Humanized anti-Nectin-4 antibodies

Abstract

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

Description

Technical Field

[0001] The present invention relates to a humanized antibody specific to nectin-4 and uses thereof. Background Art

[0002] Nectins are adhesion molecules that help organize epithelial and endothelial junctions and serve as a receptor for entry of herpes simplex, measles, and polio viruses.

[0003] Nectin belongs to the immunoglobulin superfamily and is a homolog of the poliovirus receptor (PVR / CD155), so it is also called poliovirus receptor-related protein (PRR). To date, five members have been described, namely PVR / CD155, Nectin-1 / PRR1 / CD111, Nectin-2 / PRR2 / CD112, Nectin-3 / PRR3 and Nectin-4 / PRR4. Their extracellular domains are composed of three immunoglobulin-like (Ig) V, C, C-type domains, and their amino acid sequences share between 30% and 55% identity.

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

[0005] Nectin-4 is a particularly interesting target. It is expressed during fetal development, but its expression in adult tissues is reduced and very limited compared to other members of the nectin family. Nectin-4 is a tumor-associated antigen in 83% of bladder cancers, 78% of breast cancers (mostly 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] The expression of nectin-4 in these pathologies is associated with a poor prognosis, which may be due to the ability of nectin-4 to confer tumor cells with enhanced migration, proliferation, and metastasis formation 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 Enfortumab vedotin for second-line treatment of advanced urothelial carcinoma completes the validation of nectin-4 as a target for cancer therapy. Summary of the invention

[0008] The present invention relates to a humanized antibody specific to nectin-4, an antigen binding fragment thereof and uses thereof.

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

[0010] Humanized variants derived from 5A12.2 that can be provided in high yields represent a novel approach to improve the therapeutic index of Nectin-4-positive cancer treatments 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 compared to non-humanized antibodies. This can be shown in small and large-scale production methods. This high-yield, reproducible production greatly facilitates the use of drugs.

[0012] A first aspect of the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof, which binds to a discontinuous epitope on nectin-4, which consists 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. Basically, each possible amino acid change in a given protein is first synthesized. The activity of each variant in these protein variants is detected in parallel using the barcode of each variant. The impact of each mutation is determined by comparing the activity with the wild-type protein. A detailed example of DMS is given below.

[0013] Preferably, the antibodies of the present invention have better affinity for the dimeric epitope on Nectin-4 than the prior art antibody HA22. HA22 is a human monoclonal anti-Nectin-4 antibody described in WO2012 / 047724, the contents of which are incorporated herein by reference. The "affinity" described herein describes a measurement of the overall or cumulative strength of a protein-protein complex, i.e., the total strength of all non-covalent interactions between the antibody and its ligand binding. It is determined by three parameters: the binding affinity of the complex, the valence of the protein, and the structural arrangement of the protein in the complex. The antibody of the present invention is not the antibody mAb5A12.2, which can be obtained by a hybridoma deposited in the CNCM under the deposit number CNCM I-5407, for example as described in WO2021 / 069508.

[0014] The antibodies of the present invention are monoclonal antibodies (mAbs) or monoclonal antibody fragments characterized by a specific amino acid sequence. If not indicated differently, the term "monoclonal" refers to a single species, i.e., a single amino acid composition of an 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 represents each light chain and heavy chain pair that is directly involved in binding the antibody to the antigen. The variable domain of the heavy chain is abbreviated as "VH", and the variable domain of the light chain is abbreviated as "VL".

[0016] The term "antigen binding site" refers to the region of an antibody molecule that actually binds to a ligand (eg, an antigen, ie, nectin-4 or an antigenic fragment thereof), and this region is derived from an antibody.

[0017] The antigen binding site of the antibody according to the present invention may include six complementary determining regions (CDRs), which contribute to the affinity of the binding site to the antigen to varying degrees. 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). Also included within the scope of the present invention are functional antigen binding sites containing fewer CDRs (i.e., wherein the binding specificity is determined by three, four or five CDRs). For example, less than a complete set of 6 CDRs may be sufficient for binding. In some cases, a VH or VL domain is sufficient.

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

[0019] The position of the CDR within the VH or VL region can be defined according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD (1991) or the IMGT numbering system, both of which are well known to those skilled in the art. The IMGT numbering has been defined to compare 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] Another aspect of the present invention relates to a humanized monoclonal antibody or antigen-binding fragment thereof that binds to nectin-4, comprising

[0021] (a) a heavy chain variable region (VH) comprising an amino acid sequence of any one of SEQ ID NOs: 2-5, or an amino acid sequence having at least 85%, at least 90%, at least 95%, or at least 99% identity thereto,

[0022] and comprising a complementarity determining region (CDR) of CDR-H1 of SEQ ID NO: 10 or 11, CDR-H2 of SEQ ID NO: 12 or 13, and CDR-H3 of SEQ ID NO: 14, and

[0023] (b) a light chain variable region (VL) comprising an amino acid sequence of any one of SEQ ID NOs: 6-9, or an amino acid sequence that is at least 85%, at least 90%, at least 95%, or at least 99% identical thereto,

[0024] And includes CDR-L1 of SEQ ID NO:15 or 16, CDR-L2 of SEQ ID NO:17 or 18, and CDR-L3 of SEQ ID NO:19.

[0025] The specific amino acid sequences represented by SEQ ID NO: used herein are provided in the attached sequence listing. SEQ ID NO: 10-19 define the six CDR sequences of the humanized antibodies of the present invention according to the Kabat numbering system. If not otherwise indicated, the Kabat system is used herein.

[0026] According to the present invention, 1 or 2 amino acid replacements are possible in these CDR sequences. In a specific embodiment, conservative amino acid replacements are preferred, i.e., one amino acid is replaced with another amino acid replacement with similar biochemical properties, for example, another aliphatic amino acid is replaced with an aliphatic amino acid such as Gly, Ala, Val, Leu or Ile, another basic amino acid is replaced with a basic amino acid such as His, Lys or Arg, another acidic amino acid or an amide thereof is replaced with an acidic amino acid or amide such as Asp, Glu, Asn or Gln, another aromatic amino acid is replaced with an aromatic amino acid such as Phe, Tyr or Trp, or another hydroxyl or sulphur-containing amino acid is replaced with a hydroxyl or sulphur-containing amino acid such as Ser, Thr, Met or Cys.

[0027] 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 includes fusion proteins, such as fusion proteins with non-immunoglobulin peptides or polypeptides, and conjugates with non-protein structures such as markers or toxins. The terms "antigen-binding fragment of an antibody," "antigen-binding fragment thereof," "antibody fragment," or "fragment thereof" are used interchangeably herein.

[0028] The antibody or antigen-binding fragment thereof of the present invention can be monovalent or multivalent, i.e., it can contain a single antigen binding site or multiple antigen binding sites. For example, a Fab fragment has a single antigen binding site, an IgG class antibody or a Fv or scFv fragment has two antigen binding sites, and an IgM class antibody has 5 antigen binding sites. The term "antibody" also includes heterospecific antibodies with different antigen binding sites, such as heterobispecific antibodies, particularly antibodies directed against two different epitopes on an antigen. As used herein, an "epitope" is a region in an antigen that binds to an antibody. The term "epitope" includes any polypeptide determinant that is capable of specifically binding to an antibody.

[0029] According to another preferred embodiment, the humanized antibody or antigen-binding fragment thereof comprises

[0030] (a) a VH region comprising the amino acid sequence of SEQ ID NO: 2-5, and

[0031] (b) A VL region comprising the amino acid sequence of SEQ ID NOs: 6-9.

[0032] More specifically, the antibody or antigen-binding fragment thereof may comprise

[0033] (i) a VH region comprising the amino acid sequence of 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

[0034] A VL region comprising the amino acid sequence of 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,

[0035] (ii) a VH region comprising the amino acid sequence of 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

[0036] A VL region comprising the amino acid sequence of 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,

[0037] (iii) a VH region comprising the amino acid sequence of 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

[0038] A VL region comprising the amino acid sequence of 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,

[0039] (iv) a VH region comprising the amino acid sequence of 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

[0040] A VL region comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 99% identical thereto.

[0041] "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 to the amino acid residues in that particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percentage of sequence identity. Alignments for determining percentage amino acid sequence identity can be achieved in a variety of ways within the scope of those skilled in the art, for example, using publicly available computer software such as BLAST.

[0042] 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.

[0043] According to the invention, a "chimeric" antibody is an antibody wherein a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0044] A "multispecific antibody" binds to two or more different epitopes. The epitopes can be on the same or different antigens. A preferred example of a multispecific antibody is a "bispecific antibody" that binds to two different epitopes.

[0045] The term "humanized antibody" or "humanized form of an antibody" refers to an antibody in which both the heavy and light chains have been humanized by antibody engineering. A humanized chain is typically a chain in which the amino acid sequence of the V region has been changed so that, when analyzed as a whole, it is closer in homology to the human germline sequence than to the germline sequence of the source species. For example, a mouse CDR can be transplanted into the framework region of a human antibody to prepare a "humanized antibody". See, for example, Riechmann, L. et al., Nature 332 (1988) 323-327; 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 region has been additionally modified or altered from that of the original antibody to produce the characteristics of the present invention. Humanization assessment is performed based on the resulting amino acid sequence rather than the method itself.

[0046] The antibody of the present invention can be any suitable class. The term "class" refers to the type of constant domain or constant region possessed by its heavy chain. As used herein, "constant domain" or "constant region" represents the sum of antibody domains except the variable region. The constant region does not directly participate in the binding of the antigen, but exhibits various effector functions. The antibody can be any of the five main antibody classes: 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 different classes of immunoglobulins are referred to as a, δ, ε, γ and μ, respectively. According to the present invention, IgG, IgA or IgM class antibodies or fragments thereof are particularly suitable.

[0047] According to a preferred embodiment, the antibody of the present invention is selected from the IgG class, such as IgG1, IgG2, IgG3 or IgG4; the IgM class; the IgA class; or an antigen-binding fragment thereof.

[0048] As used herein, the terms "binding" and "specific binding" refer to the binding of an antibody or fragment thereof of the invention to an epitope of the nectin-4 antigen. The measured value of the strength of antibody binding 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 using flow cytometry, immunohistochemistry and / or fluorescence is described and is particularly preferred herein.

[0049] The affinity of antibody binding to antigen is expressed in terms of Ka (rate constant for antibody binding in the antibody / antigen complex), KD (dissociation constant), and K dis (kD / ka) definition, i.e., the term "Ka" as used herein refers to the association rate of a particular antibody-antigen interaction, and the term "Kd" refers to the dissociation rate of a particular antibody-antigen interaction. These values ​​for antibodies can also be determined using methods well established in the art and the methods described herein.

[0050] The antibodies and antigen-binding fragments thereof according to the present invention preferably show a dissociation constant KD of at least 3.5, preferably at least 4.0, more preferably at least 4.2, most preferably at least 4.5 (nM).

[0051] Of course, it is preferred to specifically bind to human Nectin-4 expressed by tumors. Therefore, the antibodies provided herein preferably show specific binding to Nectin-4 and have no substantial cross-reactivity or no cross-reactivity to other proteins, particularly to proteins of the human Nectin family such as Nectin-1.

[0052] The antibody or its antigen-binding fragment can be produced in a suitable host cell, the host cell comprising a nucleic acid molecule encoding an antibody VH region or an antibody VL region, such as a DNA molecule, or encoding a complete antibody or antibody fragment, or a vector or vector system comprising the nucleic acid molecule, i.e., a plurality of vectors, preferably the nucleic acid molecule is operably linked to an expression control sequence, particularly a heterologous expression control sequence. Methods for providing antibodies of the present invention are described herein. The host cell can be any known host cell for producing antibodies or antibody fragments, such as a prokaryotic cell, such as an E. coli cell, a yeast cell, an insect cell, or a mammalian cell, such as a CHO cell or a hybridoma cell.

[0053] Surprisingly, the humanized antibodies of the present invention, particularly those characterized by a VH region comprising the amino acid sequence of SEQ ID NO: 2-5 and a VL region comprising the amino acid sequence of SEQ ID: 6-9, can be provided in high yields compared to the corresponding parent chimeric 5A12.2 antibody (see Tables 2 and 3).

[0054] According to another embodiment, the antibody or antigen binding fragment thereof of the present invention comprises a labeling group and / or an effector group coupled to the antibody or antigen binding fragment. The labeling group can be, for example, a dye, a paramagnetic group, a radioactive or fluorescent group detectable during imaging. Preferred effector groups are therapeutic groups, particularly cytotoxic agents, such as chemotherapeutic agents, drugs, anti-inflammatory agents, radioisotopes, toxins such as topoisomerase toxins, enzymes and fragments thereof, such as nuclear lytic enzymes, growth inhibitors, antibiotics and all suitable anticancer and antitumor agents known to those skilled in the art. Particularly preferred are topoisomerase toxin camptothecin and its derivatives and / or structural analogs, such as exatecan, and its derivatives, such as Deruxtecan.

[0055] Another aspect of the invention relates to an antibody-drug conjugate (ADC) comprising a humanized antibody or antigen-binding fragment thereof as described herein, 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 hydroxyl group or a sulfhydryl group, or a drug conjugated to a reactive group in the glycan structure of the antibody. Preferably, the drug is conjugated to a reactive sulfhydryl group in the side chain of a cysteine ​​residue on the antibody or antigen-binding fragment thereof.

[0056] The drug of the antibody conjugate of the present invention is preferably a topoisomerase I inhibitor, an auristatin or a maytansinoid.

[0057] A topoisomerase I inhibitor is a compound that is able to form a ternary complex with topoisomerase I and DNA, thereby preventing DNA from reconnecting and introducing DNA strand breaks in the cell genome. Topoisomerase I inhibitors can be selected, for example, from camptothecin or its analogs, indenoisoquinoline and indolecarbazole. In a specific embodiment, the topoisomerase I inhibitor is camptothecin or its analogs, i.e., a compound comprising a five-ring basic structure of camptothecin and optionally a modified substituent group that causes the presence of additional rings. Specific examples are camptothecin, topotecan, irinotecan, SN-38, belotecan, exitecan, including their derivatives, such as deruxtecan, lurtotecan or atiratecan. In a particularly preferred embodiment, the topoisomerase I inhibitor is exitecan. Exitecan is usually covalently conjugated to an antibody via its NH2 group.

[0058] A particularly preferred auristatin is monomethyl auristatin E (MMAE), a synthetic anti-tumor agent. MMAE inhibits cell division by blocking the polymerization of tubulin.

[0059] Derivatives of maytansine are called maytansines. Maytansines inhibit the assembly of microtubules by binding to tubulin at the rhizoxin binding site. Examples of maytansines are ansamycin, maytansine / entansine (DM1) and lavatansine / sorafutansine (DM4).

[0060] In principle, the drug, e.g., exatecan, can be conjugated to any suitable position of the monoclonal antibody or its antigen-binding fragment, in particular to any position that does not abolish the binding of the antibody to nectin-4. For example, a topoisomerase I inhibitor, e.g., exatecan, can be conjugated to a reactive amino acid residue on the antibody, e.g., an amino acid residue having a side chain containing an amino, hydroxyl, or sulfhydryl group, or to a reactive group in the glycan structure of the antibody. In a specific embodiment, the drug, e.g., exatecan, is conjugated to a reactive sulfhydryl group in the side chain of an accessible cysteine ​​residue on the antibody.

[0061] In certain embodiments, the antibody-drug conjugate comprises a drug-antibody / antibody fragment molar ratio (DAR) greater than 1, i.e., more than one drug molecule is attached to one antibody / antibody fragment. Typically, the conjugate has a DAR of about 2: 1 to about 16: 1, particularly about 4: 1 to about 10: 1, and more particularly about 6: 1 to about 8: 1. The DAR can be calculated from a statistical distribution according to known methods.

[0062] 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 is cleavable under physiological conditions, such as 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.

[0063] In certain embodiments, the linker is a hydrophilic polysarcosine linker, such as described in Conilh et al., "Exatecan antibody drug conjugates based on a hydrophilic polysarcosine drug-linker platform". (Pharmaceuticals 14 (2021), 247). Further preferred linkers include linkers comprising at least one ethylene glycol unit, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more ethylene glycol units, such as the linker of the antibody-drug conjugate MEDI7247, Further preferred linkers include oligopeptides, in particular dipeptides to decapeptides, for example tetrapeptide sequences, such as Further preferred linkers include highly polar spacers such as acyl, carbamoyl and / or sulfonamide groups added to at least one ethylene glycol unit, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more ethylene glycol units, such as the linker technology known as Hydraspace™.

[0064] The antibody-drug conjugates of the present invention can be prepared by known methods.

[0065] The antibody or antigen-binding fragment thereof can be reacted with a linker-drug conjugate to obtain an antibody-drug conjugate. The linker-drug conjugate comprises a drug molecule, for example, to which is attached a suitable linker, wherein the linker comprises a reactive group capable of reacting with a desired attachment site on the antibody or antigen-binding fragment thereof. For attachment to a cysteine ​​residue, the linker-drug conjugate comprises a sulfhydryl-reactive group, for example, a maleimide group.

[0066] A preferred embodiment of the anti-tumor agent relates to an anti-tumor immune stimulator, including but not limited to a toll-like receptor (TLR) agonist or a stimulator of interferon genes (STING) pathway.

[0067] 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.

[0068] According to another aspect of the invention, the antibody or antigen-binding fragment thereof is for use in medicine, in particular for therapeutic or diagnostic applications, including in vitro and in vivo diagnostic applications.

[0069] The antibody or antigen-binding fragment thereof can be used in a method for preventing and / or treating cancer and / or inflammatory diseases, wherein the cancer and / or inflammatory diseases are preferably associated with overexpression of nectin-4.

[0070] The cancer to be prevented and / or treated can be any type of cancer, wherein the term "cancer" is used herein to refer to proliferative diseases. The cancer to be prevented and / or treated according to the present invention is preferably selected from urothelial carcinoma, endometrial carcinoma, 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.

[0071] Since the antibodies and fragments thereof of the present invention are specific to nectin-4 expressed on tumors, they can also be used for diagnosis, for example, by coupling with the above-mentioned labeling groups.

[0072] According to another aspect, the antibodies or antigen binding fragments thereof described herein can be used in a method for preventing and / or treating an inflammatory disease, wherein the inflammatory disease is preferably associated with nectin-4 expression. Such treatment can preferably be combined with an anti-inflammatory agent known to those skilled in the art. Preferred anti-inflammatory agents are as described above.

[0073] Another aspect of the invention is a combination of at least 2 different monoclonal antibodies or fragments as described herein.

[0074] In addition, the present invention relates to a nucleic acid molecule, such as a DNA molecule, encoding an antibody VH region or an antibody VL region, or encoding a complete antibody or an antibody fragment as described above; a vector or a vector system, i.e., a plurality of vectors, comprising a nucleic acid molecule as described above, preferably operably linked to an expression control sequence, in particular to a heterologous expression control sequence.

[0075] In addition, the present invention relates to cells comprising nucleic acid molecules or vectors or vector systems as described above. The term "vector" as used herein refers to a nucleic acid molecule capable of proliferating another nucleic acid connected thereto. According to a preferred embodiment, the vector is an expression vector. An "expression vector" is a vector capable of directing the expression of a nucleic acid operatively connected thereto. Vectors, particularly expression vectors, used for recombinant production of antibodies are well known in the art.

[0076] The cell may be a 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 such as a CHO cell or a hybridoma cell.

[0077] Another aspect of the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described herein. Typically, the antibody or antibody fragment is administered as a pharmaceutical composition 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 buffered aqueous solutions, and are well known in the art.

[0078] Depending on the stage and severity of the disease to be treated, the pharmaceutical composition can be administered once or several times during the course of the disease. For example, it can be administered daily, every two days, twice a week, or weekly for a suitable period of time.

[0079] In certain embodiments, the pharmaceutical composition is administered parenterally, for example, by subcutaneous, intramuscular or intravenous injection or infusion. In further embodiments, the pharmaceutical composition may be administered topically, for example, orally, nasally or pulmonary, for example, as an aerosol.

[0080] The present invention is further illustrated by the following figures and examples, but they are not intended to limit the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 : Compatibility evaluation of antibodies 5A12.2_H1L0, HA22 (Enfortumab) and 15A7.5_H1L2

[0082] Yeast expressing nectin-4 fragment 32-349 was incubated with 100 nM unlabeled antibody 1 or PBS (as a positive control). After incubation for 30 minutes, 500 pM antibody 2 was added and incubated for 30 minutes. The yeast was then incubated with a fluorescent reporter molecule to detect nectin-4 expression and antibody 2 by FACS. The results are expressed as a percentage of fluorescence relative to the corresponding positive control.

[0083] Figure 2 : Sorting of libraries by flow cytometry

[0084] Two libraries covering the target Nectin-4 sequence were generated (left and right panels). Yeast expressing single mutants of Nectin-4 were incubated with 100pM biotinylated 5A12.2_H1L0 and 100pM Dy650-labeled 15A7.5_H1L2 (upper panel); or with 100pM biotinylated HA22 and 100pM Dy650-labeled 15A7.5_H1L2 (lower panel). Streptavidin PE was used to reveal the biotinylated antibody. After PBS washing, cells were analyzed and sorted on a BD FACSAria III as shown. Yeast stained with antibodies expressed single mutants of Nectin-4 that destroyed the epitopes bound by other antibodies.

[0085] Figure 3 :Positions that have a significant effect on 5A12.2_H1L0 antibody when mutated

[0086] For any amino acid in the domain of interest, the higher the numerical value for prohibited mutations, the more important the residue is for 5A12.2_H1L0 antibody binding.

[0087] Figure 4 :Positions that have a significant effect on HA22 (Enfatamab) when mutated

[0088] For any amino acid in the domain of interest, the higher the value for prohibited mutations, the more important the residue is for HA22 antibody binding.

[0089] Figure 5 :Structural reference of Nectin-4 (4FRW, pdb)

[0090] Shown is the pymol file. For each antibody, the important amino acids in the epitope are indicated. The most important residues are underlined.

[0091] Figure 6 :In vitro cytotoxic activity against SUM190PT and T47D tumor cells

[0092] HA22 (Enfortumab, circles), Ch-5A12.2 (triangles), and isotype control (open squares) monoclonal antibodies were conjugated to α-amanitin to generate antibody-drug conjugates, and the cytotoxic activity of a dose range (left: 1 pg / mL–15 μg / mL, right: 192 pg / mL–15 μg / mL) was evaluated on nectin-4 expressing SUM190PT (left) and T47D (right) cell lines. Survival after a 5-day incubation period is reported (AlamarBlue assay). EC50 values ​​were determined using nonlinear curve fitting (4 parameters) by GraphPad Prism 9 software.

[0093] Figure 7 : Apparent affinity of humanized variants for tumor cells detected by flow cytometry

[0094] T47D human tumor cells expressing nectin-4 were counted and incubated with a dose range (169 pg / mL–30 μg / mL) of Ch-5A12.2 or the indicated humanized variants. The numbers associated with H and L refer to the number of back 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 and nonlinear curve fitting (4 parameters) were used.

[0095] Figure 8 :In vitro cytotoxic activity of HCT-116 transfectants expressing nectin-4

[0096] Hu-5A12.2_H1L0 (triangle), Hu-5A12.2_H1L1 (open diamond), Hu-15A7.5_H1L2 (circle) and isotype control (ICT, open square) monoclonal antibodies were conjugated to exatecan to generate antibody-drug conjugates, and the cytotoxic activity of the dose range (left: 1pM–18.94nM) was evaluated on the HCT-116 cell line expressing nectin-4. Survival after a 7-day incubation period is reported (MTT assay). EC50 values ​​were determined by GraphPad Prism 9 software using nonlinear curve fitting (4 parameters).

[0097] Fig. 9 Treatment of SUM190PT-transplanted NSG mice with Hu-15A7.5_H1L2-isotecan or Hu-5A12.2_H1L0-isotecan ADC induced tumor regression

[0098] NSG mice (n=5 / group) were orthotopically xenografted bilaterally with SUM190PT cells embedded in Matrigel. Three different ADCs were tested: isotype control ICT exotecan (black circles), Hu-5A12.2_H1L0-exotecan (open diamonds), and Hu-15A7.5_H1L2-exotecan (black diamonds). When tumors reached approximately 70 mm 3 Treatment of mice was started at 1:10 pm (single intravenous injection of 10 mg / kg). Thereafter, tumor size was monitored twice a week with calipers (n=10 / group) and reported using the following formula (LxlxhxPi / 6).

[0099] Fig.10 : Amino acid sequence of parental 5A12.2 monoclonal antibody

[0100] Shown are the heavy and light chain amino acid sequences of the parental 5A12.2 monoclonal anti-nectin-4 antibody. CDRs were identified according to IMGT and Kabat nomenclature.

[0101] (IMGT nomenclature, Kabat nomenclature )

[0102] Fig.11 :Amino acid sequence of humanized variant of 5A12.2 monoclonal antibody

[0103] Shown are the heavy and light chain amino acid sequences of the humanized variants of the 5A12.2 monoclonal anti-nectin-4 antibody. The CDRs were identified according to IMGT and Kabat nomenclature. Back mutations were also identified.

[0104] (IMGT nomenclature / Kabat nomenclature / Anchored IMGT and / or Kabat / 回复突变 )

[0105] Fig.12 : Sequence summary of the humanized antibodies of the present invention DETAILED DESCRIPTION

[0106] Example

[0107] Principle of epitope mapping using DMS (deep mutation scanning)

[0108] DMS is a mutagenesis method that aims to perform all possible single substitutions for all selected residues in a given protein sequence. The DMS library is obtained in the form of DNA encoding the protein of interest. In this library, each DNA strand contains a single codon that is mutated relative to the parental sequence.

[0109] The DMSDNA library was integrated into an expression plasmid specifically designed to express recombinant proteins on the surface of yeast. The yeast was then transformed and induced to express single mutant proteins on the surface of yeast. This new library (called a display library) was screened by flow cytometry using fluorescent reporters to reveal the expression of the protein (anti-tag fluorescent antibody) and the binding of the protein to its partner (fluorescent partner).

[0110] For epitope mapping, it is ideal to have two antibodies with compatible epitopes that can be bound together on the same antigen. In this way, each of the two antibodies acts as a conformational control of the mutant antigen of the other antibody. In fact, a single substitution performed on an antigen can have 4 types of effects: (i) loss of affinity to the first antibody while retaining the binding to the second antibody: this is a mutation occurring in the epitope of the first antibody; (ii) loss of affinity to the second antibody while retaining the binding to the first antibody: this is a mutation occurring in the epitope of the second antibody; (iii) loss of affinity to both antibodies: this is a so-called "destructive" mutation that affects the conformation of the antigen and thereby prevents the binding of the two antibodies; (iv) no effect, the mutation is not present in the epitope of one of the two antibodies, and will not result in significant changes in the conformation of the antigen. After flow cytometry analysis, yeast populations that lose affinity to the target antibody and retain the binding to the second antibody are sorted. The plasmid contained in the yeast population is extracted and sequenced by high-throughput sequencing. Analysis of sequencing data allows identification of mutations that affect the binding of antibodies to their targets. This analysis therefore allows identification of important locations on the antigen to which the antibody of interest binds: ie, its epitope.

[0111] Construction of antigen expression plasmid

[0112] The gene corresponding to the antigen sequence Nectin-4 (32-349) was synthesized and cloned into a plasmid for expression on the surface of galactose-induced yeast. In this construct, the expressed antigen carries a C-terminal HA tag. The expression plasmid was then transformed into the yeast strain Saccharomyces cerevisiae EBY100. Two DMS libraries were generated by PCR, library 1 involving amino acid positions 32 to 91 and library 2 involving amino acid positions 92 to 151. These libraries correspond to the IgV domain of Nectin-4, which is known to be the target of the tested antibodies. Each mutation position carries a degenerate NNS or NNK codon, encoding 20 amino acids / 32 codons. Each library contains approximately 1200 single amino acid mutants and 2000 DNA codon mutants. The two libraries were converted into YSD. Unsorted yeast from the two generated libraries were sequenced to verify the efficacy of mutagenesis. 100% of the expected single mutants in library 1 were sequenced, and 99.9% of the expected single mutants in library 2 were sequenced.

[0113] Epitope compatibility

[0114] To check whether the tested antibodies were compatible with the epitope map on the nectin-4 IgV domain, i.e., whether the antibodies could bind to the nectin-4 IgV domain together, the antibodies were first biotinylated (EZ-Link TMSulfo-NHS-LC-Biotin). Induce yeast to express Nectin-4 32-349 construct, wash with PBS, BSA 0.1% for 10 5 Yeast were then incubated twice with 100 nM unlabeled antibody 1 for 30 min or left as is as a positive control. After 30 min, 500 pM antibody 2 was added and incubated for another 30 min. The yeast were then incubated on ice for 15 min with the corresponding fluorescent reporter, anti-HA APC for nectin-4 expression detection, and streptavidin PE for antibody 2 detection. After washing in PBS-BSA, the resuspended yeast were analyzed by flow cytometry. Figure 1 It was shown that 5A12.2_H1L0 and HA22 could not bind to the Nectin-4 IgV domain simultaneously. In contrast, 15A7.5_H1L2 mAb could bind to the Nectin-4 IgV domain simultaneously with 5A12.2_H1L0 or HA22 (Enfatamab).

[0115] Antigen expression induction and FACS sorting of libraries

[0116] Induce yeast expressing single mutants of nectin4 for protein expression. Induce transformed yeast 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 cell analysis / sorting, wash 10 μl of PBSF (PBS, 0.1% BSA) with 1 mL of PBSF (PBS, 0.1% BSA). 6 Up to 10 8 The cells were then resuspended in an appropriate volume of solution containing 100 pM biotinylated HA22 antibody and Dye650-labeled 15A7.5_H1L2. After incubation at 20°C with stirring for 1 to 3 hours, 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 using BD FACSdiva TM Diva software in BDFACSAria TM Analyze / sort cells on a III cytometer. Figure 2As shown, single-stained yeast in 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 amplified the target region, and the second step added the Illumina adapters required for sequencing. Sequencing was performed on an Illumina iSeq100 instrument (2x150bp, 300 cycles), with at least 150,000 reads per population. The data was then processed through an analysis pipeline using a dedicated proprietary script. Sequences of poor quality (Q<30) were removed, and single mutants were then detected and counted. For each single mutation, the frequency measured in the unsorted population was compared with the mutant frequency in the sorted population. The enrichment value of the mutant was then calculated according to the following formula:

[0117]

[0118] A high enrichment value indicates that the mutation in question causes loss of recognition of that antibody for its antigen while maintaining recognition of other antibodies for their antigen (conformational control).

[0119] Data analysis

[0120] The results of NGS sequencing data processing are presented in a bar graph that summarizes the enrichment values ​​of all single mutants present in the DMS library ( Figure 3 , Figure 4 ). 16 to 19 prohibited mutations indicate high impact, and positions classified in this category are most likely to be indirect interactions with the IgG in question. 10 to 15 prohibited mutations represent medium impact positions, and 5 to 9 prohibited mutations represent low impact positions. Figure 5 The structural analysis of Nectin-4 (4FRW, pdb) is shown to highlight the important and most significant epitope residues for each antibody tested. Table 1 lists these residues.

[0121]

[0122] Table 1: Important and most critical residues of the nectin-4 epitope of monoclonal antibodies HA22 and 5A12.2_H1L0

[0123] Humanization of chimeric antibodies

[0124] The method used is "CDR transplantation": 3 mouse CDRs (regions that determine antibody specificity) of each antibody chain (heavy chain, VH or light chain, VL) are transplanted onto the closest human germline framework. This method also takes into account the back mutation of essential amino acids in the framework region (FR) (CDR anchors, Vernier residues, interface VH / VL, etc.).

[0125] Chimeric and humanized antibody generation, production, purification and control

[0126] The light chain expression vector encodes the Vκ chain. Depending on the payload used, 2 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 anti-nectin-4 Enfortumab (HA22) was also cloned in the same vector.

[0127] The light chain vector and the heavy chain vector were transfected into the inoculated HEK293 at a ratio of 1.2 / 1. After 6 days of production, the culture supernatant was clarified and the monoclonal antibody was purified using MabSelect PrismA resin (GE Healthcare) according to the manufacturer's instructions. 0.5M glycine, 3M NaCl, pH 8.9 were used as binding buffer, and 0.1M citrate pH 3 was used for elution. Immediate neutralization was performed with 10% (V / V) 1M Tris-HCl pH 9. The monoclonal antibody was then dialyzed with PBS 1X pH 7.4 (mini dialysis device, 2mL-10k, Thermo Scientific), and then filtered on a 0.22 μM filter (Milelex GV hydrophilic PVDF, Millipore). Concentration was measured using a Nanodrop 2000 spectrophotometer (Thermo Scientific) in consideration of the specific extinction coefficient (E1% 280nm) of each monoclonal antibody. Purity was determined by UPLC-SEC using Acquity UPLC-HClass Bio (Waters), using a protein BEH200A column balanced in 0.2M NaPO4, 0.3M NaCl pH 6.9 supplemented with 10% isopropanol. The quality of the antibody was determined in a Xevo G2-S Q-Tof mass spectrometer (Waters) using a reverse phase column (PLRP-S 4000A, Agilent technologies). According to the manufacturer's instructions, all samples were analyzed after deglycosylation with PNGase F glycosidase (New England Biolabs) at 37°C. The 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).

[0128] Table 2 reports small-scale (30 mL) production yields of parental chimeric 5A12.2 and all humanized variants.

[0129] Table 3 reports the mid-scale (0.5-1.4 L) production yields of the parental chimeric 5A12.2 and the humanized variants 5A12.2_H1L0 and 5A12.2_H1L1.

[0130]

[0131]

[0132] Table 2: Small scale production yields of humanized variants vs. small scale production yields of parent chimeric 5A12.2 antibody. CHt: chimeric antibody, HUt: humanized antibody

[0133]

[0134] Table 3: Mid-scale production yields of humanized variants vs. mid-scale production yields of parent chimeric 5A12.2 antibody. CHt: chimeric antibody, HUt: humanized antibody

[0135] Antibody conjugation

[0136] The cysteine ​​reactive linker-amanitin compound from Heidelberg Pharma has a cleavable linker (valine alanine), and the cysteine ​​residues of the selected anti-Nectin-4 ThiomAb (D265C, L234A, L235A) antibody are engineered using maleimide chemistry. In brief, the ThiomAb antibody in PBS1X pH 7.4 is reduced with TCEP, and the interchain disulfide is reoxidized by dehydroascorbic acid. Subsequently, the engineered cysteine ​​is used for conjugation with the cysteine ​​reactive linker-amanitin compound. The conjugate is purified by dialysis. According to LC-MS analysis, the drug-antibody ratio (DAR) is contained between 1.44 and 1.79 toxins per conjugated mAb. As determined by SEC-HPLC, less than 2% of the material aggregates.

[0137] Cysteine-reactive linker-Ixitem compound Maleimide-Propio-PEG2-PSAR10-glucuronic acid-Ixitem (MabLink) was conjugated to the cysteine ​​residues of selected anti-Nectin-4 mAbs (P331S, L234F and L235E). In brief, the monoclonal antibody in PBS1X, 1mM EDTA was reduced with 14 molar equivalents of TCEP at 37°C for 2 hours, and then the buffer was exchanged (Amicon ultra 30kDa) to 100mM KPO4, 1mM EDTA pH 7.4. Cysteine-reactive linker-Ixitem compound was conjugated to reactive cysteine ​​for 35 minutes at room temperature using 12 molar equivalents. The final exchange buffer was performed in 20mM His pH 6.0 before filtering with a 0.22μM filter. The drug-antibody ratio (DAR) was comprised between 7.77 and 7.82 toxin per conjugated mAb according to LC-MS analysis. Less than 8% of the material was aggregated as determined by SEC-HPLC.

[0138] Cell lines

[0139] 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 supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, 100 μg / mL streptomycin. The human colorectal cancer Nectin-4 transfectant (HCT116-N4) was cultured in RPMI supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1X MEM non-essential amino acids, and 20 μg / mL hygromycin B.

[0140] In vitro cytotoxicity assay

[0141] To analyze the in vitro cytotoxic activity of α-amanitin ADC and ixitecan ADC, cell viability was assessed using the AlamarBlue staining protocol recommended by the manufacturer (Biosource, CA, USA). The test uses a fluorescent redox indicator. The fluorescence intensity is proportional to the reduction in cellular metabolism. The experiment was performed by incubating 3000 cells / well (SUM190PT, T47D or HCT116-Nectin-4) in 96-well plates in triplicate and serially diluting the ADC on day 0. On day 5, AlamarBlue was measured by incubating 1 / 10 volume of AlamarBlue solution at 37°C for 2 hours and readings were taken at 595nm (FLUOstar Optima, BMG Labtech). Figure 6 The EC of the parental 5A12.2 anti-nectin-4 monoclonal antibody was shown to be 50 Lower than HA22. Figure 8 It was shown that the humanized variants H1L0 and H1L1 of monoclonal antibody 5A12.2 showed similar EC50 compared to another humanized anti-nectin-4 antibody.

[0142] Flow cytometry

[0143] T47D cells (50,000) naturally expressing nectin-4 were incubated with the indicated antibodies at a dose range. After washing, cells were stained with phycoerythrin-conjugated goat anti-human antibody (5 μg / mL) Jackson Immuno Research). After fixation, cells were stained with cell viability dye (e780, Invitrogen) before flow cytometry acquisition. Figure 7 It was shown that all 5A12.2 humanized variants, except the H0 variant, maintained similar apparent affinities compared to the parental 5A12.2 monoclonal antibody.

[0144] Mouse experiments

[0145] NOD / SCID (non-obese diabetic / severe combined immunodeficiency) / gc null mice (NSG) were purchased from Charles River Laboratory (Margate, UK). Female mice (n=5 / group) aged 6 to 7 weeks were inoculated with SUM190PT (0.5×10 6 Bilateral orthotopic xenografts were performed with 1000 cells per group. ADC treatment was performed as described in each experiment. Thereafter, tumor size was monitored twice a week with calipers (n=10 / group) and the size was reported using the following formula (LxlxhxPi / 6). Fig. 9showed that in vivo, a single injection of the humanized variant 5A12.2_H1L0 monoclonal antibody coupled with exotecan could induce regression of the triple-negative breast cancer cell line SUM190PT.

[0146] Determination of antibody affinity on the Octet platform

[0147] The affinity of various monoclonal antibodies was determined on the Octet Red96 platform, a system based on the bio-layer interferometry (BLI) technology. Two types of analytes were used in these studies: (i) his-tagged recombinant extracellular domain of the Nectin-4 protein (R&D systems / Biotechne); (ii) homodimeric recombinant Fc protein corresponding to the human Nectin-4 IgV domain fused to the human Fc domain. The AHC sensor (anti-human Fc) was used for the recombinant his-tagged Nectin-4 EC analytes and the FAB2G sensor was used for the Fc-fused recombinant Nectin-4 IgV domain. The ligand of interest diluted in 1X kinetic buffer was loaded onto the selected sensor. The analytes were kept in solution at working concentrations ranging from 600nM to 1.56nM in 1x kinetic buffer. Each run was performed at +26°C with shaking at 1000rpm and consisted of 5 steps; loading step, equilibration step, association step, dissociation step and regeneration. To analyze 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 run through Octet software. Table 2 shows that the His-tagged recombinant nectin-4 had similar affinities for the 5A12.2 and HA22 monoclonal antibodies. In contrast, the affinities of 5A12.2 and HA22 for the Fc-fused IgV nectin-4 domain were significantly different, indicating that 5A12.2 has better affinity for the dimeric epitope on nectin-4 than HA22.

[0148]

[0149] Table 4: K of 5A12.2 and HA22 monoclonal antibodies D

[0150] Table 5 reports the affinity determinations of various humanized variants of monoclonal antibody 5A12.2. Only recombinant his-tagged Nectin-4 EC assay was used here. The affinities of the various humanized variants were similar to that of the parent antibody.

[0151]

[0152] Table 5: Affinity determination of various humanized variants of 5A12.2 compared to the parent antibody

Claims

1. A humanized 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 mutation scanning (DMS).

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

19.

3. The humanized antibody according to claim 2, comprising (i) a VH region comprising the amino acid sequence of 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 of 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 of 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 of 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 of 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 of 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 of 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 A VL region comprising the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence at least 85%, at least 90%, at least 95%, or at least 99% identical thereto.

4. The humanized antibody according to claim 2 or 3, comprising (a) a VH region comprising the amino acid sequence of SEQ ID NO: 2-5, and (b) A VL region comprising the amino acid sequence of SEQ ID NOs: 6-9.

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

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

8. The humanized antibody or antigen-binding fragment according to claim 7, wherein the labeling group is a dye, a paramagnetic group, a radioactive group or a fluorescent group detectable during imaging.

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

10. An antibody-drug conjugate comprising The 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 hydroxyl group, or a thiol group, or to a reactive group in the glycan structure of the antibody.

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 according to claim 10 or 11, wherein the drug is conjugated to the antibody or antigen-binding fragment thereof via a linker, particularly wherein the linker is a cleavable linker, such as a linker cleaved by glucuronidase and / or wherein the linker is a peptide linker.

13. The humanized antibody or fragment of 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. The humanized antibody or fragment for use according to claim 13, for use in a method for preventing and / or treating cancer and / or inflammatory diseases.

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

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 antibodies according to any one of claims 1 to 9, or encoding at least one VL and / or one VH of any one of claims 1 to 9.

18. A vector comprising the nucleic acid according to 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 carrier according to claim 18, and optionally one or more pharmaceutical excipients.

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