Novel anti-nectin-4 antibodies and antibody-drug conjugates

Abstract

The present invention relates to novel anti-Nectin-4 antibodies and antibody-drug conjugates comprising these antibodies, as well as uses thereof.

Description

[Technical Field]

[0001] The present invention relates to novel anti-Nectin-4 antibodies and antibody-drug conjugates comprising these antibodies, as well as 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] These proteins belong to the immunoglobulin superfamily and are homologs of the poliovirus receptor (PVR / CD155), hence also called poliovirus receptor-binding 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)-type V, C, and C domains that share 30–55% amino acid sequence 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 -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 the expression 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] Expression of Nectin-4 in these pathologies is associated with poor prognosis, likely as a result of Nectin-4's ability to confer increased migration, proliferation, and metastasis-forming potential to tumor cells in vitro. 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

[0007] In the present invention, it has been found that antibodies capable of binding to specific epitopes on Nectin-4 exhibit improved anti-cancer activity compared to known anti-Nectin-4 antibodies.

[0008] A first aspect of the present invention is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to a discontinuous epitope on Nectin-4 consisting of one or more of amino acids D57, S58, E60, P133, G135, F137, Q138, and R140, and optionally G56, G59, E126, R128, P133, and A134 of SEQ ID NO: 104, as determined using Deep Mutational Scanning (DMS); provided that the antibody or antibody fragment does not comprise a variable heavy chain (VH) region comprising complementarity determining regions (CDRs) CDR-H1 set forth in SEQ ID NO: 7, 21, 35, 49 or 63, CDR-H2 set forth in SEQ ID NO: 8, 22, 36, 50 or 64, and CDR-H3 set forth in SEQ ID NO: 9, 23, 37, 51 or 65, and a variable light chain (VL) region comprising complementarity determining regions (CDRs) CDR-L1 set forth in SEQ ID NO: 10, 24, 38, 52 or 66, CDR-L2 set forth in SEQ ID NO: 11, 25, 39, 53 or 67, and CDR-L3 set forth in SEQ ID NO: 12, 26, 40, 54 or 68; In particular, the antibody or antibody fragment does not comprise the VH region set forth in SEQ ID NO: 13 and the VL region set forth in SEQ ID NO: 14, In particular, the antibody is not antibody 15A7.5 disclosed in International Application No. PCT / EP2022 / 0586626.

[0009] A further aspect of the invention is an antibody-drug conjugate, a monoclonal antibody or antigen-binding fragment thereof as defined herein; and a drug conjugated to a reactive amino acid residue on the antibody or antigen-binding fragment, for example, an amino acid residue having a side chain containing an amino, hydroxy, or thiol group, or to a reactive group in the antibody glycan structure.

[0010] Furthermore, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof, a vector comprising a nucleic acid encoding said antibody or antigen-binding fragment thereof, or an antibody-drug-conjugate as defined above.

[0011] A further aspect of the invention is the use of an antibody or antibody-drug conjugate or a pharmaceutical composition as described above in medicine, particularly in human medicine.

[0012] A further aspect of the present invention is the use of the antibody-drug conjugate or pharmaceutical composition described above in a method for the prevention and / or treatment of Nectin-4-associated disorders, in particular for the prevention and / or treatment of Nectin-4-positive cancers and / or Nectin-4-associated inflammatory disorders.

[0013] A further aspect of the invention is the use of an antibody-drug conjugate or a pharmaceutical composition as described above in a method for the prevention and / or treatment of cancer, in particular for the prevention and / or treatment of a cancer selected from bladder, urothelial, endometrial, cervical, colorectal, liver, thyroid, breast, pancreatic, lung, ovarian, head and neck and / or esophageal cancer.

[0014] A further aspect of the present invention is a method for preventing and / or treating a Nectin-4-associated disorder, particularly a Nectin-4-positive cancer, comprising administering a therapeutically effective amount of the above-mentioned antibody or antibody-drug conjugate or pharmaceutical composition to a subject in need thereof.

[0015] A further aspect of the invention is a method for the prevention and / or treatment of cancer, particularly cancer selected from bladder, urothelial, endometrial, cervical, colorectal, liver, thyroid, breast, pancreatic, lung, ovarian, head and neck, and / or esophageal cancer, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody, or antibody-drug conjugate, or pharmaceutical composition as described above. DETAILED DESCRIPTION OF THE INVENTION

[0016] The inventors have discovered that anti-Nectin-4 antibodies that bind to discontinuous epitopes on Nectin-4 consisting of one or more of amino acids D57, S58, E60, P133, G135, F137, Q138, and R140 of SEQ ID NO: 104, and optionally G56, G59, E126, R128, P133, and A134, exhibit improved anti-cancer activity compared to known anti-Nectin-4 antibodies. These antibodies specifically bind to Nectin-4 expressed by tumors with higher affinity than Nectin-4 expressed by human differentiated keratinocytes. In vitro, this selectivity provides the novel antibodies with lower binding affinity, lower internalization, and lower cytotoxic activity against keratinocytes compared to tumor cells and with respect to the activity of HA22 mAb (enfortumab) in the same assays.

[0017] In vivo, this lower binding capacity to keratinocytes provides the novel antibodies with a longer half-life due to a lower absorption rate in the skin.

[0018] Therefore, the novel antibody and especially its derived humanized variants represent a new way to improve the therapeutic index of Nectin-4-positive cancer treatment through lower associated skin toxicity and higher antitumor selectivity and efficacy.

[0019] Accordingly, a first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to a discontinuous epitope on Nectin-4 consisting of one or more of amino acids D57, S58, E60, P133, G135, F137, Q138, and R140, and optionally G56, G59, E126, R128, P133, and A134 of SEQ ID NO: 104, as determined using Deep Mutational Scanning (DMS), provided that the antibody or antibody fragment does not comprise a variable heavy chain (VH) region comprising complementarity determining regions (CDRs) CDR-H1 set forth in SEQ ID NO: 7, 21, 35, 49 or 63, CDR-H2 set forth in SEQ ID NO: 8, 22, 36, 50 or 64, and CDR-H3 set forth in SEQ ID NO: 9, 23, 37, 51 or 65, and a variable light chain (VL) region comprising complementarity determining regions (CDRs) CDR-L1 set forth in SEQ ID NO: 10, 24, 38, 52 or 66, CDR-L2 set forth in SEQ ID NO: 11, 25, 39, 53 or 67, and CDR-L3 set forth in SEQ ID NO: 12, 26, 40, 54 or 68; In particular, the antibody or antibody fragment does not comprise the VH region set forth in SEQ ID NO: 13 and the VL region set forth in SEQ ID NO: 14, In particular, the antibody is not antibody 15A7.5 disclosed in International Application No. PCT / EP2022 / 058626.

[0020] Determination of binding epitopes The binding epitope on Nectin-4 was determined using deep mutational scanning (DMS). DMS is a mutagenesis method that aims to make all possible single substitutions for all selected residues in a protein sequence. A DMS library is obtained in the form of DNA encoding the protein under study. In this library, each DNA strand contains a mutated codon relative to the parent sequence.

[0021] In a specific embodiment, this DMS DNA library is incorporated into an expression plasmid specifically designed to express recombinant proteins on the yeast surface.The yeast is then transformed and induced to allow the expression of single mutant proteins on its 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).

[0022] For epitope mapping, the ideal case would be to have two antibodies with compatible epitopes that can bind together on the same antigen. In this way, each of the two antibodies serves as a conformational control for the mutated antigen of the other antibody. In fact, a single substitution made to an antigen can have four types of effects: 1. Loss of affinity for the first antibody while retaining binding to the second antibody: Mutations occurring within the epitope of the first antibody. 2. Loss of affinity for the second antibody while retaining binding to the first antibody: Mutations occurring within the epitope of the second antibody. 3. Loss of affinity for both antibodies: so-called "destructive" mutations, which affect the conformation of the antigen and therefore prevent binding of both antibodies. 4. No effect: The mutation is not present in the epitope of one of the two antibodies and does not cause a significant change in the conformation of the antigen.

[0023] Following flow cytometry analysis, a yeast population that has lost affinity for the antibody of interest but retains binding to a second antibody is selected. The plasmids contained in this yeast population are extracted and sequenced by high-throughput sequencing. Analysis of the sequencing data allows for the identification of mutations that affect antibody binding to its target. This analysis therefore allows for the identification of the critical location on the antigen for binding of the antibody of interest, i.e., its epitope.

[0024] An example of an antibody that binds to the above epitope is the monoclonal anti-Nectin-4 antibody 15A7.5 mAb. This antibody and other antibodies described in International Application No. PCT / EP2022 / 058626 are expressly excluded from the scope of protection of the present application.

[0025] antibody The antibodies of the present invention are monoclonal antibodies (mAbs) or monoclonal antibody fragments. Unless otherwise indicated, the term "monoclonal" refers to a single species, i.e., a single amino acid composition, of the antibody or antibody fragment.

[0026] The antibodies provided herein preferably exhibit specific binding to nectin-4 and do not exhibit substantial cross-reactivity or cross-reactivity with other proteins of the human nectin family, in particular nectin-1, and / or rodent nectin-4, e.g., nectin-4 from rat and / or mouse.

[0027] The antigen-binding site of an antibody of the invention comprises an antibody heavy chain variable domain / region (VH) and / or an antibody light chain variable domain / region (VL), or a VH / VL pair.

[0028] The term "antigen-binding site" refers to the region 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.

[0029] The variable domains / regions refer to each of the light and heavy chain pairs that are 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".

[0030] 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., binding specificity determined by three, four, or five CDRs) are also within the scope of the present invention. For example, fewer than a complete set of six CDRs may be sufficient for binding. In some cases, a VH or VL domain may be sufficient.

[0031] According to the present invention, only the VH region or its CDRs may constitute a complete antigen-binding site. In certain embodiments, an antibody comprises only a VH region. In other embodiments, an antibody comprises a VH region together with a VL region.

[0032] The positions of CDRs within the VH or VL region can be defined by Kabat, et al., "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991) or 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)). Unless otherwise specified, the Kabat system is used herein.

[0033] 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 with fluorescence, immunohistochemistry, and / or surface plasmon resonance are described and are particularly preferred herein.

[0034] 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 It is defined by the term (KD / Ka).

[0035] In certain embodiments, the antibodies of the invention may be chimeric antibodies, multispecific antibodies, particularly bispecific antibodies, human antibodies, humanized antibodies, or antigen-binding fragments thereof.

[0036] 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.

[0037] A "multispecific antibody" binds to two or more different epitopes. The epitopes may be on the same antigen or different antigens. A preferred example of a multispecific antibody is a "bispecific antibody" or antigen-binding fragment thereof that binds to two different epitopes. A special type of bispecific antibody is a "biparatopic" antibody or antigen-binding fragment in which each antigen-binding domain recognizes a unique, non-overlapping epitope on the same target antigen. A preferred biparatopic antibody of the present invention binds to at least two different epitopes on Nectin-4, particularly the epitope defined in claim 1, and the binding epitopes of antibodies HA22 and / or 5A12. For example, a biparatopic antibody of the present invention can bind to at least two different epitopes on Nectin-4, particularly the epitope defined above, and the binding epitopes of antibodies HA22 and / or 5A12. The anti-Nectin-4 antibodies HA22 and 5A12 are described, for example, in WO 2018 / 158398(A1), the disclosure of which is incorporated herein by reference.

[0038] Biparatopic antibodies of the present invention can be designed as described herein, for example, based on the 15A7.5 mAb, an scFv of the 5A12.2 sequence or the HA22 sequence can be attached via a linker, preferably a glycine linker, or conversely, i.e., based on the 5A12.2 mAb or the HA22 mAb, an scFv of the 15A7.5 sequence can be attached via a linker, preferably a glycine linker.

[0039] Particularly preferred are the biparatopic antibodies provided herein, namely, the 5A12.2 scFv-15A7.5 biparatopic antibody represented by SEQ ID NOs: 105 and 106, and the 15A7.5 scFv-5A12.2 biparatopic antibody represented by SEQ ID NOs: 107 and 108, respectively.

[0040] In a preferred embodiment, the antibodies of the invention are humanized antibodies.

[0041] The term "humanized antibody" or "humanized version of an 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, when analyzed as a whole, has been altered to be more homologous to human germline sequences than to the germline sequences of the originating species. For example, murine CDRs can be grafted into the framework regions of a human antibody to prepare a "humanized antibody." See, e.g., Riechmann, L., et al., Nature 332 (1988) 323-327 and Neuberger, MS, et al., Nature 314 (1985) 268-270. Another form of humanized antibody encompassed by the present invention is one in which the constant regions have been further modified or altered from those of the original antibody to produce the properties described herein. The assessment of humanization is based on the resulting amino acid sequence, not the procedure itself.

[0042] Another preferred embodiment relates to human antibodies.

[0043] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, MA, and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production.

[0044] The antibodies of the present invention may 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 entire domain 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 may be any of the five major classes of antibodies, particularly the five major classes of human 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. According to the present invention, antibodies or fragments thereof of classes, particularly human classes IgG, IgA, or IgM, are particularly suitable.

[0045] According to a preferred embodiment, the antibody of the present invention is selected from the IgG class, in particular from the class of human IgG, such as the subclasses IgG1, IgG2, IgG3 of IgG4 (IgG3 of IgG4), class IgM, class IgA, or an antigen-binding fragment thereof.

[0046] In certain embodiments, the antibody comprises a constant domain, in particular a heavy chain constant domain, more particularly a heavy chain constant domain of the IgG class, in particular of the human IgG class, e.g., IgG3 of the subclasses IgG1, IgG2, IgG4, class IgM, class IgA, which has reduced effector function compared to the wild-type sequence of the same subclass, e.g., reduced binding to Fc receptors. An example of a heavy chain constant domain with reduced effector function may contain at least one of the mutations D265C, L234A, L235A, P331S, L234F, and L235E of the human IgG, e.g., IgG1 or IgG4, sequence.

[0047] 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" and "fragment of an antibody" may be used interchangeably herein.

[0048] An antibody or antigen-binding fragment may be monovalent or multivalent, i.e., it may 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 an Fv or scFv fragment has two antigen-binding sites, and an IgM class antibody has 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.

[0049] "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 the specified 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 one in the art, for example, using publicly available computer software such as BLAST.

[0050] In addition to lower binding affinity, the antibodies and fragments of the invention provided herein also preferably exhibit lower internalization and / or cytotoxic activity against keratinocytes compared to tumor cells.

[0051] According to particularly preferred embodiments, the antibodies and fragments of the invention exhibit lower binding affinity, lower internalization, and lower cytotoxic activity towards keratinocytes compared to tumor cells.

[0052] 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.

[0053] According to a further aspect, the monoclonal antibody or antigen-binding fragment thereof is characterized in that it binds to human Nectin-4 expressed by tumors with higher affinity than Nectin-4 expressed by human differentiated keratinocytes. For comparison, binding affinity can be detected by fluorescent flow cytometry, immunohistochemistry, and / or surface plasmon resonance.

[0054] According to further embodiments, 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 is detectable by imaging. Preferred effector groups are therapeutic groups, particularly cytotoxic agents, such as chemotherapeutic agents, drugs, anti-inflammatory agents, radioisotopes, toxins, such as topoisomerase poisons, enzymes and fragments thereof, such as nucleases, growth inhibitors, antibiotics, and all suitable anticancer and antitumor agents known to those skilled in the art. Particularly preferred are the topoisomerase poison camptothecin and derivatives thereof, and / or its structural analogs, such as exatecan, and its derivatives, such as deruxtecan.

[0055] Preferred embodiments of anti-tumor agents relate to anti-tumor immune stimulators, including but not limited to toll-like receptor (TLR) agonists or stimulator of interferon genes (STING) pathway.

[0056] According to another preferred embodiment, the anti-inflammatory agent may be selected from the group comprising steroids and corticosteroids (glucocorticoids, such as cortisol and its derivatives, or mineralocorticoids, such as aldosterone and its derivatives).

[0057] Another aspect of the present invention is a combination of at least two different monoclonal antibodies or fragments described herein.

[0058] Nucleic acid molecules, vectors and cells Furthermore, the present invention relates to a nucleic acid molecule, e.g. a DNA molecule, encoding an antibody VH region or an antibody VL region, or encoding the above-mentioned complete antibody or antibody fragment, a vector or vector system, i.e. a plurality of vectors, comprising the above-mentioned nucleic acid molecule, preferably operably linked to an expression control sequence, in particular a heterologous expression control sequence.

[0059] Furthermore, the present invention relates to a cell comprising the above-described nucleic acid molecule or vector or vector system. As used herein, the term "vector" 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 a nucleic acid to which they are operably linked. Vectors, particularly expression vectors, for the recombinant production of antibodies are well known in the art.

[0060] 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.

[0061] Antibody-drug conjugates The antibody-drug conjugate of the present invention comprises: a monoclonal antibody or antigen-binding fragment thereof as defined above; and a drug conjugated to a reactive amino acid residue on the antibody or antigen-binding fragment, for example, an amino acid residue having a side chain containing an amino, hydroxy, or thiol group, or to a reactive group in the antibody glycan structure.

[0062] The drug of the antibody-drug conjugate of the present invention is preferably a topoisomerase I inhibitor. Topoisomerase I inhibitors are compounds that can form a ternary complex with topoisomerase I and DNA, thereby preventing DNA religation and introducing DNA strand breaks into the cellular genome. Topoisomerase I inhibitors can be selected from, for example, camptothecin or its analogs, indenoisoquinolines, and indolocarbazoles.

[0063] In certain embodiments, the topoisomerase I inhibitor is camptothecin or an analog thereof, i.e., a compound comprising the basic pentacyclic 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, raltotecan, or atiratecan).

[0064] In principle, a drug, such as a topoisomerase-I inhibitor such as exatecan, can be conjugated to any suitable position on a monoclonal antibody or its antigen-binding fragment, particularly any position that does not abolish the antibody's binding to Nectin-4. For example, a drug, such as a topoisomerase-I inhibitor such as exatecan, can be conjugated to a reactive amino acid residue on the antibody, such as an amino acid residue with a side chain containing an amino, hydroxy, or thiol group, or a reactive group in the antibody glycan structure. In certain embodiments, a drug, such as a topoisomerase-I inhibitor such as exatecan, is conjugated to a reactive thiol group in the side chain of an accessible cysteine residue on the antibody.

[0065] In certain embodiments, the antibody-drug conjugate has a drug-to-antibody / antibody fragment molar ratio (DAR) greater than 1, i.e., two or more drug molecules are attached 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.

[0066] The drug can be conjugated to the antibody or antigen-binding fragment thereof 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.

[0067] 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 of antibody-drug conjugate MEDI7247 or the mcc-triazole spacer-PEG7-x-Lys-PABC glycol linker of Trodelvy®. Further preferred linkers include oligopeptides, particularly di- or decapeptides, such as tetrapeptide sequences such as glycine-glycine-phenylalanine-glycine of Enhertu®. Further preferred linkers include highly polar spacers such as acyl, carbamoyl, 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™. A particularly preferred linker is a β-glucuronide linker.

[0068] In certain embodiments, the linker is a hydrophilic polysarcosine linker, e.g., comprising up to 15 sarcosine units and at least one ethylene glycol unit, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more ethylene glycol units, and the linker is subject to cleavage by a glycosidase, and in particular, by a glucuronidase.

[0069] In certain embodiments, the linker is a hydrophilic polysarcosine linker, e.g., comprising about 8-12 sarcosine units and at least one ethylene glycol unit, e.g., up to 10 ethylene glycol units, and the linker is subject to cleavage by a glycosidase, particularly a glucuronidase.

[0070] In certain embodiments, the linker is a hydrophilic polysarcosine linker comprising 10 sarcosine units and 2 ethylene glycol units, and the linker is subject to cleavage by glucuronidase.

[0071] The antibody-drug conjugates of the present invention can be prepared by known methods. The antibody or antigen-binding fragment thereof can be produced in a suitable host cell containing 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., multiple vectors, comprising the above-mentioned nucleic acid molecule, preferably operably linked to an expression control sequence, particularly a heterologous expression control sequence. 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.

[0072] An antibody or antigen-binding fragment thereof can be reacted with a linker-drug conjugate to obtain an antibody-drug conjugate. The linker-drug conjugate, for example, comprises a drug molecule attached with a suitable linker, where the linker comprises 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.

[0073] Pharmaceutical Composition A further aspect of the invention is a pharmaceutical composition comprising an active agent that is an antibody or antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0074] Examples of suitable carriers and excipients for formulating antibodies and antibody-drug conjugates include saline and aqueous buffer solutions and are well known in the art. Typically, the pharmaceutical compositions are adapted for parenteral administration, for example, subcutaneous, intramuscular, or intravenous injection or infusion. In a further embodiment, the pharmaceutical compositions are adapted for local administration, for example, intravesical instillation into the bladder.

[0075] Depending on the stage and severity of the disease, the pharmaceutical composition can be administered in a therapeutically effective dose to a subject, particularly a human subject, in need thereof once or several times, for example, once or several times daily, every two days, twice weekly, or once weekly, for an appropriate period, for example, at least one week, or at least one month.

[0076] Medical Use According to a further aspect of the invention, the antibody, antibody-drug conjugate or pharmaceutical composition is used in medicine, including human and veterinary medicine, in particular in human medicine.

[0077] In certain embodiments, the above-mentioned antibody, antibody-drug conjugate or pharmaceutical composition is used in a method for the prevention and / or treatment of Nectin-4-associated disorders such as cancer and / or inflammatory diseases, particularly for the prevention and / or treatment of Nectin-4-positive cancers and / or inflammatory diseases, and more particularly for the prevention and / or treatment of cancers and / or inflammatory diseases associated with Nectin-4 overexpression.

[0078] 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 in accordance with the present invention is preferably selected from the group consisting of bladder cancer, 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.

[0079] In therapeutic applications, active agents are administered to a subject in need thereof, particularly a human subject, in an effective amount, the dosage depending on the specific type of agent, e.g., type of antibody or antibody fragment, the type of disease, and the mode of administration, e.g., local or systemic.

[0080] In the prevention and / or treatment of cancer, the therapeutic dose of an antibody or antibody-drug conjugate is typically about 0.3 mg / kg to about 10 mg / kg.

[0081] The antibody or antibody-drug conjugate may be administered alone or with an additional active agent, which may be selected from a chemotherapeutic agent, e.g., an antimetabolite, alkylating agent, intercalating agent, or antimitotic agent; an inhibitor of a specific kinase, e.g., a tyrosine kinase inhibitor, a serine / threonine kinase inhibitor, or a phosphoinositide kinase inhibitor; an immunotherapeutic compound, e.g., an immune checkpoint inhibitor; a CAR-T cell; a therapeutic vaccine; or an oncolytic virus.

[0082] The present invention is further illustrated in more detail by the following tables, figures and examples. [Brief explanation of the drawings]

[0083] [Figure 1] Evaluation of the compatibility of antibodies 15A7.5_H1L2 and HA22. Yeast expressing Nectin-4 fragment 32-349 was 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]Library selection by flow cytometry. Two libraries covering the nectin-4 sequence of interest were generated (left and right panels). Yeast expressing nectin-4 single mutants were incubated with 100 pM biotinylated HA22 and 100 pM 15A7.5_H1L2 labeled with Dy650. The 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 any antibody express a nectin-4 single mutant that disrupts the epitope for binding of the other antibody. [Figure 3-1] Positions that show a significant effect on the 15A7.5_H1L2 antibody when mutated. 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 15A7.5_H1L2 antibody. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1] Positions that show significant effects on HA22 antibody upon mutation. For any amino acid in the domain of interest, the higher the number of prohibited mutations, the more important the residue is for HA22 antibody binding. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] A structural reference pymol file for Nectin-4 (4FRW, pdb) is shown. For each antibody, the key amino acids of the epitope are shown. The most important residues are underlined. [Figure 6] Comparative internalization between biparatopic mAbs and their parent mAbs. The indicated mAbs (mono- or biparatopic) were coupled to pHDYE (Promega). A dose range of each pHDYE-conjugated mAb was incubated with SUM190PT cells for 24 hours, after which the fluorescent signal was measured. Fluorescence intensity is proportional to the amount of internalized material acidified in the lysosomes. [Example]

[0084] Principle of epitope mapping using DMS (Deep Mutational Scanning) DMS is a mutagenesis method that aims to generate all possible single substitutions for all selected residues in a 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.

[0085] This DMS DNA library is incorporated into an expression plasmid specifically designed to express recombinant proteins on the yeast surface. The yeast is then transformed and induced to express single mutant proteins on its 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) as well as the binding of the protein to its partner (fluorescent partner).

[0086] 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 serves as a conformational control for the mutated antigen relative to the other antibody. In fact, a single substitution made to an antigen can have four types of effects: (i) loss of affinity for the first antibody while retaining binding to the second antibody: a mutation occurring within the epitope of the first antibody; (ii) loss of affinity for the second antibody while retaining binding to the first antibody: a mutation occurring within the epitope of the second antibody; (iii) loss of affinity for both antibodies: a so-called "destroying" mutation that affects the conformation of the antigen and therefore prevents binding of both antibodies; or (iv) no effect: the mutation is not present in the epitope of one of the two antibodies and does not significantly alter 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 identification of mutations that affect the binding of the antibody to its target. Thus, this analysis allows identification of the important position on the antigen for the binding of the antibody of interest, i.e., its epitope.

[0087] Construction of antigen expression plasmids A gene corresponding to amino acids 32-349 of SEQ ID NO:104 was synthesized and cloned into a plasmid that allows expression on the surface of galactose-inducible yeast. In this construct, the expressed antigen has 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: Library 1 for amino acid positions 32-91 of SEQ ID NO:104, and Library 2 for amino acid positions 92-151 of SEQ ID NO:104. 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 variants and 2,000 DNA codon variants. The two libraries were transformed into YSD. Unscreened yeast from the two generated libraries was sequenced to verify the efficiency of mutagenesis. 100% of the predicted single mutants were sequenced for library 1 and 99.9% for library 2.

[0088] 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 for a positive control. After 30 minutes, 500 pM Antibody 2 was added and incubated for an additional 30 minutes. The yeast were then incubated on ice for 15 minutes with the respective 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 the 15A7.5_H1L2 mAb can bind to the Nectin-4 IgV domain simultaneously with HA22.

[0089] Induction of antigen expression and FACS sorting of the library Yeast expressing the nectin4 single mutants were induced for protein expression. Transformed yeast were 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 8The induced cells were washed with 1 mL of PBSF (PBS, 0.1% BSA). They were then resuspended in an appropriate volume of solution containing 100 pM of 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™ 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, including the first step to amplify the region of interest and the second step 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. After removing low-quality sequences (Q<30), single variants were detected and counted. For each single mutation, the frequency measured in the unsorted population was compared with the frequency of the mutation in the sorted population. The enrichment value of the mutant was then calculated according to the following formula:

[0090]

number

[0091] 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). Between 16 and 19 forbidden mutations indicate high impact, and positions falling into this category are most likely to be indirectly interacting with the IgG under consideration. Between 10 and 15 forbidden mutations indicate positions with moderate impact, and between 5 and 9 forbidden mutations indicate positions with low impact. Figure 5 shows the structural analysis of Nectin-4 (4FRW, pdb) and highlights the critical and most important epitope residues for each of the antibodies tested. Table 1 lists these residues.

[0092] [Table 1]

[0093] Construction, production and purification of biparatopic antibodies Two biparatopic mAbs were designed: one based on the 15A7.5 mAb with the 5A12.2 sequence of scFv attached via a glycine linker, and the other based on the 5A12.2 mAb with the 15A7.5 sequence of scFv attached via a glycine linker. The corresponding cDNAs were cloned into dedicated vector systems using conventional (non-PCR-based) cloning techniques. Vector plasmids were gene synthesized. Plasmid DNA was prepared under low-endotoxin conditions using anion-exchange chromatography. DNA concentration was determined by measuring absorbance at 260 nm. Sequence accuracy was verified by Sanger sequencing (up to two sequencing reactions per plasmid, depending on the size of the cDNA).

[0094] The heavy and light chain sequences of the 5A12.2 scFv-15A7.5 biparatopic antibody are represented by SEQ ID NOs: 105 and 106, and the heavy and light chain sequences of the 15A7.5 scFv-5A12.2 biparatopic antibody are represented by SEQ ID NOs: 107 and 108, respectively.

[0095] Suspension-adapted CHO K1 cells were used for production. The cells were grown in a chemically defined, animal-component-free, serum-free medium. The cells were transfected with a transfection reagent, and after transfection, the cells were grown in an animal-component-free, serum-free medium.

[0096] The supernatant was collected by centrifugation and subsequent filtration (0.2 μm filter). Antibodies were purified using MabSelect™ SuRe™.

[0097] Coupling of pHAB thiol-reactive dye (Promega G9835) Five antibodies (isotype control, humanized 15A7.5 and 5A12.2, biparatopic 5A12.2 scFv-15A7.5, and 15A7.5 scFv-5A12.2) were dialyzed overnight at 4°C in 0.1 M phosphate buffer, pH 7. Seven hundred micrograms of each antibody was reduced with 2.5 mM DTT for 1 hour at room temperature. Excess DTT was removed using a desalting column, and the antibodies were resuspended in 0.1 M phosphate buffer, pH 7. pHDYE was added at the following ratio: 1.2 μL pHDYE for 100 μg of antibody and 133 μg of biparatopic antibody for 1 hour at room temperature, away from light. Excess pHDYE was removed using a desalting column, and the antibodies were resuspended in 0.1 M phosphate buffer, pH 7.

[0098] Internalization assay On day -1, SUM190PT cells were plated in a 96-well black plate (10 4 Cells / 75 μL medium).

[0099] On day 0, 75 μL of pHDye antibody was added to the wells at 2x concentrations ranging from 0.005344 nM to 16.7 nM (final concentration). After gentle mixing, the plates were incubated at 37°C in a 5% CO atmosphere for 24 hours. After washing three times with 150 μL of PBS, fluorescence was monitored on a Clariostar+ fluorometer plate reader (Cy3 setting). The results are shown in Figure 6.

Claims

1. a monoclonal antibody or antigen-binding fragment thereof, which specifically binds to a discontinuous epitope on Nectin-4 consisting of one or more of amino acids D57, S58, E60, P133, G135, F137, Q138, and R140, and optionally G56, G59, E126, R128, P133, and A134 of SEQ ID NO: 104, as determined using Deep Mutational Scanning (DMS); provided that the antibody or antibody fragment does not comprise a variable heavy chain (VH) region comprising complementarity determining regions (CDRs) CDR-H1 set forth in SEQ ID NO: 7, 21, 35, 49 or 63, CDR-H2 set forth in SEQ ID NO: 8, 22, 36, 50 or 64, and CDR-H3 set forth in SEQ ID NO: 9, 23, 37, 51 or 65, and a variable light chain (VL) region comprising complementarity determining regions (CDRs) CDR-L1 set forth in SEQ ID NO: 10, 24, 38, 52 or 66, CDR-L2 set forth in SEQ ID NO: 11, 25, 39, 53 or 67, and CDR-L3 set forth in SEQ ID NO: 12, 26, 40, 54 or 68; In particular, the antibody or antibody fragment does not comprise the VH region set forth in SEQ ID NO: 13 and the VL region set forth in SEQ ID NO: 14, In particular, the antibody or antigen-binding fragment is not the antibody 15A7.5 disclosed in International Application No. PCT / EP2022 / 0586626.

2. 2. The antibody or antigen-binding fragment of claim 1, which is a chimeric antibody, a multispecific antibody, in particular a bispecific antibody, a human antibody, a humanized antibody or an antigen-binding fragment thereof.

3. The antibody or antigen-binding fragment of claim 1 or 2, which is a biparatopic antibody or antigen-binding fragment that binds to at least two different epitopes on Nectin-4, in particular the epitope of claim 1 and the binding epitope of antibody HA22 and / or 5A12.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which is an antibody or antigen-binding fragment thereof of class IgG, for example IgG3 of subclass IgG1, IgG2, IgG4, class IgM, class IgA, or which is a single-chain antibody or an antibody Fv fragment.

5. 5. The antibody or antigen-binding fragment of any one of claims 1 to 4, which binds to Nectin-4 expressed by tumors with higher affinity compared to Nectin-4 expressed by human differentiated keratinocytes.

6. 6. The antibody or antigen-binding fragment of claim 5, wherein the binding affinity is determined by fluorescence flow cytometry, immunohistochemistry and / or surface plasmon resonance.

7. The antibody or antigen-binding fragment of 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. 8. The 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 antibody or antigen-binding fragment of claim 7 , wherein the effector group is a therapeutic group, in particular a cytotoxic agent.

10. The antibody or antigen-binding fragment according to any one of claims 1 to 9 for use in medicine, in particular for therapeutic or diagnostic applications, including in vitro and in vivo diagnostic applications.

11. 11. The antibody or antigen-binding fragment for use according to claim 10 in a method for preventing and / or treating cancer and / or inflammatory disorders.

12. The antibody or antigen-binding fragment for use according to claim 11, wherein the cancer is associated with Nectin-4 overexpression.

13. 13. The antibody or antigen-binding fragment for use according to any one of claims 11 to 12, 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.

14. A nucleic acid encoding the antibody or antigen-binding fragment of any one of claims 1 to 9.

15. A vector, particularly an expression vector, comprising a nucleic acid according to claim 14.

16. A host cell comprising the vector of claim 15.

17. 1. An antibody-drug conjugate comprising: The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, an antibody-drug conjugate comprising: a drug conjugated to a reactive amino acid residue on the antibody or antigen-binding fragment, for example, an amino acid residue having a side chain comprising an amino, hydroxy, or thiol group, or to a reactive group in an antibody glycan structure.

18. 18. The antibody-drug conjugate of claim 17, 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.

19. The antibody-drug conjugate of claim 17 or 18, 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 peptide linker.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the vector according to claim 15, or the antibody-drug-conjugate according to any one of claims 17 to 19.

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