, the topoisomerase I inhibitor is exatecan. Exatecan is typically covalently attached to the antibody via its NH group.

[0045] A particularly preferred auristatin is the synthetic antitumor agent monomethyl auristatin E (MMAE), which inhibits cell division by blocking tubulin polymerization.

[0046] Derivatives of maytansine are known as maytansinoids. Maytansinoids inhibit microtubule assembly by binding to tubulin at the rhizoxin binding site. Examples of maytansinoids are ansamitocin, mertansine / emtansine (DM1), and ravtansine / soravtansine (DM4).

[0047] In principle, a drug, such as exatecan, may be conjugated to any suitable position on a monoclonal antibody or its antigen-binding fragment, particularly any position that does not abolish the binding of the antibody to Nectin-4. For example, a topoisomerase-I inhibitor, such as exatecan, may be conjugated to a reactive amino acid residue on the antibody, such as an amino acid residue having a side chain containing an amino, hydroxy, or thiol group, or a reactive group in the antibody glycan structure. In certain embodiments, the drug, such as exatecan, is conjugated to a reactive thiol group in the side chain of an accessible cysteine residue on the antibody.

[0048] In certain embodiments, the antibody-drug conjugate has a drug-antibody / antibody fragment molar ratio (DAR) greater than 1, i.e., two or more drug molecules are conjugated to the antibody / antibody fragment. Typically, the conjugate has a DAR of about 2:1 to about 16:1, specifically about 4:1 to about 10:1, and more specifically about 6:1 to about 8:1. The DAR can be calculated from statistical distributions according to known methods.

[0049] In certain embodiments, the drug is conjugated to the antibody or antigen-binding fragment via a linker. In certain embodiments, the linker is a cleavable linker, i.e., a linker that can be cleaved under physiological conditions, for example, by a physiological enzyme. Specific examples of cleavable linkers are peptide-based linkers that can be cleaved by proteases or glycoside-based linkers that can be cleaved by glycosidases.

[0050] In certain embodiments, the linker is a hydrophilic polysarcosine linker, such as those described by Conilh et al., "Exatecan antibody drug conjugates based on a hydrophilic polysarcosine drug-linker platform" (Pharmaceuticals 14 (2021), 247). Further preferred linkers include linkers containing at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units, such as the linker in the antibody-drug conjugate MEDI7247, or the mcc-triazole spacer-PEG7-x-Lys-PABC glycol linker from Trodelvy®. Further preferred linkers include oligopeptides, particularly dipeptides to decapeptides, e.g., tetrapeptide sequences such as glycine-glycine-phenylalanine-glycine from Enhertu®. Further preferred linkers include highly polar spacers such as acyl groups, carbamoyl groups, and / or sulfamide groups attached to at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units, e.g., linker technology known as Hydraspace™.

[0051] The antibody-drug conjugate of the present invention may be prepared by known methods.

[0052] The antibody or antigen-binding fragment thereof may be reacted with a linker-drug conjugate to obtain an antibody-drug conjugate. The linker-drug conjugate, for example, comprises a drug molecule attached to a suitable linker, the linker comprising a reactive group capable of reacting with a desired binding site on the antibody or antigen-binding fragment thereof. For attachment to a cysteine residue, the linker-drug conjugate comprises a thiol-reactive group, for example, a maleimide group.

[0053] A preferred embodiment of the anti-tumor agent relates to an anti-tumor immune stimulator, including but not limited to, toll-like receptor (TLR) agonists or stimulator of interferon gene (STING) pathway agonists.

[0054] According to another preferred embodiment, the anti-inflammatory agent may be selected from the group comprising steroids and corticosteroids such as glucocorticoids, for example cortisol and its derivatives, or mineralocorticoids such as aldosterone and its derivatives.

[0055] According to a further aspect of the invention, the antibody or antigen-binding fragment thereof is for use in medicine, in particular for therapeutic or diagnostic uses, including in vitro and in vivo diagnostic uses.

[0056] The antibody or antigen-binding fragment thereof may be useful in methods for preventing and / or treating cancer and / or inflammatory disorders, wherein the cancer and / or inflammatory disorders are preferably associated with Nectin-4 overexpression.

[0057] The cancer to be prevented and / or treated may be any type of cancer, and the term "cancer" is used herein to refer to a proliferative disease. The cancer to be prevented and / or treated according to the present invention is preferably selected from the group consisting of urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, bladder cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer, and / or esophageal cancer.

[0058] Being specific for Nectin-4 expressed on tumors, the antibodies and fragments thereof of the present invention may also be used for diagnosis, for example, by binding them to a labeling group as described above.

[0059] According to a further aspect, the antibodies or antigen-binding fragments thereof described herein may be used in methods for preventing and / or treating inflammatory disorders, preferably associated with Nectin-4 expression. Such treatment may preferably be combined with an anti-inflammatory agent known to those skilled in the art. Preferred anti-inflammatory agents are described above.

[0060] Another aspect of the present invention is a combination of at least two different monoclonal antibodies or fragments described herein.

[0061] Furthermore, the present invention relates to nucleic acid molecules, e.g., DNA molecules, encoding antibody VH or VL regions or encoding the above-mentioned complete antibodies or antibody fragments, vectors or vector systems, i.e., vectors comprising the above-mentioned nucleic acid molecule(s), preferably operably linked to expression control sequences, in particular heterologous expression control sequences.

[0062] Furthermore, the present invention relates to a cell comprising the above-mentioned nucleic acid molecule or vector or vector system. The term "vector", as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. According to a preferred embodiment, the vector is an expression vector. "Expression vectors" are vectors that are capable of directing the expression of nucleic acids to which they are operatively linked. Vectors, particularly expression vectors, for the recombinant production of antibodies are well known in the art.

[0063] The cell may be any host cell known for producing antibodies or antibody fragments, for example a prokaryotic cell such as an E. coli cell, a yeast cell, an insect cell or a mammalian cell, for example a CHO cell or a hybridoma cell.

[0064] A still further aspect of the present invention relates to pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof described herein. Typically, the antibodies or antibody fragments are administered as pharmaceutical compositions comprising an active agent and a pharmaceutically acceptable carrier or excipient. Examples of suitable carriers and excipients for formulating antibodies or antibody fragments include saline and aqueous buffer solutions and are well known in the art.

[0065] Depending on the stage and severity of the disorder being treated, the pharmaceutical composition may be administered once or several times during the course of the disorder, for example daily, every other day, twice a week, or weekly for a suitable period of time.

[0066] In certain embodiments, the pharmaceutical composition is administered parenterally, e.g., by subcutaneous, intramuscular, or intravenous injection, or by infusion. In further embodiments, the pharmaceutical composition may be administered topically, e.g., orally, intranasally, or pulmonally, e.g., as an aerosol.

[0067] The present invention is further illustrated by the following figures and examples, which are not intended to limit the scope of the invention. [Brief explanation of the drawings]

[0068] [Figure 1] Figure 1. Evaluation of the compatibility of antibodies 5A12.2_H1L0, HA22 (enfortumab), and 15A7.5_H1L2. Yeast expressing Nectin-4 fragment 32-349 were incubated with 100 nM unlabeled Antibody 1 or PBS (as a positive control). After 30 minutes of incubation, 500 pM of Antibody 2 was added for 30 minutes. Yeast were then incubated with a fluorescent reporter, and Nectin-4 expression and Antibody 2 were detected by FACS. Results are expressed as a percentage of fluorescence relative to the respective positive control. [Figure 2]Flow cytometric library sorting. Two libraries covering the nectin-4 sequence of interest were generated (left and right panels). Yeast expressing nectin-4 monomutants were incubated with either 100 pM biotinylated 5A12.2_H1L0 and 100 pM Dy650-labeled 15A7.5_H1L2 (upper panel) or 100 pM biotinylated HA22 and 100 pM Dy650-labeled 15A7.5_H1L2 (lower panel). Biotinylated antibodies were revealed using streptavidin PE. After washing with PBS, cells were analyzed and sorted on a BD FACSAria III as indicated on the graph. Yeast single-stained with antibodies express nectin-4 monomutants that disrupt the epitope for binding of other antibodies. [Figure 3]

[0023] Figure 1 is a diagram of positions that, when mutated, show significant effects on the 5A12.2_H1L0 antibody. For any amino acid in the domain of interest, the higher the number of prohibited mutations, the more important the residue is for binding of the 5A12.2_H1L0 antibody. [Figure 4]

[0033] Figure 1 shows the positions that, when mutated, show significant effects on the HA22 (enfortumab) antibody. For any amino acid in the domain of interest, the higher the number of prohibited mutations, the more important the residue is for binding of the HA22 antibody. [Figure 5] This is a structural reference diagram of Nectin-4 (4FRW, pdb). The pymol file is shown. For each antibody, the key amino acids of the epitope are shown. The most important residues are underlined. [Figure 6]Figure 1 shows in vitro cytotoxic activity against SUM190PT and T47D tumor cells. mAbs HA22 (enfortumab, circles), Ch-5A12.2 (triangles), and an isotype control (open squares) were conjugated to α-amanitin to form antibody-drug conjugates. Cytotoxic activity over a dose range (left: 1 pg / mL to 15 μg / mL, right: 192 pg / mL to 15 μg / mL) was assessed in the nectin-4-expressing cell lines SUM190PT (left) and T47D (right). Viability (AlamarBlue assay) after a 5-day incubation period is reported. EC50 values were determined by GraphPad Prism 9 software using nonlinear curve fitting (4 parameters). [Figure 7] Figure 1 shows the apparent affinity of humanized variants for tumor cells. Detection by flow cytometry. Nectin-4-expressing T47D human tumor cells were numbered and incubated with a range of doses (169 pg / mL to 30 μg / mL) of Ch-5A12.2 or the indicated humanized variants. The numbers accompanying H and L indicate the number of reversion mutations introduced. Cells were then stained with phycoerythrin-conjugated goat anti-human Fc antibody and analyzed by flow cytometry. Mean fluorescence intensity is reported. GraphPad Prism 9 software was used with nonlinear curve fitting (4 parameters). [Figure 8] Figure 1 shows in vitro cytotoxic activity against HCT-116 transfectants expressing nectin-4. The Hu-5A12.2_H1L0 (triangles), Hu-5A12.2_H1L1 (open diamonds), Hu-15A7.5_H1L2 (circles), and isotype control (ICT, open squares) mAbs were conjugated to exatecan to generate antibody-drug conjugates, and their cytotoxic activity over a dose range (left: 1 pM to 18.94 nM) was evaluated in the HCT-116 nectin-4-expressing cell line. Viability (MTT assay) after a 7-day incubation period is reported. EC50 values were determined by GraphPad Prism 9 software using nonlinear curve fitting (4 parameters). [Figure 9]This figure shows that treatment of SUM190PT-implanted NSG mice with Hu-5A7.5_H1L0-exatecan ADC or Hu-15A12.2_H1L2-exatecan ADC induces tumor regression. NSG mice (n=5 / group) were orthotopically xenografted bilaterally with SUM190PT cells embedded in Matrigel. Three different ADCs were tested: isotype control, ICT-exatecan (closed circles), Hu-5A12.2_H1L0-exatecan (open diamonds), and Hu-15A7.5_H1L2-exatecan (closed diamonds). Treatment of mice (single intravenous injection of 10 mg / kg) began when tumors reached approximately 70 mm. Tumor size (n=10 / group) was then monitored twice weekly using calipers and the size was reported using the following formula (LxlxhxPi / 6). [Figure 10] 1 is a diagram of the amino acid sequence of the parental 5A12.2 monoclonal antibody. The heavy and light chain amino acid sequences of the parental 5A12.2 monoclonal anti-Nectin-4 antibody are shown. CDRs are identified according to the IMGT and Kabat nomenclature systems (IMGT nomenclature, Kabat nomenclature). [Figure 11] 1 is a diagram of the amino acid sequence of a humanized variant of the 5A12.2 monoclonal antibody. The amino acid sequences of the heavy and light chains of the humanized variant of the 5A12.2 monoclonal anti-Nectin-4 antibody are shown. CDRs are identified according to the IMGT nomenclature and Kabat nomenclature. Back mutations are also identified. (IMGT nomenclature / Kabat nomenclature / anchor-IMGT and / or Kabat / back mutation) [Figure 12] FIG. 1 summarizes the sequences of the humanized antibodies of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0069] Example Principles of epitope mapping using DMS (Comprehensive Mutation Scanning) DMS is a mutagenesis method that aims to generate all possible monosubstitutions for all selected residues in a given protein sequence. A DMS library is obtained in the form of DNA that encodes the protein under study. In this library, each DNA strand contains a mutated codon relative to the parent sequence.

[0070] This DMS DNA library is incorporated into an expression plasmid specifically designed to express recombinant proteins on the yeast surface. Yeast is then transformed and induced to allow expression of monomutated proteins on the yeast surface. This new library (called a display library) is screened by flow cytometry using a fluorescent reporter to reveal the expression of the protein (anti-tag fluorescent antibody) and the binding of the protein to its partner (fluorescent partner).

[0071] For epitope mapping, the ideal case is to have two antibodies with compatible epitopes that can bind together on the same antigen. In this way, each of the two antibodies acts as a conformational control for the mutated antigen against the other antibody. In fact, monosubstitutions made on an antigen can have four types of effects: (i) loss of affinity for the first antibody while retaining binding to the second antibody. This is a mutation made within the epitope of the first antibody; (ii) loss of affinity for the second antibody while retaining binding to the first antibody. This is a mutation in the epitope of the second antibody; (iii) loss of affinity for both antibodies. This is a so-called "destroying" mutation that affects the conformation of the antigen and thus prevents binding of both antibodies; and (iv) no effect; the mutation is absent from the epitope of one of the two antibodies and does not cause a significant change in the conformation of the antigen. Following flow cytometry analysis, yeast populations that have lost affinity for the antibody of interest but retained binding to the second antibody are selected. The plasmids contained in this yeast population are extracted and sequenced by high-throughput sequencing. Analysis of the sequencing data allows the identification of mutations that affect the binding of the antibody to its target. This analysis therefore allows the identification of the critical position on the antigen for binding of the antibody of interest, i.e., its epitope.

[0072] Construction of antigen expression plasmids A gene corresponding to the antigen sequence Nectin-4 (32-349) was synthesized and cloned into a plasmid that allows expression on the surface of galactose-inducible yeast. In this construct, the expressed antigen possesses a C-terminal HA tag. The expression plasmid was then transformed into the yeast strain S. cerevisiae EBY100. Two DMS libraries were generated by PCR, for amino acid positions 32-91 (Library 1) and 92-151 (Library 2). The libraries correspond to the IgV domain of Nectin-4, known to be the target of the test antibody. Each mutation position contains a degenerate NNS or NNK codon encoding 20 amino acids / 32 codons. Each library contained approximately 1,200 single amino acid mutants and 2,000 DNA codon mutants. The two libraries were transformed into YSD. Unsorted yeast from the two libraries were sequenced to verify the efficiency of mutagenesis. 100% of the predicted single variants were sequenced for library 1 and 99.9% for library 2.

[0073] Epitope compatibility To confirm whether the tested antibodies matched the epitope mapping on the Nectin-4 IgV domain, i.e., whether the antibodies could bind together on the Nectin-4 IgV domain, the antibodies were first biotinylated (EZ-Link™ Sulfo-NHS-LC-Biotin). Yeast was induced to express the Nectin-4 32-349 construct and incubated for 10 min. 5The yeast cells were washed twice with PBS, 0.1% BSA. Then, the yeast cells were incubated with 100 nM unlabeled antibody 1 for 30 minutes or left alone as a positive control. After 30 minutes, 500 pM antibody 2 was added and incubated for another 30 minutes. The yeast were then incubated on ice for 15 minutes with the corresponding fluorescent reporters: anti-HA APC for detecting Nectin-4 expression and streptavidin-PE for detecting Antibody 2. After washing in PBS-BSA, the resuspended yeast were analyzed by flow cytometry. Figure 1 shows that 5A12.2_H1L0 and HA22 cannot simultaneously bind to the Nectin-4 IgV domain. In contrast, the 15A7.5_H1L2 mAb can simultaneously bind to the Nectin-4 IgV domain with either 5A12.2_H1L0 or HA22 (enfortumab).

[0074] Induction of antigen expression and FACS sorting of the library Yeast expressing the nectin-4 monomutant was induced for protein expression. Transformed yeast was induced in SG-CAA induction medium [6.7 g / L yeast nitrogen base without casamino acids, 20 g / L glucose, 5 g / L casamino acids, 100 mM sodium phosphate, pH 6.0]. For cytometric analysis / sorting, 10 6 ~10 pieces 8The induced cells were washed with 1 mL of PBSF (PBS, 0.1% BSA). They were then resuspended in an appropriate volume of a solution containing 100 pM biotinylated HA22 antibody and Dye650-labeled 15A7.5_H1L2. After incubation at 20°C for 1–3 hours with agitation, the cells were washed with 1 mL of ice-cold PBSF and then incubated with streptavidin on ice for 15 minutes. The cells were then analyzed and sorted on a BD FACSAria™ III cytometer using BD FACSdiva™ Diva software. As shown in Figure 2, single-stained yeast from each experiment were sorted, and plasmids were extracted from each sorted yeast population and prepared for sequencing. A two-step PCR was performed: the first step was to amplify the region of interest, and the second step was to add the Illumina adapters required for sequencing. Sequencing was performed on an Illumina iSeq100 instrument (2 × 150 bp, 300 cycles) with at least 150,000 reads per population. The data were then processed through an analysis pipeline using a dedicated proprietary script. Low-quality sequences (Q<30) were removed, and then monomutants were detected and counted. For each monomutation, the measured frequency in the unsorted population was compared with the frequency of the variant in the sorted population. The variant enrichment value was then calculated according to the following formula:

[0075]

number

[0076] Data interpretation The results of processing the NGS sequencing data are presented as bar graphs summarizing enrichment values for all single variants present in the DMS library (Figures 3 and 4). A value of 16–19 prohibitive mutations indicates a high impact; positions falling into this category are most likely to be indirectly interacting with the IgG under consideration. A value of 10–15 prohibitive mutations indicates a moderate impact position, and a value of 5–9 prohibitive mutations indicates a low impact position. Figure 5 shows an analysis of the structure of Nectin-4 (4FRW, pdb) to highlight the critical and most important epitope residues for each of the antibodies tested. Table 1 lists these residues.

[0077] [Table 1]

[0078] Humanization of chimeric antibodies The methodology used is "CDR grafting." The three mouse CDRs (regions that determine antibody specificity) of each antibody chain (heavy chain, VH or light chain, VL) are grafted onto the closest human germline framework. This methodology also allows for back-mutation of essential amino acids in the framework regions (FR) (e.g., anchors for CDRs, vernier residues, VH / VL interface, etc.).

[0079] Creation, production, purification and control of chimeric and humanized antibodies The light chain expression vector encodes the Vκ chain. Depending on the payload used, two different heavy chain expression vectors were used: one encoding an Fc fragment with D265C (ThiomAb) L234A and L235A mutations, and the other encoding an Fc fragment with P331S, L234F, and L235E mutations. Both Fc fragments are "Fc silent." The sequence of the anti-nectin-4 antibody Enfortumab (HA22) was also cloned into the same vector.

[0080] The light and heavy chain vectors were transfected into HEK293 cells seeded at a 1.2 / 1 ratio. After 6 days of production, the culture supernatant was clarified, and the mAb was purified using MabSelect PrismA resin (GE Healthcare) according to the manufacturer's instructions. 0.5 M glycine, 3 M NaCl, pH 8.9 was used as the binding buffer, and 0.1 M citrate solution, pH 3, was used for elution. Immediate neutralization was performed with 10% (V / V) 1 M Tris-HCl, pH 9. The monoclonal antibody was then dialyzed against PBS 1X, pH 7.4 (Mini Dialysis Device, 2 mL-10k, Thermo Scientific) and subsequently filtered through a 0.22 μM filter (Milelex GV Hydrophilic PVDF, Millipore). Taking into account the specific extinction coefficient (E1% 280 nm) of each monoclonal antibody, the concentration was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific). Purity was determined by UPLC-SEC using an Acquity UPLC-HClass Bio (Waters) with a Protein-BEH 200A column equilibrated in 0.2 M NaPO4, 0.3 M NaCl pH 6.9 supplemented with 10% isopropanol. Antibody mass was determined on a Xevo G2-S Q-Tof mass spectrophotometer (Waters) using a reversed-phase column (PLRP-S 4000A, Agilent Technologies). All samples were analyzed after deglycosylation with PNGase F glycosidase (New England Biolabs) at 37°C according to the manufacturer's instructions. Fragmentation and / or aggregation of the final material was assessed by SDS-PAGE. Endotoxin load was determined using a chromogenic LAL kinetic assay (Charles River Endosafe).

[0081] Table 2 reports the small scale (30 mL) production yields of the parent chimeric 5A12.2 and all humanized variants.

[0082] Table 3 reports the medium-scale (0.5-1.4 L) production yields of the parent chimeric 5A12.2 and the humanized variants 5A12.2_H1L0 and 5A12.2_H1L1.

[0083] [Table 2]

[0084] [Table 3]

[0085] Antibody conjugation A cysteine-reactive linker-amanitin compound (Heidelberg Pharma) with a cleavable linker (valine-alanine) to the engineered cysteine residue of the selected anti-nectin-4 thiomAb (D265C, L234A, L235A) antibody was used using maleimide chemistry. Briefly, the thiomAb antibody in PBS 1X pH 7.4 was reduced with TCEP, and the interchain disulfides were reoxidized with dehydroascorbic acid. The engineered cysteine was subsequently used for conjugation with the cysteine-reactive linker-amanitin compound. The conjugate was purified by dialysis. The drug-to-antibody ratio (DAR) by LC-MS analysis was 1.44 to 1.79 toxin per conjugated mAb. Less than 2% of the material aggregated as determined by SEC-HPLC.

[0086] The cysteine-reactive linker-exatecan compound, maleimide-propioPEG2-PSAR10-glucuronide-exatecan (MabLink), was conjugated to the cysteine residues of selected anti-nectin-4 mAbs (P331S, L234F, and L235E). Briefly, the mAb in PBS 1X, 1 mM EDTA was reduced with 14 molar equivalents of TCEP for 2 hours at 37°C, followed by buffer exchange (Amicon Ultra 30 kDa) into 100 mM KPO4, 1 mM EDTA pH 7.4. 12 molar equivalents of the cysteine-reactive linker-exatecan compound were used to conjugate with the reactive cysteine for 35 minutes at room temperature. The final exchange buffer was performed in 20 mM His, pH 6.0, followed by filtration through a 0.22 μM filter. The drug-antibody ratio (DAR) by LC-MS analysis ranged from 7.77 to 7.82 toxin per conjugated mAb. Less than 8% of the material was aggregated as determined by SEC-HPLC.

[0087] cell line The human triple-negative breast cancer cell line SUM190PT (BioIVT, Westbury, NY) was cultured in Ham's F12 medium containing 5% fetal bovine serum, 1% non-essential amino acids, 1% Hepes, 1% insulin, 1 μg / mL hydrocortisone, 6.8 ng / mL triiodo-L-tyrosine, 100 IU / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine. The human breast cancer T47D cell line was cultured in RPMI medium supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, and 100 μg / mL streptomycin. The human colorectal cancer Nectin-4 transfectant (HCT116-N4) was cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1x MEM non-essential amino acids, and 20 μg / mL hygromycin B.

[0088] In vitro cytotoxicity assay To analyze the in vitro cytotoxic activity of α-amanitin and exatecan ADCs, cell viability was assessed using the AlamarBlue staining protocol recommended by the manufacturer (Biosource, CA, USA). This test incorporates a fluorescent redox indicator. Fluorescence intensity is proportional to the reduction in cell metabolism. Experiments were performed by incubating 3,000 cells / well (SUM190PT, T47D, or HCT116-nectin-4) in triplicate with serial dilutions of the ADCs on day 0 in a 96-well plate. AlamarBlue was measured on day 5 by incubating 1 / 10 volume of AlamarBlue solution at 37°C for 2 hours and reading at 595 nm (FLUOstar Optima, BMG Labtech). Figure 6 shows that the parental 5A12.2 anti-nectin-4 monoclonal antibody exhibited a lower EC than HA22. 50 Figure 8 shows that the humanized variants H1L0 and H1L1 of monoclonal antibody 5A12.2 exhibit similar EC50 values compared to other humanized anti-Nectin-4 antibodies.

[0089] Flow cytometry T47D cells (50,000 cells) naturally expressing Nectin-4 were incubated with the indicated antibody dose range. After washing, the cells were then stained with phycoerythrin-conjugated goat anti-human antibody (5 μg / mL) (Jackson Immuno Research). After fixation, the cells were stained with viability dye (e780, Invitrogen) prior to flow cytometry acquisition. Figure 7 shows that all 5A12.2 humanized variants, except for the H0 variant, retain similar apparent affinity compared to the parent 5A12.2 monoclonal antibody.

[0090] Mouse experiments NOD / SCID (non-obese diabetic / severe combined immunodeficiency) / gc null mice (NSG) were obtained from Charles River Laboratory (Margate, UK). Six to seven-week-old female mice (n = 5 / group) were injected with SUM190PT (0.5 × 10) cells embedded in Matrigel. 6) cells were orthotopically xenografted bilaterally. Treatment with ADC was performed as described in the corresponding experiment. Tumor size (n=10 / group) was then monitored twice weekly using calipers, and size was reported using the following formula: (LxlxhxPi / 6). Figure 9 shows that a single injection of the humanized variant 5A12.2_H1L0 monoclonal antibody conjugated to exatecan can induce regression of the triple-negative breast cancer cell line SUM190PT in vivo.

[0091] Antibody affinity determination on the Octet platform The affinities of various monoclonal antibodies were determined on the Octet Red 96 platform, a system based on biolayer interferometry (BLI) technology. Two types of analytes were used in these studies: (i) a his-tagged recombinant extracellular domain of the nectin-4 protein (R&D systems / Biotechne) and (ii) a homodimeric recombinant Fc protein corresponding to the IgV domain of human nectin-4 fused to a human Fc domain. The AHC sensor (anti-human Fc) was used for the recombinant his-tagged nectin-4 EC analyte, and the FAB2G sensor was used for the Fc-fused recombinant IgV domain of nectin-4. The ligand of interest, diluted in 1x kinetic buffer, was loaded onto the selected sensor. The analyte remained in solution at working concentrations in 1x kinetic buffer ranging from 600 nM to 1.56 nM. Each experiment was performed at 26°C with shaking at 1,000 rpm and consisted of five steps: loading, equilibration, association, dissociation, and regeneration. For analysis of the generated data, a standard 1:1 Langmuir model was selected for the AHC sensor, and a 1:2 bivalent analyte model was selected for the FAB2G sensor. Both analytical models were implemented using Octet software. Table 2 shows that the affinity for His-tagged recombinant nectin-4 was similar for the 5A12.2 and HA22 monoclonal antibodies. In contrast, the affinity for the Fc-fused IgV nectin-4 domain differed significantly between 5A12.2 and HA22, suggesting that 5A12.2 has better avidity for its dimeric epitope on nectin-4 than HA22.

[0092] [Table 4]

[0093] Table 5 reports the affinity determinations for various humanized variants of monoclonal antibody 5A12.2. Here, only recombinant His-tagged Nectin-4 EC analyte was used. The affinities of the various humanized variants are similar to those of the parent antibody.

[0094] [Table 5]

Claims

1. A humanized monoclonal antibody or an antigen-binding fragment thereof that binds to a discontinuous epitope on Nectin-4 consisting of amino acids L81, H83, Y86, G87, H89, S91, P92 and E95 of SEQ ID NO: 1, as determined using comprehensive mutation scanning (DMS).

2. (a) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; a variable heavy chain (VH) region comprising complementarity determining regions (CDRs) CDR-H1 set forth in SEQ ID NO: 10 or 11, CDR-H2 set forth in SEQ ID NO: 12 or 13, and CDR-H3 set forth in SEQ ID NO: 14; (b) comprising an amino acid sequence set forth in any one of SEQ ID NOs: 6-9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; 2. The humanized antibody of claim 1, comprising a variable light chain (VL) region comprising CDR-L1 set forth in SEQ ID NO: 15 or 16, CDR-L2 set forth in SEQ ID NO: 17 or 18, and CDR-L3 set forth in SEQ ID NO:

19.

3. (i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; (ii) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 7 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; (iii) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 4 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; (iv) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 5 or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto; and The humanized antibody of claim 2, comprising a VL region comprising the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having at least 85%, at least 90%, at least 95% or at least 99% identity thereto.

4. (a) a VH region comprising an amino acid sequence set forth in SEQ ID NOs: 2 to 5; (b) a VL region comprising an amino acid sequence set forth in SEQ ID NOs: 6 to 9. The humanized antibody of claim 2 or 3.

5. The humanized antibody or antigen-binding fragment according to any one of claims 1 to 4, which is a multispecific antibody, in particular a bispecific antibody such as a biparatopic antibody, or an antigen-binding fragment thereof.

6. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which is an antibody or antigen-binding fragment thereof of class IgG, for example subclass IgG1, IgG2, IgG3 or IgG4, class IgM, class IgA, or which is a single-chain antibody or an antibody Fv fragment.

7. The humanized antibody or antigen-binding fragment of any one of claims 1 to 6, comprising a labeling group and / or an effector group linked to the antibody or antigen-binding fragment.

8. 8. The humanized antibody or antigen-binding fragment of claim 7, wherein the labeling group is a dye, a paramagnetic group, a radioactive group, or a fluorogenic group that is detectable upon imaging.

9. The humanized antibody or antigen-binding fragment of claim 8 , wherein the effector group is a therapeutic group, in particular a cytotoxic agent.

10. An antibody-drug conjugate comprising: A humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a drug conjugated to a reactive amino acid residue on the antibody or antigen-binding fragment, such as an amino acid residue having a side chain containing an amino group, a hydroxy group, or a thiol group, or to a reactive group in an antibody glycan structure.

11. 11. The antibody-drug conjugate of claim 10, wherein the drug is conjugated to a reactive thiol group in the side chain of a cysteine residue on the antibody or antigen-binding fragment thereof.

12. The antibody-drug conjugate of claim 10 or 11, wherein the drug is conjugated to the antibody or antigen-binding fragment thereof via a linker, in particular the linker is a cleavable linker such as a linker that is subject to cleavage by glucuronidase, and / or the linker is a peptidic linker.

13. The humanized antibody or fragment according to any one of claims 1 to 10 for use in medicine, in particular for therapeutic or diagnostic applications, including in vitro and in vivo diagnostic applications.

14. 14. The humanized antibody or fragment for use according to claim 13 in a method for the prevention and / or treatment of cancer and / or inflammatory disorders.

15. The humanized antibody or fragment for use according to claim 13 or 14, wherein the cancer is associated with Nectin-4 overexpression.

16. 16. The humanized antibody or fragment for use according to any one of claims 13 to 15, wherein the cancer is urothelial cancer, endometrial cancer, cervical cancer, colorectal cancer, liver cancer, thyroid cancer, breast cancer, pancreatic cancer, lung cancer, ovarian cancer, head and neck cancer and / or esophageal cancer.

17. A nucleic acid encoding one or more of the antibodies of any one of claims 1 to 9, or at least one VL and / or one VH of any of the antibodies of claims 1 to 9.

18. A vector comprising the nucleic acid of claim 17.

19. A host cell comprising the vector of claim 18.

20. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the antibody-drug conjugate according to any one of claims 10 to 12, or the vector according to claim 18, and optionally one or more pharmaceutical excipients.

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