Nectin-4 binding agent

Abstract

The present invention relates to antibodies and antibody fragments that bind to Nectin-4 polypeptides. The present invention also relates to antibody-drug conjugates comprising said antibodies or antibody fragments, as well as methods for producing said conjugates, pharmaceutical compositions and methods for using them for the diagnosis, treatment or prevention of diseases, such as cancers characterized by Nectin-4-expressing tumor cells.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application seeks the benefit of priority to U.S. Provisional Application No. 63 / 345,453, filed May 25, 2022, which is incorporated by reference in its entirety, including any drawings.

[0002] (Reference to sequence listing) This application is submitted with an electronic sequence listing. The sequence listing is provided under the file name "Nectin-4-3 PCT" created on May 22, 2023, and is 97 KB in size. The information set forth in the electronic sequence listing is incorporated herein by reference in its entirety.

[0003] (Technical field) The present invention relates to antibodies and fragments thereof that bind to Nectin-4 polypeptides. The present invention also relates to cells producing such compounds, methods for producing said compounds, conjugates and methods for producing the conjugates, pharmaceutical compositions containing them, and methods for diagnosing, treating or preventing diseases (e.g., cancers characterized by Nectin-4-expressing tumor cells) using said compounds. [Background technology]

[0004] Nectin-4 is a surface molecule of the nectin protein family that plays important roles in various biological processes such as polarity, proliferation, differentiation and migration of epithelial, endothelial, immune and neuronal cells during development and adulthood. Nectin-4 was first cloned from human trachea by Lopez's group in 2001 (see Reymond et al. (2001) J. Biol. Chem. 276(46):43205-15). Nectin is the main receptor for polio, herpes simplex and measles viruses and is further involved in several pathological processes in humans. Nectin-4 is expressed at significantly higher levels in several tumors, and is overexpressed in breast cancer, e.g., triple-negative breast cancer (TNBC) (see M-Rabet et al. 2017 Apr 1;28(4):769-776), pancreatic cancer and urothelial cancer. Furthermore, nectin-4 is also expressed in tumor specimens from non-small cell lung cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer. Challita-Eid et al. reported moderate to strong immunohistochemical staining (H-score ≥ 100) in bladder (60%) and breast (53%) tumor tissues (Challita-Eid, et al. (2016) Cancer Res. 76(10):3003-3013). Zeindler et al. reported high expression of nectin-4 in 86 (58%) of 148 TNBC cases (Zeindler et al. 2019 Front. Med. 6:200). Detection of a soluble form of nectin-4 in the serum of these cancer patients is associated with poor prognosis. Serum nectin-4 levels increase during metastatic cancer progression and decrease after treatment. These results suggest that nectin-4 may be a reliable target for cancer therapy.

[0005] Thus, several anti-nectin-4 antibodies have been described in the prior art. In particular, enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting nectin-4 that is currently undergoing clinical trials for the treatment of patients with solid tumors. Challita-Eid et al. (2016) (supra) developed an anti-nectin-4 antibody conjugated with the highly active and potent microtubule disrupting agent MMAE based on the antibody AGS-22. This work has led to the ADC drug candidate enfortumab vedotin (see U.S. Pat. No. 8,637,642 and PCT Publication No. WO2012 / 047724; Agensys Inc.), which has provided promising results in human clinical trials in the treatment of patients with locally advanced or metastatic urothelial carcinoma who have previously received neoadjuvant / adjuvant platinum-containing chemotherapy and PD-1 / PD-L1 checkpoint inhibitors in the locally advanced or metastatic setting. Several other groups have also proposed anti-nectin-4 agents conjugated to various toxic drugs. In PCT patent application WO2018 / 158398 (INSERM), several anti-nectin-4 antibodies are reported and the possibility of conjugation with various cytotoxic agents is proposed. Similarly, US Patent No. 8,637,642 (Agensys Inc.) also shows anti-nectin-4 antibodies and the possibility of conjugation with various cytotoxic agents is proposed. In addition, Bicycle Therapeutics has reported the development of an anti-nectin-4 targeting agent comprising a nectin-4 binding protein conjugated to a cytotoxic auristatin (MMAE) payload via a valine-citrulline (val-cit) cleavable linker. To date, all anti-nectin-4 antibodies reported to be active when conjugated to chemotherapeutic agents target the Ig-like V-type domain of nectin-4, and as a result, the Ig-like V-type domain is believed to result in the strongest internalization of anti-nectin-4 ADCs.

[0006] Enfortumab vedotin (anti-nectin-4 ADC) binds to the Ig-like V-type domain of nectin-4 and is associated with high internalization of anti-nectin-4 antibodies. Although enfortumab vedotin showed impressive therapeutic efficacy in UC with an ORR (objective response rate) of 44% and a CR (complete response rate) of 12% in the EV-201 Phase 2 study (2019), approximately half of the patients discontinued treatment. Most of the reasons for discontinuation were due to RECIST-assessed progression (48%) or clinical findings (5%). In addition, 18% of patients who discontinued treatment experienced adverse events, especially neuropathy. Thus, nectin-4-targeted ADCs have limitations, and there is a need in the art to improve their benefits to patients suffering from UC and other cancers.

[0007] Summary of the Invention Provided herein are variable heavy (VH) and variable light (VL) domains for use in Nectin-4 binding proteins. One object of the present disclosure is to provide an anti-Nectin-4 antibody or antibody fragment, comprising a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) with the heavy chain comprising the amino acid sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57, and a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) with the light chain comprising the amino acid sequence of SEQ ID NO: 59, 61, 63 or 65.

[0008] In one embodiment, an anti-Nectin-4 antibody or antibody fragment is provided, comprising a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a heavy chain comprising the amino acid sequence of SEQ ID NO:69, and a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:70.

[0009] In one embodiment, an anti-Nectin-4 antibody or antibody fragment is provided, comprising: a heavy chain variable region (VH) comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 21, 22 and 23, respectively, and the amino acid sequences of framework FR1, FR2 and FR3 derived from the human IGHV1-46*01 gene (and, optionally, the amino acid sequence of an additional framework FR4 derived from the human IGHJ4*01 gene); and a light chain variable region (VL) comprising CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively, and the amino acid sequences of framework FR1, FR2 and FR3 derived from the human IGKV2-28*01 gene (and, optionally, the amino acid sequence of an additional framework FR4 derived from the human IGKJ4*01 gene).

[0010] In one embodiment, an anti-Nectin-4 antibody or antibody fragment is provided that comprises a heavy chain variable region that comprises 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions at Kabat positions selected from 28, 38, 40, 48, 69, 71, 73, 78. Optionally, the threonine residue at position 28 may be substituted with an isoleucine residue. Optionally, the arginine residue at position 38 may be substituted with a lysine residue. Optionally, the alanine residue at position 40 may be substituted with an arginine residue. Optionally, the methionine residue at position 48 may be substituted with an isoleucine residue. Optionally, the methionine residue at position 69 may be substituted with a leucine residue. Optionally, the arginine residue at position 71 may be substituted with a leucine residue. Optionally, the threonine residue at position 73 may be substituted with a lysine residue. Optionally, the valine residue at position 78 may be substituted with a threonine residue.

[0011] In one embodiment, an anti-Nectin-4 antibody or antibody fragment is provided that comprises a light chain variable region that comprises one, two, three or four amino acid substitutions at Kabat positions selected from 2, 8, 11, 64. Optionally, the isoleucine residue at position 2 may be substituted with a valine residue. Optionally, the proline residue at position 8 may be substituted with an alanine residue. Optionally, the leucine residue at position 11 may be substituted with an asparagine residue. Optionally, the glycine residue at position 64 may be substituted with a serine residue.

[0012] In one embodiment, an anti-Nectin-4 antibody is provided, comprising a heavy chain having at least about 80% sequence identity (e.g., at least about 70%, 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a heavy chain comprising the amino acid sequence of SEQ ID NO: 77; and a light chain having at least about 80% sequence identity (e.g., at least about 70%, 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0013] In one embodiment, an anti-Nectin-4 antibody or antibody fragment is provided, comprising a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a heavy chain comprising the amino acid sequence of SEQ ID NO:47; and a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:65.

[0014] In one embodiment, a Nectin-4 binding protein is provided that comprises an antibody or antibody fragment of the present disclosure, and optionally further comprises another antigen binding domain.

[0015] In any embodiment, the antibody or antibody fragment may be characterized as monoclonal, humanized and / or in isolated form, as desired.

[0016] In one embodiment, the anti-Nectin-4 antibody or antibody fragment binds to the VC1 cross-linking domain of the Nectin-4 polypeptide.

[0017] In any embodiment, the antibody or antibody fragment of the disclosure is for use in the manufacture of an antibody drug conjugate (ADC). In any embodiment, the antibody of the disclosure is for use in reducing cell-cell adhesion between Nectin-4-expressing tumor cells, inhibiting Nectin-4:Nectin-1 interaction, inhibiting Nectin-4:Nectin-4 interaction, reducing proliferation of Nectin-4-expressing tumor cells, and / or reducing clustering of Nectin-4-expressing tumor cells (e.g., for use in the methods described below).

[0018] According to one aspect of the present invention, there is provided an antibody-drug conjugate comprising an anti-Nectin-4 antibody or antibody fragment linked to a cytotoxic agent, optionally wherein the cytotoxic agent is conjugated to the antibody or antibody fragment via a linker, and optionally further wherein the linker or the linker-toxic agent comprises any one of formulae III to XIV.

[0019] In one embodiment, an antibody drug conjugate (ADC) comprises an anti-Nectin-4 antibody or antibody fragment linked to at least one cytotoxic agent moiety, wherein the antibody or antibody fragment comprises a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:69, and a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:70.

[0020] In one embodiment, an antibody drug conjugate (ADC) comprises: (a) at least one antigen-binding domain that binds to a Nectin-4 polypeptide, wherein the antigen-binding domain comprises: (i) a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, or 57; and (ii) a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO: 59, 61, 63, or 65; and (b) at least one cytotoxic agent moiety.

[0021] In a preferred embodiment, the antibody drug conjugate (ADC) comprises (a) an anti-Nectin-4 humanized antibody comprising: (i) a heavy chain variable region having at least 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:47; and (ii) a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO:65.

[0022] In some embodiments, an antibody (e.g., full-length antibody, antibody fragment) that binds to human Nectin-4 polypeptide for use in manufacturing an antibody drug conjugate can reduce cell-cell adhesion between Nectin-4-expressing tumor cells and / or reduce the proliferation and / or clustering of Nectin-4-expressing tumor cells (e.g., as assessed using three-dimensional or non-attached tumor cell culture; tumor spheroid assays).

[0023] In some embodiments, the antibody (e.g., a full-length antibody, an antibody fragment) is capable of inhibiting nectin-4:nectin-1 interaction and / or nectin-4:nectin-4 interaction (e.g., the antibody is capable of reducing the interaction of nectin-4 on a first cell with nectin-1 and / or nectin-4 on a second cell; optionally, the cell is a tumor cell).

[0024] In any of the embodiments herein, the anti-Nectin-4 antibody or antibody fragment, or an antibody-drug conjugate comprising said antibody or fragment, is capable of undergoing intracellular internalization upon binding to Nectin-4 on the surface of tumor cells.

[0025] In some embodiments, a method for producing an antibody drug conjugate is provided, comprising conjugating an anti-Nectin-4 antibody or antibody fragment (e.g., an antibody or antibody fragment of the present disclosure) to a cytotoxic agent (e.g., a linker or linker-toxic agent of the present disclosure).

[0026] In some embodiments, the preparation of an antibody drug conjugate includes conjugating the antibody to a cytotoxic agent (e.g., via a linker moiety; the linker moiety further comprises a moiety that is cleavable intracellularly).

[0027] In one embodiment, the anti-Nectin-4 antibody or antibody fragment is conjugated to a cytotoxic agent via an intracellularly cleavable (e.g., protease-cleavable) oligopeptide (e.g., di-, tri-, tetra-, or pentapeptide). In one embodiment, the anti-Nectin-4 antibody or antibody fragment is conjugated to a cytotoxic agent (e.g., camptothecin derivative) via an intracellularly cleavable (e.g., protease-cleavable) di-, tri-, tetra-, or penta-peptide and a self-eliminating spacer. In one embodiment, the anti-Nectin-4 antibody or antibody fragment is conjugated to a cytotoxic agent (e.g., camptothecin derivative) via an intracellularly cleavable (e.g., protease-cleavable) tetra- or penta-peptide and a self- or non-self-eliminating spacer.

[0028] In one embodiment, the cytotoxic agent is a highly potent chemotherapeutic agent, optionally selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mitomycins, vinca alkaloids, nitrogen mustards, maytansinoids, duocarmycins, tubulysins, dolastatins and auristatins, enediynes (e.g., calicheamicin, esperamicin, shishijimycin, and namenamycin), pyrrolobenzodiazepines (e.g., pyrrolobenzodiazepine dimers and indolino-pyrrolobenzodiazepine dimers), amatoxins, and ethylenimines. In one embodiment, the cytotoxic agent is a DNA damaging agent, such as a DNA intercalating agent, e.g., an agent that inserts itself into the DNA structure of a cell and binds to DNA, causing DNA damage (e.g., daunorubicin). Compounds include topoisomerase inhibitors, i.e. compounds that inhibit the action of topoisomerase (topoisomerase I and II). Such compounds are used in a wide range of solid tumors and hematological malignancies, especially lymphomas. Topoisomerase I inhibitors include camptothecins, such as irinotecan (approved for colon cancer), topotecan (approved for ovarian and lung cancer), camptothecin, lamellarin D, indenoisoquinoline, indimitecan. Further camptothecans include ciratecan, cositecan, exatecan, lurtotecan, gimatecan, belotecan and rubitecan. Examples of topoisomerase II inhibitors include etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticine, aurintricarboxylic acid, and HU-331, a quinolone synthesized from cannabidiol.

[0029] Optionally, the antibody or antibody fragment is functionalized (eg, conjugated, covalently attached) with a linker-toxin of any one of Formulas III-XIV.

[0030] In one embodiment, the cytotoxic agent is a camptothecin analogue, such as exatecan, Dxd or an SN-38 molecule.

[0031] In one aspect, the present invention provides a Nectin-4 binding antibody or antibody fragment of the disclosure conjugated (e.g., covalently attached) to a camptothecin (e.g., a camptothecin analog, exatecan or exatecan derivative, a Dxd molecule, or an SN-38 molecule).

[0032] In any of the embodiments herein, a Nectin-4 binding antibody or antibody fragment of the present disclosure conjugated (e.g., covalently attached) to a camptothecin analog (e.g., an exatecan or SN-38 molecule) may be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine, lysine, glutamine residues, unnatural amino acid residues) functionalized via a linker, together with a molecule comprising the structure of Compound 1 or 2. In any of the embodiments herein, a Nectin-4 antibody or antibody fragment may be characterized as being functionalized with a linker-camptothecin molecule having the structure of Formula III, IV, V, VI, VII, IX, X, XI, XII, XIII or XIV, or any of Compounds 3-16.

[0033] In one embodiment, the Nectin-4 antibody or antibody fragment conjugated to a camptothecin derivative is a Nectin-4 antibody or antibody fragment conjugated to an Exatecan molecule (e.g., a molecule having the structure of Compound 1 (1a or 1b)). In any embodiment, the cleavable linker or immunoconjugate or ADC comprising a linker may be characterized as releasing an Exatecan molecule, e.g., a molecule having the structure of Compound 1 (1a or 1b), upon, for example, enzymatic cleavage of the linker. In one embodiment, the humanized Nectin-4 antibody or antibody fragment conjugated to a camptothecin derivative is a Nectin-4 antibody or antibody fragment conjugated to a SN-38 molecule (e.g., a molecule having the structure of Compound 2). In one embodiment, the Nectin-4 antibody or antibody fragment is conjugated to a linker (e.g., a cleavable linker molecule with or without an additional spacer, e.g., a spacer (Y') as described herein), having the following structure: [ka] The present invention can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide having one or more amino acid residues (e.g., cysteine, lysine, glutamine, or a non-natural amino acid residue) functionalized with a molecule having the formula:

[0034] In one embodiment, the Nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent has formula (I): Ab-XZ Formula (I) [In the formula, Ab is an antibody or antibody fragment that specifically binds to a human nectin-4 polypeptide (e.g., any antibody or antibody fragment of the disclosure, which, optionally, may bind to the VC1 cross-linking domain of human nectin-4 and / or may exhibit reduced binding to a mutant human nectin-4 polypeptide comprising amino acid substitutions at residues K197 and / or S199 (see SEQ ID NO:1) compared to binding to a wild-type human nectin-4 polypeptide); X is a linker molecule linking Ab and Z (e.g., covalently binding each of Ab and Z), X comprising a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions, optionally, X may further comprise a self-eliminating or non-self-eliminating spacer system (Y') positioned between the cleavable moiety and Z, optionally, X may further comprise a spacer (Y) positioned between the Ab and the cleavable moiety; and Z is a cytotoxic agent, optionally Z is a camptothecin analog, optionally Z is exatecan, optionally Z is exatecan, and cleavage of the linker releases a compound having the structure of Compound 1 (exatecan). The immunoconjugate may be identified as having the formula:

[0035] In one embodiment, the Nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent has formula (I): Ab-XZ Formula (I) [In the formula, Ab is an antibody or antibody fragment of the disclosure that specifically binds to a human Nectin-4 polypeptide; X is a linker molecule linking Ab and Z (e.g., covalently binding each of Ab and Z), where X comprises a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide, X further comprises a self-eliminating or non-self-eliminating spacer system (Y') positioned between the cleavable moiety and Z, and X further comprises a spacer (Y) positioned between the Ab and the cleavable moiety; and Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule. The immunoconjugate may be identified as having the formula:

[0036] In one embodiment, there is provided a method of delivering or targeting a cytotoxic agent (optionally a camptothecin analog) to a tumor, releasing a cytotoxic agent (optionally a camptothecin analog, Dxd, exatecan) to a tumor (e.g., a cancer patient), or sensitizing a tumor or cancer to a cytotoxic agent (optionally a camptothecin analog), comprising administering to a subject having cancer a compound represented by formula (I): Ab-XZ Formula (I) [In the formula, Ab is an antibody or antibody fragment that specifically binds to a human Nectin-4 polypeptide; X is a linker molecule linking Ab and Z (e.g., covalently binding each of Ab and Z), X comprising a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions, optionally, X may further comprise a self-eliminating or non-self-eliminating spacer system (Y') positioned between the cleavable moiety and Z, optionally, X may further comprise a spacer (Y) positioned between the Ab and the cleavable moiety; and Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exatecan molecule or an SN-38 molecule, optionally where Z is exatecan and cleavage of the linker releases a compound having the structure of Compound 1 (exatecan). The method is characterized in that an immunoconjugate represented by the formula:

[0037] In one embodiment, the antibody or antibody fragment conjugated to a cytotoxic agent, optionally a camptothecin analog, has the formula (II): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is an antibody or antibody fragment of the disclosure that specifically binds to a human Nectin-4 polypeptide; X is a linker molecule linking Ab and Z, X comprises a cleavable moiety (optionally a protease cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions, and optionally X further comprises a self-eliminating or non-self-eliminating spacer system (Y') located between the cleavable moiety and Z, and optionally X further comprises a spacer (Y) located between the Ab and the cleavable moiety; Z is a cytotoxic agent (optionally a camptothecin analog), optionally Z is a molecule including an exatecan molecule or an SN-38 molecule, e.g., a molecule having the structure of compound 1 or 2; n is 1; and m is 4 to 8, or optionally, m is an integer selected from 4, 5, 6, 7, or 8. The immunoconjugate may be identified as having the formula:

[0038] In one embodiment, the Nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent, optionally a camptothecin analog, has the formula (II): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is an antibody or antibody fragment of the disclosure that specifically binds to a human Nectin-4 polypeptide; X is a molecule linking Ab and Z, X comprising a cleavable moiety (optionally a protease cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions, optionally X further comprising a self-eliminating or non-self-eliminating spacer system (Y') located between the cleavable moiety and Z, optionally X further comprising a spacer (Y) located between the Ab and the cleavable moiety; Z is a cytotoxic agent (optionally a camptothecin analog), optionally Z can be a molecule comprising an exatecan molecule or an SN-38 molecule; n is 1 and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the composition have m (the number of XZ sites) between 2 and 4, between 4 and 8 (optionally between 6 and 8), and optionally at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the composition have m of 4, 6, 7 or 8, or may be at least 4, 6, 7 or 8. The composition may be characterized as an immunoconjugate composition represented by the formula:

[0039] In formula I or II, the (XZ) moiety can optionally be characterized as having the structure of any of formulas III-XI or the structure of any of compounds 3-12.

[0040] In formula I or II, molecule X or spacer Y can be identified as optionally comprising a reactive group (R), or a residue that reacts with a reactive group (R) and a complementary reactive group (R') that is attached to an amino acid of an antigen binding protein (e.g., an antibody) or an amino acid of an antibody or antibody fragment.

[0041] In any embodiment herein, the exatecan molecule can be defined as being linked to the linker (X) via the amine at position 1 of the exatecan (when the exatecan molecule is part of the linker, the NH at position 1 is 2 is replaced by NH). In one embodiment, exatecan is attached to the linker (X), for example, to the carbonyl of the p-aminobenzyloxycarbonyl (PAB) self-eliminating spacer moiety of X. In any embodiment herein, the SN-38 molecule can be defined as being attached to the linker (X) via the OH at position 9 (when the SN-38 molecule shown in compound 2 becomes part of the linker, the OH at position 9 is replaced by O).

[0042] In one embodiment, the Nectin-4 binding agent conjugated to Exatecan has one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues) linked via a spacer (Y) to the following structure: [ka] The present invention can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide functionalized with a linker-exatecan molecule comprising:

[0043] In one embodiment, the humanized Nectin-4 antibody or antibody fragment conjugated to Exatecan has one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues) linked via a spacer (Y) to the following structure: [ka] The present invention can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide functionalized with a linker-exatecan comprising the linker

[0044] In one embodiment, the humanized Nectin-4 antibody or antibody fragment conjugated to Exatecan has one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues) linked via a spacer (Y) to the following structure: [ka] The present invention can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide functionalized with a linker-exatecan molecule comprising:

[0045] In one embodiment, the humanized Nectin-4 antibody or antibody fragment conjugated to Exatecan has one or more amino acid residues (e.g., cysteine ​​residues, glutamine residues) linked via a spacer (Y) to the following structure: [ka] The present invention can be characterized as comprising an antibody that specifically binds to a human Nectin-4 polypeptide functionalized with a linker-exatecan molecule comprising:

[0046] The spacer (Y) may be defined as being or comprising a substituted or unsubstituted alkyl or heteroalkyl chain, optionally having a chain length of 2 to 100 atoms, 2 to 40 atoms, optionally 2 to 30, 2 to 20, 4 to 40, 4 to 30 or 4 to 20 atoms, optionally one or more atoms may be other than carbon, e.g. oxygen, sulfur, nitrogen or other atoms, optionally any carbon of the chain may be substituted by alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide. For example, Y may comprise one or more ethylene oxide monomers, optionally Y may comprise a polyethylene oxide moiety, optionally Y has the structure -(CH 2 CH 2 O) x - (wherein x is 1 to 24, and may be 1 to 12, 1 to 8, or 1 to 6 as desired).

[0047] In some embodiments, the compound has the structure: [ka] A linker-exatecan molecule is provided having the following structure:

[0048] In certain embodiments, an immunoconjugate that binds to a human Nectin-4 polypeptide is provided, said immunoconjugate having the following structure: [ka] or [ka] or [ka] [In the formula, n is 1 to 15, 5 to 15, 5 to 23 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23); and The Ab is an antibody or antibody fragment thereof that specifically binds to a human Nectin-4 polypeptide, optionally wherein said antibody or antibody fragment comprises an amino acid sequence that is at least 60%, optionally at least 70%, optionally 80% or optionally 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 19, 47, 69 or 77; and optionally comprises an amino acid sequence that is at least 60%, optionally at least 70%, optionally 80% or optionally 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 65, 70 or 78; S can be defined as the atom of a cysteine ​​residue of an antibody (Ab). It is indicated by one of the following:

[0049] In one embodiment, an immunoconjugate that binds to a human Nectin-4 polypeptide is provided, the immunoconjugate having the following structure: [ka] wherein Ab is an antibody or antibody fragment thereof that specifically binds to a human nectin-4 polypeptide. In one embodiment, n is 15, and the immunoconjugate is characterized in that the DAR is 6 to 8, optionally the DAR is 6, and optionally the DAR is 8; and S is an atom of a cysteine ​​residue of the antibody (Ab). In one embodiment, Ab comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 65, and optionally the antibody or antibody fragment comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 77; and a light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0050] In one embodiment, a method of treatment is provided that exhibits improved efficacy and / or improved (lower) drug resistance compared to existing anti-Nectin-4 ADC therapies (e.g., anti-Nectin-4 antibodies or antibody fragments linked to auristatins; enfortumab vedotin). In one embodiment, a method of treating and / or preventing cancer and / or killing tumor cells in an individual in need of treatment is provided, the treatment being characterized by 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more administrations of a Nectin-4 binding agent conjugated to a camptothecin derivative (e.g., exatecan or SN-38 molecule) at a frequency of 1-2 times a month (e.g., once every 2 weeks, once every 3 weeks, once every 4 weeks).

[0051] In one aspect, the present invention provides a method of using the antibody or antibody fragment thereof (optionally conjugated to a cytotoxic moiety) in the treatment and / or prevention of a disease (e.g., cancer) in an individual in need thereof. In one aspect, the antibody or antibody fragment thereof (optionally conjugated to a cytotoxic moiety) is administered to an individual suffering from cancer in an amount and frequency sufficient to kill tumor cells expressing Nectin-4. In one embodiment, the individual has a tumor expressing Nectin-4, and optionally, the tumor is a HER-2 expressing tumor or a HER2 negative tumor (e.g., urothelial carcinoma, head and neck squamous cell carcinoma, or esophageal cancer). In one embodiment, the cancer or tumor is an advanced recurrent or metastatic cancer, and optionally, an advanced recurrent or metastatic urothelial carcinoma. In one embodiment, the cancer or tumor is triple negative breast cancer (TNBC).

[0052] In one embodiment, the therapeutic methods herein can be used on individuals with cancers that express Nectin-4, regardless of the expression level of Nectin-4 on tumor cells.

[0053] In one embodiment, the therapeutic methods herein may be advantageously used in individuals whose tumor cells express P-glycoprotein (Pgp).

[0054] In one aspect, the therapeutic methods of the present disclosure may be advantageously used in individuals who have undergone prior treatment with chemotherapeutic agents (e.g., chemotherapeutic agents delivered by P-glycoprotein (Pgp), platinum agents (e.g., oxaliplatin, cisplatin, carboplatin, nedaplatin, phenanthriplatin, picoplatin, satraplatin), taxanes (e.g., paclitaxel (Taxol®) and docetaxel (Taxotere®)).

[0055] The increased anti-tumor efficacy and broadened therapeutic window provided by the anti-Nectin-4 antibodies of the present disclosure (e.g., particularly when conjugated with camptothecin derivatives) offers the potential for improved therapeutic outcomes in individuals with tumors that are resistant to, unresponsive to, or progressed after treatment with a composition comprising an anti-HER2 agent (e.g., trastuzumab; ADC comprising trastuzumab) or a composition comprising another anti-Nectin-4 agent (e.g., ADC comprising enfortumab; enfortumab vedotin). This improved therapeutic window offers the potential for combination treatment with other agents, particularly chemotherapeutic agents and / or anti-HER2 agents.

[0056] In one embodiment, the therapeutic methods herein may be advantageously used in individuals whose tumors or cancers are resistant to, do not respond to, or have progressed following treatment with a composition comprising an anti-HER2 antibody (e.g., trastuzumab; an ADC comprising trastuzumab) or a composition comprising another anti-Nectin-4 agent (e.g., an ADC comprising enfortumab; enfortumab vedotin).

[0057] In one aspect of the embodiments herein, the individual has undergone prior treatment with radiation therapy, surgery, chemotherapy and / or biologic agents.

[0058] In one aspect of the embodiments herein, the individual has been prior treated with an anti-Nectin-4 agent, optionally an anti-Nectin-4 agent that includes a cytotoxic moiety other than a topoisomerase inhibitor, such as enfortumab vedotin.

[0059] In one aspect of the embodiments herein, provided herein is a method of treating cancer, killing tumor cells, and / or delivering a cytotoxic agent to a tumor in an individual in need of treatment, the method comprising administering to the individual, who has been pre-treated with a Nectin-4 binding agent conjugated to an auristatin, optionally enfortumab vedotin, a therapeutically effective amount of an immunoconjugate that binds to a human Nectin-4 polypeptide, the immunoconjugate having the following formula: [ka] [In the formula, n is 5 to 23, optionally n is 7 to 15, optionally n is 15, and Ab is an antibody or antibody fragment thereof that specifically binds to human Nectin-4 polypeptide. Optionally, the immunoconjugate is characterized by a DAR of 6 to 8, optionally a DAR of 6, or optionally a DAR of 8.

[0060] In one embodiment, the present invention provides a therapeutic method that can be used to achieve an anti-tumor effect in an individual at a lower or smaller fixed dose than that used for conventional anti-Nectin-4 ADCs, e.g., less than 3 mg / kg body weight, less than 1.25 mg / kg body weight, less than 1 mg / kg body weight, or less than 125 mg.

[0061] In one aspect, the therapeutic methods of the present disclosure may be used in individuals with pre-existing neuropathy, diabetes or hyperglycemia, heart failure, eye pathologies.

[0062] In one embodiment, the therapeutic method of the present disclosure may be used for individuals having a Nectin-4-expressing cancer characterized by low or moderate tumor cell expression levels of Nectin-4 polypeptide (e.g., expression of Nectin-4 polypeptide at the tumor cell membrane).

[0063] In any embodiment herein, the anti-Nectin-4 antibody conjugated to a cytotoxic agent is used in combination with another cytotoxic agent (e.g., a chemotherapeutic agent, a chemotherapeutic agent delivered by P-glycoprotein (Pgp, the product of the human MDR1 gene), a platinum agent, a taxane), and the other cytotoxic agent is administered separately from the anti-Nectin-4 antibody conjugated to a cytotoxic agent.

[0064] In another aspect of the present invention, pharmaceutical compositions and kits are provided that include an anti-Nectin-4 antibody or antibody fragment (optionally conjugated to a cytotoxic agent moiety) and one or more additional components (e.g., various carriers), which may generally be active or inactive ingredients that facilitate formulation, delivery, stabilization or other properties of the composition.

[0065] These aspects are described more fully in the description of the invention provided herein, and additional aspects, features and advantages will become apparent from the description of the invention provided herein. [Brief description of the drawings]

[0066] [Figure 1] Figure 1 shows the expression levels of HER2 and Nectin-4 polypeptides on the surface of SUM190 human breast cancer tumor cells as determined by FACS (MFI: mean fluorescence intensity). SUM190 tumor cells expressed HER2 at low to moderate levels (median fluorescence units 1777) and Nectin-4 at lower levels (median fluorescence units 991). [Diagram 2]Figure 2 shows the expression levels of HER2 and Nectin-4 polypeptides on the surface of SUM185 human breast cancer tumor cells as determined by FACS (MFI: mean fluorescence intensity). SUM185 cells expressed HER2 at medium to high levels (median fluorescence units 2880) and Nectin-4 at higher levels (median fluorescence units 4326). [Figure 3A] FIG. 3A shows the killing of human breast cancer cells by 5E7 antibody conjugated to camptothecin analog Dxd (via the GGFG-Dxd linker shown in Example 9) or exatecan [via the (PEG(8U)-Val-Ala-PAB-exatecan) linker shown in Example 9], all at the same drug-antibody ratio (DAR=8), along with an isotype control antibody (IC) compared to V-domain-bound Enhertu® (trastuzumab deruxtecan (anti-HER2)]. [Figure 3B] FIG. 3B shows the efficacy of "5E7-exatecan" [5E7 conjugated to the exatecan linker (PEG(8U)-Val-Ala-PAB-exatecan) shown in Example 9] in causing HER-2 and nectin-4 expressing SUM185, SUM190, MDA-MB-468 (TNBC) human tumor cells and MC38 (colon cancer) and B16F10 (melanoma) mouse tumor cell death, as well as the EC50 value at which 5E7-exatecan can cause cell death. [Figure 4] Figure 4 shows the efficacy of 5E7 antibody as a camptothecin ADC at a single dose of 3 mg / kg compared to the same dose of enfortumab, N41 and isotype control antibody (IC) in a mouse model of human breast cancer. Each antibody is conjugated with the camptothecin analog (Dxd) and tetrapeptide-containing linker (GGFG) at the same drug-antibody ratio (DAR=8). Only 5E7 was able to effectively control tumor growth. [Diagram 5]Figure 5 shows the killing of SUM185 human breast cancer cells expressing relatively high levels of Nectin-4 by the 5E7 antibody compared to Padcev® (enfortumab vedotin) (enfortumab conjugated with the auristatin compound MMAE at DAR=4) and Enhertu®. This setting is used as an anti-HER2 resistance model. [Figure 6A] FIG. 6A shows the binding of anti-Nectin-4 antibodies to rat and cynomolgus Nectin-4-expressing CHO cell lines, respectively, as determined by flow cytometry. [Figure 6B] FIG. 6B shows the binding of anti-Nectin-4 antibodies to rat and cynomolgus Nectin-4-expressing CHO cell lines, respectively, as determined by flow cytometry. [Figure 7] 7A and 7B show the structure of human nectin-4 protein with substitution shading; white regions corresponding to C1 domain residues that are substituted in variants 7 (12A) and 7bis (12B) are shown. [Figure 8] 8A and 8B show some views of the structure of the human Nectin-4 protein with substitution shading; white areas corresponding to residues that are substituted in variants 1, 2, 3, 4, 5, 6, 7, 8 and 9 are shown. [Figure 9] Figures 9A, 9B and 9C show the progression of tumor growth over time (days after tumor implantation) in mice treated with ADC. Figures 9A and 9B show treatment with various anti-nectin-4 antibodies (5E7 or 6A7) conjugated to the same linker-payload at the same DAR, iv at a dose of 3 mg / kg body weight. Figure 9C shows treatment with antibody 5E7 conjugated to a linker designed to release either Dxd or exatecan upon cleavage of the linker, iv at a dose of 10 mg / kg body weight. [Figure 10A]Figure 10A shows the luminescence (indicating cell viability) of cells treated with Padcev® (enfortumab vedotin), antibody 5E7 conjugated to Dxd, or 5E7 conjugated to exatecan. The cells are MC-38 cells that endogenously express MDR1 p-glycoprotein and have been engineered to express nectin-4. The ADC with exatecan as the payload was so potent that it reduced cell viability in this drug-resistant setting. [Figure 10B] FIG. 10B shows tumor growth (area under the curve) of MC38 cells treated with Padcev® (enfortumab vedotin), antibody 5E7 conjugated to Dxd, or 5E7 conjugated to exatecan at 150 nM ADC in the presence or absence of the Pgp inhibitor cyclosporine, normalized to control antibody, suggesting that the anti-tumor activity of Padcev® and antibody 5E7 bound to Dxd is negatively affected by Pgp. [Figure 11] Figure 11 shows the results of in vivo evaluation in which free toxin and ADC were administered to mice at a dose of 3 mg / kg. The results for IC (free toxin) are shown in Figure 11. [Figure 12] Figure 12 shows the results of an in vivo evaluation of free toxin and ADC administered to mice at a dose of 3 mg / kg. The results for the ADC are shown in Figure 12. [Figure 13] Figure 13 shows the in vivo antitumor effect of anti-Nectin-4 ADC in mice and the plasma concentration of ADC over time. The top left panel shows that PBS did not prevent tumor volume increase. The top right panel shows that a 1 mg / kg dose of ADC showed a strong antitumor effect. The bottom panel shows the plasma ADC concentration over time. [Figure 14] Figure 14 shows the in vivo plasma concentrations of ADC in rats: Figure 14A shows the results for the 3 mg / kg dose (top) and the 10 mg / kg dose (bottom), and Figure 14B shows the results for the 30 mg / kg dose. [Figure 15] Figure 15 shows the in vivo plasma concentrations of ADC in non-human primates: Figure 15A shows the results for the 3 mg / kg dose (top) and the 10 mg / kg dose (bottom), and Figure 15B shows the results for the 30 mg / kg dose.

[0067] Table 15 shows the nucleic acid and amino acid sequences disclosed in the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] definition As used herein, "a" or "an" may mean one or more. When used in the claims, when used with the word "comprising," the word "a" or "an" may mean one or more than one. As used herein, "another" may mean at least a second or more.

[0069] Where "comprising" is used, this may be replaced with "consisting essentially of" or "consisting of," as appropriate.

[0070] "Nectin-4" and "Nectin-4 polypeptide" refer to a protein or polypeptide encoded by the NECTIN4 gene (see Uniprot Accession No. Q96NY8) or by a cDNA prepared from such a gene. Any naturally occurring isoforms, alleles, or variants are encompassed by the term Nectin-4 polypeptide (e.g., a Nectin-4 polypeptide that is 95%, 98%, or 99% identical to SEQ ID NO: 1, or to a contiguous sequence of at least 100, 200, 300, 400, or 500 amino acid residues thereof). The sequence of 510 amino acid residues of authentic human Nectin-4 (isoform 1), including a 31 amino acid signal peptide, is shown below: MPLSLGAEMW GPEAWLLLLL LLASFTGRCP AGELETSDVV TVVLGQDAKL PCFYRGDSGE QVGQVAWARV DAGEGAQELA LLHSKYGLHV SPAYEGRVEQ PPPPRNPLDG SVLLRNAVQA DEGEYECRVS TFPAGSFQAR LRLRVLVPPL PSLNPGPALE EGQGLTLAAS CTAEGSPAPS VTWDTEVKGT TSSRSFKHSR SAAVTSEFHL VPSRSMNGQP LTCVVSHPGL LQDQRITHIL HVSFLAEASV RGLEDQNLWH IGREGAMLKC LSEGQPPPSY NWTRLDGPLP SGVRVDGDTL GFPPLTTEHS GIYVCHVSNE FSSRDSQVTV DVLDPQEDSG KQVDLVSASV VVVGVIAALL FCLLVVVVVL MSRYHRRKAQ QMTQKYEEEL TLTRENSIRR LHSHHTDPRS QPEESVGLRA EGHPDSLKDN SSCSVMSEEP EGRSYSTLTT VREIETQTEL LSPGSGRAEE EEDQDEGIKQ AMNHFVQENG TLRAKPTGNG IYINGRGHLV (SEQ ID NO:1)

[0071] SEQ ID NO:1 corresponds to the Uniprot KB identifier Q96NY8-1, the disclosure of which is incorporated herein by reference.

[0072] Certain aspects of the present disclosure provide anti-Nectin-4 antibodies that bind to human Nectin-4, or homologs thereof, including, but not limited to, mammalian Nectin-4 proteins and Nectin-4 orthologs from other species (e.g., non-human primates, cynomolgus monkeys).

[0073] The term "HER2" (also known as HER2 / neu and ErbB-2) stands for "human epidermal growth factor receptor 2." The term includes variants and isoforms of HER2.

[0074] The terms "immunoconjugate" and "antibody conjugate" are used interchangeably and refer to an antigen-binding agent, such as an antibody bound to a polypeptide, or an antibody conjugated to another molecule (e.g., a camptothecin derivative, an exatecan molecule, an SN-38 molecule). When the immunoconjugate comprises an antigen-binding agent conjugated to a therapeutic agent, such as a cytotoxic or anticancer agent, the immunoconjugate can also be referred to as an "antibody drug conjugate" or "ADC". Examples of cytotoxic agents include camptothecins, taxanes, anthracyclines, camptothecins, epothilones, mitomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamicins, duocarmycins, tubulysins, dolastatins and auristatins, enediynes, pyrrolobenzodiazepines, and ethylenimines.

[0075] As used herein, "treatment" and "treating" generally refer to obtaining a desired pharmacological and physiological effect. The effect may be preventive in that it prevents or partially prevents the disease, its symptoms or condition, and / or it may be therapeutic in that it partially or completely cures the disease, the condition, symptoms or adverse effects caused by the disease. The term "treatment" as used herein includes any treatment of disease in a mammal, particularly a human, and includes (a) preventing the onset of disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, e.g., preventive early asymptomatic intervention, (b) inhibiting the disease, e.g., preventing its onset, or relieving the disease, e.g., causing the regression of the disease and / or its symptoms or condition, e.g., amelioration of damage or treatment, in a subject who has been diagnosed with the disease, etc. Optionally, the treatment may cause (e.g., may be characterized as a method for causing) a reduction in tumor burden, a reduction in the size and / or number of lesions, a reduction or delay in the progression of cancer (e.g., an increase in progression-free survival), a delay or prevention of cancer metastasis, and / or an increase in survival. Optionally, the treatment may cause or bring about (e.g., may be characterized as a method for causing or bringing about) stable disease, partial response, or complete response in the subject according to standard criteria, which may be, for example, RECIST criteria.

[0076] Whenever "treatment of cancer" or the like is mentioned in connection with a Nectin-4 binding agent (e.g., an antibody or antibody fragment, an immunoconjugate), (a) a method for the treatment of cancer, comprising the step of administering (for at least one treatment) a Nectin-4-binding agent to an individual, a mammal, in particular a human, in need of such treatment, in a dose (therapeutically effective amount) that allows for the treatment of cancer, optionally in the doses (amounts) defined herein; (b) use of a Nectin-4 binding agent for the treatment of cancer; (c) a Nectin-4 binding agent for use in the treatment of cancer (particularly in humans); (d) use of a Nectin-4 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer; (e) a method of using a Nectin-4 binding agent for the manufacture of a pharmaceutical preparation for the treatment of cancer, the method comprising admixing the Nectin-4 binding agent with a pharma- ceutical acceptable carrier; (f) a pharmaceutical formulation comprising an effective dose of a Nectin-4 binding agent suitable for treating cancer; (g) Any combination of (a), (b), (c), (d), (e) and (f), depending on patentable subject matter in the country in which the application is filed. Includes:

[0077] The term "biopsy" as used herein is defined as the removal of tissue for the purpose of examination, such as to establish a diagnosis. Examples of types of biopsy include, for example, application of suction with a needle attached to a syringe; removal of tissue fragments with an instrument; removal with a suitable instrument via an endoscope; surgical removal of, for example, the entire lesion, etc.

[0078] The term "antibody" as used herein refers to polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are assigned to one of five major classes, namely IgA, IgD, IgE, IgG and IgM. Some of these are further divided into subclasses or isotypes, e.g., IgG1, IgG2, IgG3, IgG4, etc. An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDA) and one "heavy" chain (about 50-70 kDA). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The variable light chain (VLC) is a tetramer that is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDA) and one "heavy" chain (about 50-70 kDA). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. L ) and variable heavy chain (V HThe terms "antibody" and "antibody" refer to these light and heavy chains, respectively. The heavy chain constant domains corresponding to the different classes of immunoglobulins are termed "alpha", "delta", "epsilon", "gamma" and "mu", respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgG is the exemplary class of antibody used herein because it is the most common antibody in the physiological situation and is the most easily made in a laboratory setting. The antibody may be a monoclonal antibody. Particular examples of antibodies are humanized, chimeric, human or otherwise human-suitable antibodies. "Antibody" also includes any fragment or derivative of any of the antibodies described herein.

[0079] The amino acid residues of an antibody which are responsible for antigen-binding are sometimes referred to as hypervariable region. The hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" [e.g., residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al. 1991] and / or amino acid residues from the "hypervariable loops" [e.g., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917], or a similar system for determining essential amino acids responsible for antigen binding. Typically, the numbering of amino acid residues in this region is performed according to the method described by Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering in Kabat" and "according to Kabat" herein refer to this numbering system for the heavy chain variable domain or the light chain variable domain. When using the Kabat numbering system, the actual linear amino acid sequence of the peptide may contain fewer or additional amino acids corresponding to shortening or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of CDR H2 (residue 52A according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the "standard" Kabat numbered sequence with the antibody sequence in the homology region.

[0080] The term "specifically binds to" means that the antibody can bind to a binding partner, e.g., Nectin-4, preferably in a competitive binding assay, when assessed using either a recombinant form of the protein, an epitope therein, or a native protein present on the surface of an isolated target cell. Competitive binding assays and other methods for determining specific binding are well known in the art. For example, binding can be detected via physical methods such as radiolabeling, mass spectrometry, or direct or indirect fluorescent labeling, e.g., detected using cytofluorometry (e.g., FACScan). Binding that exceeds the amount seen with a control, i.e., a non-specific agent, indicates that the agent is binding to the target.

[0081] When an antibody is said to "compete with" a particular monoclonal antibody, this means that the antibody competes with the monoclonal antibody in a binding assay using either a recombinant molecule (e.g., Nectin-4) or a surface-expressed molecule (e.g., Nectin-4). For example, if a test antibody reduces the binding of an antibody having a heavy chain variable region of either SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 20 to a Nectin-4 polypeptide or a Nectin-4-expressing cell in a binding assay, the test antibody is said to "compete with" said antibody, respectively.

[0082] The term "internalization" is used interchangeably with "intracellular internalization" and refers to the molecular, biochemical and cellular events associated with the process of transferring a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for intracellular internalization of molecules are well known and may involve, among others, the internalization of extracellular molecules (e.g., hormones, antibodies and small organic molecules); membrane-associated molecules (e.g., cell surface receptors); and complexes of membrane-associated molecules bound to extracellular molecules (e.g., ligands bound to transmembrane receptors or antibodies bound to membrane-associated molecules). Thus, "inducing and / or increasing internalization" includes events that initiate intracellular internalization and / or events that increase the rate and / or extent of intracellular internalization.

[0083] The term "affinity" as used herein means the strength of binding of an antibody to an epitope. The affinity of an antibody is indicated by the dissociation constant Kd, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant K A is defined by 1 / Kd. Methods for determining the affinity of monoclonal antibodies can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entirety. One standard method well known in the art for determining the affinity of monoclonal antibodies is the use of surface plasmon resonance (SPR) screening (e.g., by analysis using a BIAcore® SPR analyzer).

[0084] In the present context, a "determinant" refers to an interacting or binding site on a polypeptide.

[0085] The term "epitope" refers to an antigenic determinant, the location or region of an antigen to which an antibody binds. A protein epitope may include amino acid residues directly involved in binding and those that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., within the "footprint" of an antibody. An epitope is the simplest form or smallest structural part in a complex antigen molecule that can bind, for example, to an antibody or receptor. An epitope may be a linear epitope or a conformational / structural epitope. The term "linear epitope" is defined as an epitope composed of amino acid residues that are contiguous in a linear sequence of amino acids (primary structure). The term "conformation or structural epitope" is defined as an epitope composed of amino acid residues that represent separate portions of a linear sequence of amino acids that are not all contiguous and thus are in close proximity to each other due to folding of the molecule (secondary, tertiary and / or quaternary structure). A conformational epitope is dependent on a three-dimensional structure. Thus, the term "conformation" is often used interchangeably with "structure".

[0086] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological polymer, or an extract prepared from biological material. The term "therapeutic agent" refers to an agent that has biological activity.

[0087] The terms "Fc domain," "Fc portion," and "Fc region" refer to a C-terminal fragment of an antibody heavy chain, e.g., derived from about amino acid (aa) 230 to about aa 450 of the human gamma (gamma) heavy chain or the corresponding sequences in other types of antibody heavy chains (e.g., alpha, delta, epsilon, and mu of human antibodies), or naturally occurring allotypes thereof. Unless otherwise specified, the generally accepted Kabat amino acid numbering for immunoglobulins is used throughout this disclosure (see Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0088] "Framework" or "FR" residues, as used herein, refer to the regions of an antibody variable domain excluding the regions defined as the CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3 and FR4) separated by the CDRs.

[0089] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany the material as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein that is the predominant species present in a preparation is substantially purified.

[0090] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0091] The term "recombinant," when used in reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein, or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise aberrantly expressed, under-expressed, or not expressed at all.

[0092] In the present context, the term an antibody that "binds" to a polypeptide or epitope denotes an antibody that binds to said determinant with specificity and / or affinity.

[0093] The term "identity" or "identical", when used in the context of the sequences of two or more polypeptides, refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between two or more strings of amino acid residues. "Identity" measures the percent of exact matches between the smaller of two or more sequences, with gap alignments (if any), as processed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIaM J. Applied Math. 48, 1073 (1988).

[0094] Methods for determining identity are designed to give the greatest match between the sequences tested. Methods for determining identity are described in publicly available computer programs. Computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0095] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that includes a fully saturated (no double or triple bonds) hydrocarbon group. An alkyl group can have, for example, 1 to 20 carbon atoms (whenever expressed herein, a numerical range such as "1 to 20" refers to each integer in the given range, e.g., "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkyl" where no numerical range is specified). The alkyl group of a compound is "C 1 -C 4 By way of example only, "C 1 -C 4"Alkyl" indicates that 1 to 4 carbon atoms are present in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, and hexyl. Alkyl groups may be substituted or unsubstituted.

[0096] As used herein, the term "heteroalkyl" refers to a straight- or branched-chain alkyl group that contains one or more heteroatoms, i.e., elements other than carbon (including, but not limited to, oxygen, sulfur, nitrogen, phosphorous), replacing one or more carbon atoms.

[0097] Whenever a group is described as being "substituted," the group is substituted with one or more of the specified substituents. When no substituents are indicated, it is meant that the indicated "substituted" group can be substituted with one or more groups individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamyl, thiocarbamyl, amido, sulfonamido, sulfonamido, carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, amino, mono- and di-substituted amino groups, and protected derivatives thereof.

[0098] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C 1 -C 3 The "alkoxyphenyl" may include one or more of the same or different alkoxy groups containing 1, 2 or 3 atoms.

[0099] Reference to a "compound" or "formula" having a particular number (e.g., "compound 1," "compound 2," "formula I," or "formula II") refers to all compounds derived from the particular number or formula, unless the context clearly dictates otherwise. For example, compound 1 includes reference to compounds 1A and 1B.

[0100] Production of antibodies and antibody fragments The hypervariable region, heavy and light chain CDR, heavy and light chain variable region, and proteins comprising them, such as full-length antibodies or antibody fragments, multispecific antigen-binding proteins, chimeric antigen receptors, can bind to human Nectin-4 expressed on the surface of cells (e.g., tumor cells). In one embodiment, the Nectin-4 binding by the antibody is mediated by one antigen-binding domain or two identical antigen-binding domains, and optionally, each antigen-binding domain may comprise a pair of VH and VL domains. In some embodiments, what is provided is one VH or VL variable domain; the domains can be incorporated into a polypeptide that is, for example, expressed and produced separately, and then combined to form the Nectin-4 binding domain.

[0101] When used as either immunoconjugates or unconjugated ("naked") antibodies (e.g., optionally in combination with a separately administered cytotoxic agent) in therapies to eliminate Nectin-4-expressing tumor cells, Nectin-4-binding antibodies (e.g., full-length antibodies, antibody fragments) or proteins, conjugates or complexes comprising such antibodies may advantageously inhibit cell-cell interactions mediated by Nectin-4, as determined, for example, by assessing cell cluster formation of Nectin-4-expressing cells (e.g., by assessing tumor spheroid formation or growth in a three-dimensional cell culture system) and / or by assessing anchorage-independent growth of Nectin-4-expressing cells. When used in a therapy for eliminating Nectin-4-expressing tumor cells, either as an immunoconjugate with a cytotoxic agent or as an unconjugated ("naked") antibody combined with a separately administered cytotoxic agent, the Nectin-4-binding antibody may advantageously be able to sensitize the tumor to the cytotoxic agent, e.g., the antibody may enhance the ability of the cytotoxic agent to inhibit the proliferation or cause the death of tumor cells, e.g., to inhibit the proliferation or cause the death of tumor cells in clusters (e.g., spheroids), or to inhibit the formation or growth of tumor cell clusters (e.g., spheroids). When used as an immunoconjugate in a therapy for eliminating Nectin-4-expressing tumor cells, the anti-Nectin-4 immunoconjugate, when conjugated to a cytotoxic molecule disclosed herein, may advantageously be able to cause the death of Nectin-4-expressing tumor cells.

[0102] Tumor cells that express Pgp may be less sensitive to certain cytotoxic agents. Antibodies and cytotoxic agents (whether or not the cytotoxic agent is conjugated to the antibody) can, if desired, be tested using cells (e.g., tumor cells) that express Pgp.

[0103] Tumor spheroids are generally less sensitive to chemotherapy, partly due to the protection provided by their structure, but also due to their slower growth rate, so they can be useful for evaluating the antitumor effect of antibodies or immunoconjugates.Antibody or immunoconjugates can be tested for their ability to inhibit spheroid formation in a concentration-dependent manner or to prevent cancer cells from forming spheroids.Antibody or immunoconjugates can be tested for their ability to change spheroid formation in various cancer cell lines, for example, using real-time digital photography.

[0104] The antibody or immunoconjugate can be characterized, for example, as being capable of maintaining (or improving) the maintenance of cancer cells as single cells, which can render the cells more sensitive to cytotoxic agents (e.g., chemotherapy). Promotion of the maintenance of cancer cells as single cells, which improves sensitivity to chemotherapy, can be tested in vitro by adding different chemotherapeutic agents (e.g., doxorubicin, cisplatin, paclitaxel, camptothecin or camptothecin analogs) at different concentrations, either separately or in combination, in the presence of the antibody or immunoconjugate, and optionally further compared to an antibody or immunoconjugate that binds to the Ig-like V domain or the Ig-like C2 type 1 or 2 domain alone. The maintenance of cancer cells as single cells, which improves sensitivity to chemotherapy, can also be tested in vitro, for example, by adding different immunoconjugates targeting different domains of Nectin-4 with different toxins, where the IC50 of chemotherapy or ADCs whose antibodies bind to the VC1 domain of Nectin-4 is lowered, or the dose of ADCs whose antibodies bind to the VC1 domain of Nectin-4 is lowered to control tumor growth. Sensitivity to chemotherapy can be evaluated as cell viability, cell proliferation or cytotoxicity, and can be monitored using various readouts, such as CTG by luminescence, Incucyte, or confluence using caspase 3 / 7 or Annexin V. In one embodiment, the anti-Nectin-4 antibody or antibody fragment binds to wild-type human Nectin-4 polypeptide, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 1, as well as a modified human Nectin-4 polypeptide having the amino acid sequence of SEQ ID NO: 10 (a Nectin-4 protein containing Ig-like C2 type 1 and 2 domains but lacking the Ig-like V type domain).Optionally, the antibody may retain partial binding to a polypeptide lacking an Ig-like V-type domain, and optionally, the antibody may retain binding to a Nectin-4 protein lacking an Ig-like V-type domain at a reduced level compared to binding to a wild-type Nectin-4 protein, for example, a reduced level may be between 5-50% of binding to wild-type Nectin-4, optionally 5-30%, optionally 5-25%, optionally 5-15%. Furthermore, the anti-Nectin-4 antibody may not bind to a Nectin-4 polypeptide lacking both an Ig-like V-type domain and an Ig-like C2-type 1 domain, for example, a polypeptide having the amino acid sequence of SEQ ID NO: 11. Optionally, binding may be assessed by flow cytometry using cells engineered to express the Nectin-4 polypeptide, and fluorescence intensity levels (e.g., MFI) determined.

[0105] In one embodiment, the anti-Nectin-4 antibody or antibody fragment does not bind to any of human Nectin 1 protein, human Nectin 2 protein, human Nectin 3 protein and human PVR protein. Each Nectin or PVR protein can be defined as being expressed on the surface of a cell.

[0106] In one embodiment, the anti-Nectin-4 antibody or antibody fragment is A Nectin-4 polypeptide having the amino acid sequence of SEQ ID NO:1; and 10 (optionally at a lower level compared to binding to SEQ ID NO: 1, optionally at a level between 5-50%), and said anti-Nectin-4 antibody or antibody fragment binds to a Nectin-4 polypeptide having the amino acid sequence of SEQ ID NO: 10 (optionally at a lower level compared to binding to SEQ ID NO: 1, optionally at a level between 5-50%), A polypeptide having the amino acid sequence of SEQ ID NO:13; A polypeptide having the amino acid sequence of SEQ ID NO: 16; A polypeptide having the amino acid sequence of SEQ ID NO: 17, and A polypeptide having the amino acid sequence of SEQ ID NO: 18, does not bind to any of the

[0107] Optionally, the anti-Nectin-4 antibody or antibody fragment may not bind to a polypeptide having the amino acid sequence of SEQ ID NO:11.

[0108] Optionally, the anti-Nectin-4 antibody or antibody fragment may further bind to a rat Nectin-4 polypeptide (eg, the amino acid sequence of SEQ ID NO:13).

[0109] In one embodiment, the anti-Nectin-4 antibody or antibody fragment binds to the VC1 cross-linking domain, epitope or determinant of Nectin-4.

[0110] In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to human nectin-4 at one, two, three, four or five or more residues selected from the group consisting of A72, G73, S195, K197, S199, L150, S152, Q234 and I236 (according to SEQ ID NO: 1). In one embodiment, the anti-nectin-4 antigen binding protein or antibody binds to human nectin-4 at one, two or three residues selected from the group consisting of K197, S199 and Q234, optionally residues K197 and / or S199. In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to residue K197 of human nectin-4. In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to residue S199 of human nectin-4. In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to residue Q234 of human nectin-4. Binding can be determined, for example, by assessing whether the antibody or antibody fragment exhibits reduced or lost binding to a mutant Nectin-4 polypeptide (e.g., expressed on the surface of a cell) in which the residues are replaced by different residues.

[0111] In one embodiment, the anti-Nectin-4 antibody or antibody fragment can bind to both the Ig-like V-type domain and the Ig-like C2-type 1 domain, e.g., to a determinant or epitope on the Ig-like V-type domain and a determinant or epitope on the Ig-like C2-type 1 domain.

[0112] In one embodiment, the anti-Nectin-4 antibody or antibody fragment retains at least partial binding to a modified Nectin-4 polypeptide that includes an Ig-like C2-type 1 domain but lacks an Ig-like V-type domain, e.g., a Nectin-4 polypeptide lacking all or part of the amino acid sequence of SEQ ID NO: 3. Partial binding can be characterized as retaining binding, but at a lower level (e.g., as assessed by flow cytometry) compared to binding to a wild-type human Nectin-4 polypeptide having the amino acid sequence of residues of SEQ ID NO: 1. A Nectin-4 polypeptide can be defined as being expressed on the surface of a cell (e.g., a host cell engineered to express the polypeptide).

[0113] Antibodies can be produced by various techniques known in the art. Typically, antibodies are produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a Nectin-4 polypeptide, preferably a human Nectin-4 polypeptide. The Nectin-4 polypeptide may comprise the full-length sequence of the human Nectin-4 polypeptide, or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of the polypeptide comprising an epitope exposed on the surface of a cell expressing the Nectin-4 polypeptide, e.g., an epitope recognized by the 5E7 antibody. The Nectin-4 polypeptide may be defined as comprising or consisting of the VC1 cross-linking domain, or a fragment or subsequence thereof. Such a fragment typically contains at least about 7 consecutive amino acids of the mature polypeptide sequence, even more preferably at least about 10 consecutive amino acids thereof. The fragment typically is essentially derived from the extracellular domain of the receptor. In one embodiment, the immunogen comprises a wild-type human Nectin-4 polypeptide in a lipid membrane, typically on the surface of a cell. In a specific embodiment, the immunogen comprises an intact cell, which may be treated or lysed, in particular an intact human cell. In another preferred embodiment, the polypeptide is a recombinant Nectin-4 polypeptide. In a specific embodiment, the immunogen comprises intact Nectin-4-expressing cells.

[0114] Immunizing a non-human mammal with an antigen can be carried out by any method known in the art for stimulating the production of antibodies in mice (see, e.g., E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988), the entire disclosure of which is incorporated herein by reference).

[0115] Antibodies can also be produced by selection of combinatorial immunoglobulin libraries, for example as disclosed in Ward et al. Nature, 341 (1989) p. 544, the disclosure of which is incorporated herein by reference in its entirety.

[0116] Provided herein is a modified human acceptor framework sequence into which antibody CDRs can be incorporated so that the resulting anti-Nectin-4 variable region has high binding and internalization potency in Nectin-4 expressing cells.The antibody has the advantage of low or reduced immunogenicity in humans, e.g., low ability or likelihood of eliciting undesirable anti-Nectin-4 antibody-directed immune responses when administered to humans.Examples of such antibodies of the present disclosure include antibodies comprising a combination of H0, H1, H2, H3, H4, H5, H6, H7, H8, H9 or H10 VH domain and L0, L1, L2 or L3 VL domain. Examples of antibodies of the disclosure include antibodies that comprise the set of VH and VL domains of any one of antibodies H0+L0, H1+L0, H2+L0, H3+L0, H3+L1, H3+L2, H3+L3, H4+L1, H4+L2, H4+L3, H5+L1, H5+L2, H5+L3, H6+L1, H6+L2, H6+L3, H7+L1, H7+L2, H7+L3, H8+L1, H9+L1, and H10+L1.

[0117] In one embodiment, the antibody or antibody fragment that binds to human Nectin-4 polypeptide comprises VH and VL frameworks (e.g., FR1, FR2, FR3, FR4) of human origin (e.g., derived from a human amino acid sequence). In one embodiment, the antibody or antibody fragment comprises: HCDR1 (heavy chain CDR1) comprising the amino acid sequence SYWMH set forth in SEQ ID NO: 21; HCDR2 (heavy chain CDR2) comprising the amino acid sequence EIDPSDSYTNYNQKFKG set forth in SEQ ID NO: 22; HCDR3 (heavy chain CDR3) comprising the amino acid sequence GYGNYGDY set forth in SEQ ID NO: 23; LCDR1 comprising the amino acid sequence RSSKSLLHSNGITYLY set forth in SEQ ID NO: 24; LCDR2 comprising the amino acid sequence QMSNLAS set forth in SEQ ID NO: 25; LCDR3 comprising the amino acid sequence AQNLELPWT set forth in SEQ ID NO: 26.

[0118] In one embodiment, the antibody is a human IGHV subgroup IGHV1-46 (optionally IGHJ4, preferably IGHJ4 * 01), and optionally IGHV1-46 comprises a heavy chain framework derived from IGHV1-46. * In one embodiment, the antibody comprises a light chain framework from the human subgroup IGKV2-28 (optionally together with IGKJ, preferably IGKJ4*01), optionally comprising IGKV2-28 * The number is 01.

[0119] The antibodies can further include one, two, three, four, five, or more amino acid substitutions throughout the human heavy and / or light chain frameworks to, e.g., enhance the affinity, stability, or other properties of the antibody.

[0120] Optionally, in any embodiment, the antibody may be defined as being an antibody other than a murine parent antibody, for example a murine parent antibody having the VH and VL of SEQ ID NOs: 19 and 20, respectively.

[0121] Optionally, the human framework comprises one or more mutations, e.g., backmutations to introduce residues present at particular positions in a non-human mammal (e.g., mouse). Optionally, the human framework of the heavy chain variable region of the antibody comprises 1, 2, 3, 4, 5, 6, 7 or 8 mutations at positions selected from Kabat positions 28, 38, 40, 48, 69, 71, 73 or 78.

[0122] In one aspect of any embodiment herein, the threonine residue at Kabat heavy chain position 28 is substituted with a threonine residue.

[0123] In one aspect of any embodiment herein, the arginine residue at Kabat heavy chain position 38 is substituted with a lysine residue.

[0124] In one aspect of all embodiments herein, an alanine residue at Kabat heavy chain position 40 is substituted with an arginine residue.

[0125] In one aspect of all embodiments herein, the methionine residue at Kabat heavy chain position 48 is substituted with an isoleucine residue.

[0126] In one aspect of any embodiment herein, a methionine residue at Kabat heavy chain position 69 is substituted with a leucine residue.

[0127] In one aspect of any embodiment herein, the arginine residue at Kabat heavy chain position 71 is substituted with a leucine residue.

[0128] In one aspect of any embodiment herein, the threonine residue at Kabat heavy chain position 73 is substituted with a lysine residue.

[0129] In one aspect of any embodiment herein, a valine residue at Kabat heavy chain position 78 is substituted with a threonine residue.

[0130] Optionally, the human framework of the light chain variable region of the antibody comprises 1, 2, 3 or 4 mutations at positions selected from Kabat positions 2, 8, 11 or 64.

[0131] In one aspect of any embodiment herein, the isoleucine residue at light chain Kabat position 2 is replaced by a valine residue.

[0132] In one aspect of any embodiment herein, the proline residue at Kabat position 8 of the light chain is substituted with an alanine residue.

[0133] In one aspect of any embodiment herein, the leucine residue at light chain Kabat position 11 is substituted with an asparagine residue.

[0134] In one aspect of any embodiment herein, the glycine residue at light chain Kabat position 64 is substituted with a serine residue.

[0135] Positions within the VH and VL domains herein are described using the Kabat numbering system (Kabat et al. (1991) Sequences of Protein of Immunological Interest, 5th ed., United States Public Health Service, National Institute of Health, Bethesda, MD).

[0136] In one embodiment, the isolated anti-Nectin-4 humanized monoclonal antibody or fragment thereof comprises a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of any of SEQ ID NOs: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57, and a light chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of any of SEQ ID NOs: 59, 61, 63 or 65.

[0137] In one embodiment, the isolated anti-Nectin-4 humanized monoclonal antibody or fragment thereof comprises a heavy chain variable region having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to the amino acid sequence of SEQ ID NO: 47 (H5), and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 65 (L3).

[0138] In one embodiment, an anti-Nectin-4 antibody is provided, comprising a heavy chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a heavy chain comprising the amino acid sequence of SEQ ID NO: 77; and a light chain having at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) to a light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0139] DNA encoding the antibody can be prepared and inserted into a suitable expression vector for transfection into a suitable host that is used for recombinant production of the antibody or variants thereof, such as humanized forms of the monoclonal antibody, active fragments of the antibody, chimeric antibodies containing the antigen recognition portion of the antibody, or forms containing a detectable moiety.

[0140] DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the mouse antibody). In one aspect, a nucleic acid is provided that encodes the heavy or light chain of the anti-Nectin-4 antibody or antibody fragment of any embodiment herein. Once isolated, the DNA can be incorporated into an expression vector, which can then be transfected into host cells, such as E. coli cells, Simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, to synthesize the monoclonal antibody in the recombinant host cell. Such DNA sequences can be modified for various purposes, such as, for example, humanizing the antibody, producing fragments or derivatives, modifying the antigen-binding site to optimize the binding specificity of the antibody, and the like. In one embodiment, an isolated nucleic acid sequence encoding the light and / or heavy chain of the antibody, as well as a recombinant host cell comprising said nucleic acid (e.g., in its genome) is provided. Recombinant expression of DNA encoding the antibody in bacteria is well known in the art (e.g., Skerra et al., Curr. Opinion in Immunol., 5, 256 (1993); and Pluckthun, Immunol. 130, p. 151 (1992).

[0141] Fragments and derivatives of antibodies (as encompassed by the term antibody or antibody as used in this application; unless otherwise stated or clearly contradicted by the context) can be produced by techniques known in the art. A "fragment" includes a portion of an intact antibody, generally an antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; antibody fragments that are polypeptides having a primary structure consisting of one uninterrupted sequence of consecutive amino acid residues (referred to herein as single-chain antibody fragments or single-chain polypeptides); and multispecific (e.g., bispecific) antibodies formed from antibody fragments. For example, a multispecific protein can include the hypervariable regions (e.g., VH and VL) of any of the antibodies of the embodiments herein and the hypervariable regions (e.g., VH and VL) of a different antibody (e.g., an antibody that binds to an antigen of interest other than Nectin-4).

[0142] Typically, the anti-Nectin-4 antibodies provided herein are administered in a concentration of about 10 4 ~about 10 11 M -1 (For example, about 10 7 ~about 10 10 M -1 ) for Nectin-4 polypeptide (e.g., the Nectin-4 polypeptide produced in the Examples herein). For example, in certain embodiments, the present disclosure provides a method for the production of Nectin-4 polypeptides having an affinity of 1×10 for Nectin-4, as determined, for example, by surface plasmon resonance (SPR) screening. -7 The average dissociation constant (K D ) (e.g., by analysis using a Biacore® SPR analyzer). In a more specific exemplary embodiment, the present disclosure provides an anti-Nectin-4 antibody having a specific binding affinity of about 1×10 -7 M ~ approx. 1×10 -10 K of M D The present invention provides an anti-Nectin-4 antibody having the formula:

[0143] The antibodies may have an average K of, for example, about 200, 150, 100, 80, 70, 60, 40, 30, or 25 nanomolar or less. D (i.e., better affinity). D can be determined, for example, by immobilizing recombinantly produced human Nectin-4 protein on a chip surface and then applying the antibody to be tested in solution. In one embodiment, the method further comprises a step (d) of selecting an antibody from (b) that can compete with the control antibody for binding to Nectin-4.

[0144] Anti-Nectin-4 antibodies may or may not have substantial binding to human Fcγ receptors. In one embodiment, anti-Nectin-4 antibodies comprise an Fc domain of human IgG1 isotype that retains binding to human Fcγ receptors. In one embodiment, anti-Nectin-4 antibodies comprise an Fc domain of human IgG1 isotype that has been modified (e.g., by introducing additional modifications) to increase binding to human Fcγ receptors (e.g., CD16A). In another embodiment, anti-Nectin-4 antibodies can be produced such that they do not substantially bind to any one or more of human Fcγ receptors, e.g., CD16A, CD16B, CD32A, CD32B, and / or CD64. Such antibodies can comprise various heavy chain constant regions that are known to be unable to bind, or to bind weakly to, Fcγ receptors. Alternatively, to avoid Fc receptor binding, F(ab') 2Antibody fragments that do not contain constant regions (or portions thereof), such as fragments, can also be used. Fc receptor binding can be evaluated according to methods known in the art, for example, by testing the binding of antibodies to Fc receptor proteins in a BIACORE assay. Also, in general, any antibody IgG isotype whose Fc portion has been modified (e.g., modified by introducing 1, 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, for example, WO 03 / 101485; the disclosure of which is incorporated herein by reference) can be used. Such assays to evaluate Fcγ receptor binding are well known in the art and are described, for example, in WO 03 / 101485. An example of a silent Fc lgG1 antibody is the LALA mutant, which contains the L234A and L235A mutations in the lgG1 Fc amino acid sequence. Another example of an Fc silent mutation is a mutation at residues D265 or D265 and P329, such as the DAPA (D265A, P329A) mutation used in IgG1 antibodies (US 6,737,056). Another silent IgG1 antibody contains a mutation at residue N297 (e.g., N297A, N297S mutation), resulting in an aglycosylated / non-glycosylated antibody.Other silent mutations include substitutions of residues L234 and G237 (L234A / G237A); substitutions of residues S228, L235, and R409 (S228P / L235E / R409K,T,M,L); substitutions of residues H268, V309, A330, and A331 (H268Q / V309L / A330S / A331S); substitutions of residues C220, C226, C229, and P238 (C220S / C226S / C229S / P23 8S); substitutions of residues C226, C229, E233, L234 and L235 (C226S / C229S / E233P / L234V / L235A; substitutions of residues K322, L235 and L235 (K322A / L234A / L235A); substitutions of residues L234, L235 and P331 (L234F / L235E / P331S); substitutions of residues 234, 235 and 297; substitutions of residues E318, K320 and K322 (L235E substitutions of residues (V234A, G237A, P238S); substitutions of residues 243 and 264; substitutions of residues 297 and 299; substitutions such that residues 233, 234, 235, 237 and 238 as defined by the EU numbering system comprise a sequence selected from PAAAP, PAAAS and SAAAS (see WO2011 / 066501). The antibody has a substitution in the heavy chain constant region at any one, two, three, four, five, or more of residues selected from the group consisting of 220, 226, 229, 233, 234, 235, 236, 237, 238, 243, 264, 268, 297, 298, 299, 309, 310, 318, 320, 322, 327, 330, 331, and 409 (numbering of residues in the heavy chain constant region is according to EU numbering according to Kabat).

[0145] In the abbreviations used herein, the format is as follows: wild type residue; position in polypeptide; mutant (mutant) residue; where said residue positions are according to EU numbering according to Kabat.

[0146] In a specific embodiment, an antibody is provided that binds essentially the same epitope or determinant as monoclonal antibody 5E7, which antibody may optionally include the hypervariable region of antibody 5E7. In any embodiment herein, antibody 5E7 may be characterized by its amino acid sequence and / or the nucleic acid sequence encoding it. In one embodiment, the monoclonal antibody is a Fab or F(ab') of 5E7. 2 The antibody or antibody fragment further comprises a portion of the heavy chain variable region of 5E7. Also provided is an antibody or antibody fragment comprising the heavy chain variable region of 5E7. In one embodiment, the antibody or antibody fragment comprises the three CDRs of the heavy chain variable region of 5E7. Also provided is an antibody or antibody fragment further comprising the light chain variable region of 5E7 or one, two or three CDRs of the light chain variable region of 5E7. HCDR1, 2, 3 and LCDR1, 2, 3 sequences can be optionally all (or each independently) specified as being in the Kabat numbering system, the Chotia numbering system, the IMGT numbering system, or any other suitable numbering system. Optionally, any one or more of the light or heavy chain CDRs can contain one, two, three, four or five or more amino acid modifications (e.g., substitutions, insertions or deletions).

[0147] In one embodiment, the anti-Nectin-4 antibody or antibody fragment has a VH domain having at least about 60%, 70% or 80% sequence identity, optionally at least about 85%, 90%, 95%, 97%, 98% or 99% identity to the VH domain of SEQ ID NO: 19. H In another embodiment, the anti-Nectin-4 antibody comprises a VL domain having at least about 60%, 70% or 80% sequence identity, optionally at least about 85%, 90%, 95%, 97%, 98% or 99% identity to the VL domain of SEQ ID NO:20. L Includes the domain.

[0148] The VH and VL comprise (e.g., are modified to incorporate) human acceptor frameworks. In one embodiment, an anti-Nectin-4 antibody of the present disclosure comprises a VH CDR1, CDR2 and / or CDR3 (e.g., according to Kabat numbering) of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19. In one embodiment, an anti-Nectin-4 antibody comprises a VL CDR1, CDR2 and / or CDR3 (e.g., according to Kabat numbering) of a light chain variable region having the amino acid sequence of SEQ ID NO: 20. In one embodiment, an anti-Nectin-4 antibody of the present disclosure comprises a VH comprising Kabat CDR1, CDR2 and / or CDR3 of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19, and a VL comprising Kabat CDR1, CDR2 and / or CDR3 of a light chain variable region having the amino acid sequence of SEQ ID NO: 20. 5E7 VH: QVQLQQPGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTLDKSSSTTYMQLSSLTSEDSAVYYCVRGYGNYGDYWGQGTTLTVSS (SEQ ID NO: 19) 5E7 VL: DVVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSSSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTFGGGTKLEIK (SEQ ID NO: 20) [ka]

[0149] In one embodiment, the anti-Nectin-4 antibody may comprise, for example, a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 37, 39, 41, 43, 45, 47, 49, 51, 53, 55 or 57, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 59, 61, 63 or 65. Anti-Nectin-4 antibodies include, for example, the following: HCDR1 comprising the amino acid sequence: SYWMH (SEQ ID NO: 21) or a sequence of at least four consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with different amino acids); HCDR2 comprising the amino acid sequence: EIDPSDSYTNYNQKFKG (SEQ ID NO: 22) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with different amino acids); HCDR3 comprising the amino acid sequence: GYGNYGDY (SEQ ID NO: 23) or a sequence of at least 4, 5 or 6 consecutive amino acids thereof (optionally, one or more of these amino acids may be substituted with different amino acids). DR3; LCDR1 comprising the amino acid sequence: RSSKSLLHSNGITYLY (SEQ ID NO: 24) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof (optionally, one or more of these amino acids may be replaced with different amino acids); LCDR2 region comprising the amino acid sequence: QMSNLAS (SEQ ID NO: 25) or a sequence of at least 4, 5 or 6 consecutive amino acids thereof (optionally, one or more of these amino acids may be replaced with different amino acids); and / or LCDR3 region comprising the amino acid sequence: AQNLELPWT (SEQ ID NO: 26) or a sequence of at least 4, 5, 6, 7 or 8 consecutive amino acids thereof (optionally, one or more of these amino acids may be replaced with different amino acids). The CDR positions may be according to Kabat numbering.

[0150] Advantageously, the antibody of the present disclosure can be used in a method for producing an antibody conjugate. In one embodiment, the method for producing an antibody conjugate comprises conjugating a cytotoxic agent (Z) to an anti-Nectin-4 antibody or antibody fragment of the present disclosure. In one embodiment, the cytotoxic agent (Z) can be defined as being conjugated to the antibody or fragment via a linker (X). X is a linker that connects the antibody or fragment (Ab) and the cytotoxic agent (Z), e.g., when conjugated, X is a residue of the linker following covalent attachment to one or both of Ab and Z.

[0151] In an embodiment herein, a method for producing an antibody-drug conjugate comprises contacting and / or reacting an anti-Nectin-4 antibody or antibody fragment (Ab) of the present disclosure with a cytotoxic agent (Z). Contacting can be performed under suitable conditions such that an antibody-drug conjugate of an embodiment of the present disclosure is formed or obtained. Z may be included in, for example, a compound comprising a cytotoxic agent (Z) and a linker (X) or a portion of a linker (X), and thus, the step comprises contacting an anti-Nectin-4 antibody or antibody fragment of the present disclosure with a compound comprising a cytotoxic agent (Z) and a linker (X) or a portion of a linker (X). The method can optionally provide for isolating or recovering the formed antibody-drug conjugate, and optionally further processing the composition for use as a pharmaceutical, optionally formulating the antibody (e.g., with a pharmaceutical excipient) for administration to a human subject.

[0152] Optionally, the method of making an ADC may include conjugating the antibody or antibody fragment to 2, 3, 4, 5, 6, 7, or 8 molecules of a cytotoxic agent. Optionally, the resulting composition may be characterized by a DAR between 2 and 4, between 4 and 6, between 6 and 8. In one embodiment, the method includes conjugating the antibody to 4 molecules of a cytotoxic agent. In one embodiment, the method includes conjugating the antibody to 8 molecules of a cytotoxic agent. Optionally, the method may further include evaluating the DAR, and if the DAR corresponds to a predetermined specification (e.g., a DAR or DAR range disclosed herein, i.e., a DAR of about 2, 4, 6, or 8, etc.), further processing the composition for use as a pharmaceutical, and optionally formulating the antibody (e.g., with a pharmaceutical excipient) for administration to a human subject.

[0153] In certain embodiments, the linker (X)-(Z) elements are prepared and isolated prior to forming an antibody drug conjugate by contacting (and reacting) a compound comprising (X) and (Z) with (Ab).

[0154] In one embodiment, the method comprises: (a) contacting and / or reacting a linker (X) or a portion of a linker (X) with (Ab) to form an Ab-X conjugate; and (b) contacting and / or reacting Ab-X of step (a) with a cytotoxic agent (Z), or a compound comprising a second portion of the linker (X) and (Z), thereby forming an antibody-drug conjugate.

[0155] X may represent a molecule comprising a cleavable moiety, for example, under physiological conditions, optionally under intracellular conditions. In one embodiment, X represents a molecule comprising (i) a spacer (Y); (ii) a cleavable moiety; and (iii) an optional self-eliminating or non-self-eliminating spacer system (Y'). The cleavable moiety may be, for example, an oligopeptide (e.g., a di-, tri-, tetra- or pentapeptide). The spacer Y may be located between Ab and the cleavable moiety, and the spacer system (Y') may be located between the cleavable moiety and Z.

[0156] In certain embodiments, the linker X or spacer Y can be defined as comprising a reactive group (R) that can react (e.g., under appropriate conditions, optionally after deprotection) with an amino acid of the antibody or a complementary reactive group (R') attached to an amino acid of the antibody. Optionally, R can be a group reactive with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody. For example, R can be the following structure, which reacts with a thiol group (also referred to as a sulfhydryl group) on an antibody: [ka] It may be a maleimido-N-yl group having the formula:

[0157] In certain embodiments, the linker X or spacer Y can be defined as optionally comprising the residue of a reactive group R for reaction with an amino acid of the antibody or a complementary reactive group (R') attached to an amino acid of the antibody. R can also be the residue of a group reactive with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody for reaction with said free amino, hydroxyl, sulfhydryl or carboxyl group.

[0158] In any embodiment, prior to the step of contacting and / or reacting the anti-Nectin-4 antibody or antibody fragment with a compound (e.g., a linker and / or a cytotoxic agent), the method includes a step of preparing, selecting or providing an anti-Nectin-4 antibody or antibody fragment. In one embodiment, this step includes preparing, selecting or providing an anti-Nectin-4 antibody or antibody fragment, and determining or testing whether the antibody or antibody fragment has the characteristics of an anti-Nectin-4 antibody or antibody fragment of the present disclosure.

[0159] For example, an anti-nectin-4 antibody or antibody fragment can be tested for its ability to bind to the VC1 cross-linking domain of nectin-4. An antibody or antibody fragment determined to bind to the VC1 cross-linking domain of nectin-4 is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a cytotoxic agent (Z)). For example, an anti-nectin-4 antibody or antibody fragment can be tested for its ability to bind to a mutant nectin-4 polypeptide (e.g., a mutant nectin-4 polypeptide containing a substitution at residue K197 and / or S199). An antibody or antibody fragment determined to have reduced or lost binding to a mutant nectin-4 polypeptide (e.g., compared to binding to a wild-type nectin-4 polypeptide) is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a cytotoxic agent (Z)).

[0160] In another example, anti-Nectin-4 antibodies or antibody fragments can be tested for their ability to reduce cell-cell adhesion between Nectin-4-expressing tumor cells (e.g., adherent tumor cells) and / or for their ability to reduce the growth of Nectin-4-expressing tumor cells (e.g., in a tumor spheroid formation assay in a three-dimensional cell culture). Antibodies or antibody fragments assessed for their ability to reduce cell-cell adhesion between Nectin-4-expressing tumor cells and / or for their ability to reduce the growth of Nectin-4-expressing tumor cells (e.g., adherent tumor cells) are then contacted and / or reacted with a compound (e.g., a linker (X) and / or a cytotoxic agent (Z)).

[0161] In another example, an anti-Nectin-4 antibody or antibody fragment can be tested for the ability to sensitize a tumor to a cytotoxic agent (e.g., cytotoxic agent Z, or a cytotoxic agent of the same drug class as Z, e.g., a camptothecin drug). An antibody or antibody fragment determined to have the ability to sensitize a tumor to a cytotoxic agent is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a cytotoxic agent (Z)).

[0162] Optionally, the method can include any two or more antibody testing steps prior to contacting the antibody with a compound (eg, a linker (X) and / or a cytotoxic agent (Z)).

[0163] As further described herein, some well-known methods for conjugating cytotoxic agents to antibodies involve multiple reaction steps in which the antibody is first modified with a linker or portion of a linker, followed by a reaction in which the cytotoxic agent is conjugated to the antibody-linker composition.

[0164] In one embodiment, a method for producing an antibody conjugate, the method comprising the steps of: (i) culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody; and obtaining an anti-Nectin-4 antibody by recovering and purifying the antibody of interest from the culture obtained in the above step; (ii) contacting an anti-Nectin-4 antibody or antibody fragment with a compound (L) that comprises (a) a first reactive group capable of reacting with an amino acid of the antibody (e.g., a side chain or glycan of the amino acid, or a group attached to the amino acid or glycan of the amino acid), and (b) a second reactive group (R') to obtain a modified antibody that comprises one or more amino acids functionalized with compound (L); and (iii) reacting the modified antibody of step (i) with a compound comprising: (a) a reactive group (R) complementary to the reactive group (R'); (b) an amino acid unit (e.g., a di-, tri-, tetra- or pentapeptide) that is cleaved by an intracellular peptidase or protease enzyme; (c) optionally a non-self-eliminating or self-eliminating spacer (Y'); and (d) a cytotoxic agent (Z); Optionally, the compound of step (ii) may further comprise a spacer (Y) disposed between R and the amino acid unit.

[0165] If desired, the process of obtaining anti-Nectin-4 antibody can further include the steps of preparing antibody-producing cells by immunizing an animal by injecting an antigen, taking blood and assaying its antibody titer to determine when to remove the spleen; preparing myeloma cells; fusing the antibody-producing cells with myelomas; screening a group of hybridomas that produce the desired antibody; splitting the hybridomas into single cell clones (cloning); culturing the hybridomas or raising animals transplanted with hybridomas to produce large amounts of monoclonal antibodies; and / or testing the monoclonal antibodies so produced for biological activity and binding specificity or assaying them for their properties as labeling reagents, etc.

[0166] If desired, the step of obtaining an anti-Nectin-4 antibody can further include a step of preparing a cell library.

[0167] In one embodiment, R and R' are capable of undergoing a Click reaction or cycloaddition, where optionally R comprises or may be an alkyne moiety and R' comprises or may be an azide moiety; or R' comprises or may be an alkyne moiety and R comprises or may be an azide moiety, and where the reaction in step (ii) is a 1,3-dipolar cycloaddition.

[0168] In one embodiment, the reaction of step (i) is carried out in the presence of a catalyst, which may be an enzyme (e.g., transglutaminase).

[0169] In one embodiment, step (i) comprises modifying the anti-nectin-4 antibody or antibody fragment before contacting the anti-nectin-4 antibody or antibody fragment with compound (L). For example, the antibody or antibody fragment can be modified by reacting or contacting it with an enzyme capable of modifying antibody glycosylation (e.g., at Kabat residue N297). In one example, the modification includes deglycosylation of the antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, where the core N-acetylglucosamine and the core N-acetylglucosamine substituent may be fucosylated. Examples of endoglycosidases include EndoS, EndoA, EndoE, Endo18A, EndoF, EndoM, EndoD, EndoH, EndoT and EndoSH and / or combinations thereof.

[0170] The antigen binding protein (e.g., antibody) molecule and the cytotoxic agent (e.g., camptothecin derivative molecule) are linked by a linker. In such embodiments, the immunoconjugate may be, for example, represented by Formula (II): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is an anti-Nectin-4 antibody or antibody fragment, X is a linker connecting Ab and Z (e.g., the residue of a linker following covalent bonds to one or both of Ab and Z); Z is a cytotoxic agent (e.g., a camptothecin analog), and optionally Z may include the structure of compound 1 or 2 (an exatecan or SN-38 molecule); n is 1 or 2; When n is 1, m is 1 to 8, or optionally m is an integer selected from among 1 to 8 or 1 to 6, optionally m is an integer selected from among 1 to 4, optionally m is 2 or 4; optionally m is 2, 3, 4, 5, 6, 7 or 8; when n is 2, m is 1 to 4, or optionally m is an integer selected from among 1 to 4 or 1 to 3, optionally m is an integer selected from among 1 to 4, optionally m is 2 or 4; optionally m is 1, 2, 4 or 4. Optionally, "n" can be defined to represent the degree of branching or polymerization. "n" and "m" can be defined to represent averages in a composition comprising multiple antibodies.

[0171] In one embodiment, X represents a molecule comprising a moiety that is cleavable, e.g., under intracellular conditions, optionally under physiological conditions. In one embodiment, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable moiety, and (iii) an optional self-eliminating or non-self-eliminating spacer system (Y'). The spacer Y can be located between Ab and the cleavable moiety, and the spacer system (Y') can be located between the cleavable moiety and Z. The molecule X or the spacer Y can be defined as optionally comprising a reactive group (R) or a residue for reaction of the reactive group R with an amino acid of the antibody or a complementary reactive group (R') attached to an amino acid of the antibody.

[0172] The variable m is the -X-(Z) n In a composition comprising multiple anti-Nectin-4 ADCs, the number "m", which is the number of -XZ moieties per antibody molecule, can vary. Thus, in an exemplary composition comprising multiple immunoconjugates of the formulas herein, m represents the number of -X-(Z) moieties per Ab. n m is the average number of moieties, in which case m can also be referred to as the average drug loading number or drug:antibody ratio (DAR). The average drug loading number or DAR is advantageously 1 to about 8 (-X-(Z) n ) moieties. "n", the number of Z moieties attached to moiety X, can be, for example, 1 or 2. Typically, n is 1. In some embodiments, n is 1, m represents the average drug loading, and m is between 2 and 8. In some embodiments, n is 1, m represents the average drug loading, and m is between 2 and 6. In some embodiments, n is 1, m represents the average drug loading, and m is between 4 and 8. In some embodiments, n is 1, m represents the average drug loading, and m is between 6 and 8, optionally about 6, 7 or 8. In some embodiments, n is 1, m represents the average drug loading, and m is between 4 and 6, optionally about 4, 5 or 6.

[0173] The number of (-XZ) moieties per Ab can be characterized by conventional means such as mass spectrometry, ELISA assays, and HPLC. The quantitative distribution of the immunoconjugates with respect to m can also be determined. In some cases, separation, purification, and characterization of homogeneous immunoconjugates with a particular value of m, as distinguished from immunoconjugates with other drug-loading numbers, can be achieved by means such as reverse-phase HPLC or electrophoresis.

[0174] In one embodiment, the anti-Nectin-4 composition used in the treatment method of the present disclosure has formula (I): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment); X is a molecule linking Ab and Z, e.g., a residue of a linker following covalent attachment to one or both of Ab and Z; Z is a cytotoxic agent (optionally a topoisomerase inhibitor, optionally a camptothecin analog, optionally a camptothecin analog including an exatecan or SN-38 molecule, e.g., a molecule having the structure of Compound 1 or 2); n is 1 or 2; At least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the antibody sample have m (the number of XZ moieties) of 2 or 4, at least 2, between 2 and 4, at least 4, between 4 and 6, or between 4 and 8; optionally, n is 1 and at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the antibody sample have m (the number of XZ moieties) of 2 or 4, at least 2, between 2 and 4, at least 4, between 4 and 6, or between 4 and 8. The immunoconjugate is characterized as comprising a plurality of immunoconjugates represented by:

[0175] In one embodiment, the anti-Nectin-4 composition used in the treatment method of the present disclosure has formula (I): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is an anti-Nectin-4 antigen-binding protein (e.g., an antibody or antibody fragment); X is a molecule linking Ab and Z, e.g., a residue of a linker following covalent attachment to one or both of Ab and Z; Z is a camptothecin analog, including an exatecan or SN-38 molecule, e.g., a molecule containing the structure of compound 1 or 2; n is 1; and At least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the antibody sample have m (the number of XZ moieties) of 6, at least 6, between 6 and 8, or 8. The immunoconjugate is characterized as comprising a plurality of immunoconjugates represented by:

[0176] It will be appreciated that a variety of methods can be used to non-specifically or specifically covalently attach a linker comprising a cytotoxic agent to an antibody or antigen-binding protein to a particular amino acid residue. The linker (X) can include a moiety that is cleavable under physiological conditions, which may be intracellular conditions, for example, as shown in the Examples, such that cleavage of the linker releases the cytotoxic agent (e.g., Compound 1, Compound 2, Compound 13, etc.) into the intracellular environment. The linker can be attached to a chemically reactive group on the antibody molecule, such as a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., at the N- or C-terminus, the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the sulfhydryl group of one or more cysteinyl residues), a carbohydrate, or generally any reactive group that has been introduced or engineered into the antibody. The site at which the linker is attached may be a natural residue in the amino acid sequence of the antibody molecule, or may be introduced into the antibody molecule, for example, by recombinant DNA techniques (e.g., by introducing a cysteine ​​or protease cleavage site into the amino acid sequence, i.e., by introducing a non-natural amino acid residue) or by protein biochemistry (e.g., reduction, pH adjustment or proteolysis by glycomodification, which is the enzymatic modification of amino acid-linked glycans).

[0177] In certain embodiments, the linker (X) comprises a peptide residue comprising an amino acid selected from phenylalanine, glycine, valine, alanine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally, the linker (X) may comprise a dipeptide, tripeptide or tetrapeptide residue.

[0178] In certain embodiments, an intermediate that is a precursor of the linker (X) is reacted with a cytotoxic agent (Z) under appropriate conditions. In certain embodiments, reactive groups on the cytotoxic agent and / or intermediate are used. In certain embodiments, the reaction product of the cytotoxic agent and intermediate, or a derivatized cytotoxic agent, is then reacted with an antibody molecule under appropriate conditions. In other embodiments, a precursor of the linker (X) is first reacted with an antibody molecule under appropriate conditions to obtain an antibody bound to the precursor of the linker (X), and the antibody is then reacted with a molecule comprising a cytotoxic agent (Z).

[0179] In some embodiments, the linker (X) is cleavable by a cleavage agent present in the intracellular environment (e.g., in a lysosome or endosome or caveolea). The linker can include, for example, a peptidyl linker or amino acid unit that is cleaved by an intracellular peptidase or protease enzyme, including but not limited to a lysosomal or endosomal protease. In some embodiments, the peptidyl linker moiety is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, resulting in the release of the active drug inside the target cell. Peptidyl linkers that are cleavable by enzymes present in cells are most typical. In a specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin with a valine-citrulline linker). The valine-citrulline (Val-Cit) element has the structure shown below: [ka] may have the following structure:

[0180] In another specific embodiment, the peptidyl linker cleavable by an intracellular protease is a valine-alanine (Val-Ala) linker. The Val-Ala element has the structure shown below: [ka] may have the following structure:

[0181] In another specific embodiment, the peptidyl linker cleavable by an intracellular protease is a glycine-containing oligopeptide linker, e.g., a glycine- and phenylalanine-containing oligopeptide linker, optionally a GGF, DGGF, (D-)D-GGF, EGGF, SGGF, KGGF, DGGFG (SEQ ID NO: 71), DDGGFG (SEQ ID NO: 72), KDGGFG (SEQ ID NO: 73), GGFGGGF (SEQ ID NO: 74), GGFG, GGFGG (SEQ ID NO: 75) or GGFGGG (SEQ ID NO: 76) linker (see, e.g., U.S. Pat. No. 6,835,807, the disclosure of which is incorporated herein by reference), where (D-)D represents D-aspartic acid.

[0182] In certain embodiments, the linker may function to act as a spacer or stretcher to distance the antibody from Z to avoid interference with the antibody's ability to bind to nectin-4 and / or inhibit cell-cell interactions mediated by nectin-4. The linker may comprise a spacer unit (Y) and / or a spacer or spacer system (Y'). Thus, the spacer Y may be located between the Ab and the cleavable moiety. The spacer system (Y') may be located between the cleavable moiety and Z. The molecule X or the spacer Y may optionally be defined as comprising a reactive group (R) or a residue of reaction of the reactive group R with an amino acid of the antibody or a complementary reactive group (R') attached to an amino acid of the antibody. The spacer Y can be, for example, a molecule that forms a bond (e.g., via its reactive group R) with an amino acid of an antibody, such as a sulfur atom, a primary or secondary amino group, or a carbohydrate group of the antibody, and this spacer or stretcher (Y) links the antibody to a cytotoxic agent (Z) or a cleavable amino acid unit (e.g., a peptidyl linker, a cleavable di-, tri-, tetra- or pentapeptide) that may further have a self-eliminating and / or non-self-eliminating spacer (Y') linked to Z. Thus, when the spacer (Y) is linked at one end to an amino acid unit (e.g., a cleavable di-, tri-, tetra- or pentapeptide), the cleavable amino acid unit can be directly linked to Z or can include a further spacer (Y'), such as a non-self-eliminating or self-eliminating spacer, that links the amino acid unit to Z.

[0183] The spacer (Y) may be defined as being or comprising an optionally substituted or unsubstituted alkyl or heteroalkyl chain, where optionally Y may have a chain length of from 2 to 100 atoms, optionally from 2 to 40, 2 to 30, 2 to 20, 4 to 40, 4 to 30 or 4 to 20 atoms, where optionally one or more atoms may be other than carbon, e.g., oxygen, sulfur, nitrogen, or other atoms, and where optionally any carbon in the chain may be substituted by alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0184] Spacer (Y) can be defined as optionally comprising a stability enhancing moiety. For example, spacer Y can comprise an orthogonal poly-ethylene glycol (PEG) moiety or a polysarcosine (poly-N-methylglycine or PSAR) moiety in a linker design (see, for example, WO 2019 / 081455, WO 2015 / 057699 and WO 2016 / 059377, the disclosures of which are incorporated herein by reference).

[0185] In a specific embodiment, the spacer (Y) may comprise one or more ethylene oxide monomers, optionally Y may comprise a polyethylene oxide moiety, optionally Y may comprise 1 to 24, optionally 1 to 12, optionally 1 to 8, optionally 6 to 24 polyethylene oxide moieties, and Y may be a group having the structure -(CH 2 CH 2 O) x - (wherein x is 1 to 24, optionally 1 to 12, optionally 1 to 6, optionally 6 to 24).

[0186] An example of a suitable stability enhancing moiety, spacer chain Y, can include a stability enhancing moiety disclosed in PCT Publication No. WO2015 / 057699 or WO2019 / 081455. For example, spacer chain Y can include an orthogonal connector moiety and a stability enhancing moiety. The stability enhancing moiety can be a PEG homopolymer, or generally any single molecular weight homopolymer (e.g., a PEG or polysarcosine homopolymer) linked to an orthogonal connector moiety. The homopolymer can have, for example, 1-4, 1-6, 1-7, 1-8, 1-10, 1-12 units of PEG or other monomer, at least 6, 8, or 10 units, or 6-12, 6-24, 6-72 units of PEG or other monomer, or no more than 6, 7, or 8 units of PEG or other monomer. The term orthogonal connector refers to a branched linker unit component that links a linker moiety (e.g., a chain of spacer Y) with a homopolymeric unit and is linked to a cytotoxic agent (Z) via a linker [e.g., a cleavable oligopeptide (Pep) and a spacer Y'] such that the homopolymeric unit is in a parallel (not serial) arrangement with respect to the cytotoxic agent (the homopolymer is in parallel with the Pep-Y'-Z moiety). The orthogonal connector moiety can be, for example, one or more natural or unnatural amino acids that may be selected from glutamic acid, lysine, and glycine. Optionally, an amino acid orthogonal connector moiety can be located at the end of the spacer chain Y such that an amino acid residue of the orthogonal connector moiety is attached to an amino acid residue of the peptidyl linker [e.g., (Pep) in Formula V or VI] via a peptide bond between the α-carboxyl group of one amino acid and the α-amino group of the other amino acid. Y can be, for example, an orthogonal connector moiety and a spacer chain of Formula D: [ka] [In the formula, R 1 and R 2 is different, R 1 and R 2 is H or an inert group, and R 1and R 2 The other of Z is a functionalized reactive group, which is reactive for covalent bonding to a bondable group of the orthogonal connector moiety under reaction conditions in which the inert group is non-reactive, and may be the same or different. 1 and Z 2 is an optional spacer, n is 1 or more, and k is 2 or more. may include the result of a reaction with a moiety of

[0187] Optionally, the orthogonal connector is derived from glutamic acid.

[0188] In another example, the spacer Y is a group disclosed in U.S. Patent Application Publication No. 2017 / 0072068A1, the disclosure of which is incorporated herein by reference, such as a group represented by formula (E): [ka] [In the formula, a is 0 or 1, R 1 is hydrogen, C 1 -C 24 Alkyl group, C 3 -C 24 Cycloalkyl groups, C 2 -C 24 (Hetero)aryl groups, C 3 -C 24 Alkyl(hetero)aryl groups and C 3 -C 24 (hetero)arylalkyl groups, 1 -C 24 Alkyl group, C 3 -C 24 Cycloalkyl groups, C 2 -C 24 (Hetero)aryl groups, C 3 -C 24 Alkyl(hetero)aryl groups and C 3 -C 24 (Hetero)arylalkyl groups are O, S and NR 3wherein R 3 is hydrogen and C 1 -C 4 alkyl groups] or a salt thereof, wherein a group of formula E or a salt thereof is located between the first and second ends of the spacer chain Y.

[0189] In this embodiment, the spacer Y is -(succinimid-3-yl-N)-CH 2 CH 2 -C(=O)-, -(succinimid-3-yl-N)-CH 2 CH 2 CH 2 -C(=O)-, -(succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 -C(=O)-, -(succinimid-3-yl-N)-CH 2 CH 2 CH 2 CH 2 CH 2 It may contain -C(=O)-.

[0190] Optionally, such a spacer Y can have the following structure: -NH-(CH 2 CH 2 -O)n-CH 2 CH 2 It may further include -C(=O)- [wherein n is an integer of 1 to 6, preferably 2 to 4]. Examples of such a structure include -NH-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)-, -NH-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)-, -NH-CH 2 CH 2 -O-CH 2 CH2 -O-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)-, -NH-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)-, -NH-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)-, -NH-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -O-CH 2 CH 2 -C(=O)- is exemplified.

[0191] The spacer or spacer system (Y') located may be a self-eliminating spacer or a non-self-eliminating spacer. The spacer Y' may, for example, comprise a substituted or unsubstituted alkyl or heteroalkyl chain, Y having a chain length of 2 to 30 atoms, optionally 2 to 20, 4 to 20, 2 to 10 or 4 to 20 atoms, optionally one or more atoms may be other than carbon, e.g. oxygen, sulfur, nitrogen or other atoms, optionally any carbon of the chain is substituted by alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide. In one embodiment, Y' comprises a p-aminobenzyloxycarbonyl group. In one embodiment, Y' is a non-self-eliminating spacer, -(CH 2 )nC(=O)-, where n is an integer from 0 to 5, which is linked to the cleavable moiety by -O- or a single bond. For example, Y' includes -C(=O)-, -OC(=O)-, -O-CH 2 -C(=O)-, -O-CH 2 CH 2 -C(=O)-, -O-CH 2 CH 2 CH 2 -C(=O)-, -O-CH 2 CH 2 CH 2 CH 2 -C(=O)-, -O-CH 2 CH 2 CH 2 CH 2 CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 CH 2 -C(=O)-, -CH 2 CH2 CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2 -C(=O)- or -CH 2 CH 2 -O-CH 2 It may be or contain a -C(=O)- group.

[0192] A "self-eliminating" spacer unit allows the release of the drug moiety without a separate hydrolysis step. When a self-eliminating spacer is used, after cleavage or transformation of the amino acid unit, the side of the spacer linked to the amino acid unit becomes unblocked, resulting in the eventual release of one or more moieties Z. Self-eliminating spacer systems can be, for example, those described in WO 02 / 083180 and WO 2004 / 043493, the disclosures of which are incorporated herein by reference in their entirety, as well as other self-eliminating spacers known to those skilled in the art. In certain embodiments, the spacer unit of the linker comprises a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit via an amide bond, resulting in a carbamate, methyl carbamate or carbonate between the benzyl alcohol and the cytotoxic agent. In one embodiment, the spacer unit can be, for example, a structure: [ka] and p-aminobenzyloxycarbonyl (PAB) having the formula:

[0193] Examples of self-eliminating spacer units include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, for example, US Patent Application Publication No. 2005 / 0256030A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and ortho- or para-aminobenzyl acetals. Spacers that can be used may undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al.. Chemistry Biology, 1995, 2, 223) and 2-aminophenylpropionic acid amides (Amsberry, et al., J. Org. Chem., 1990, 55. 5867). Elimination of amine-containing drugs substituted at the a-position of glycine (Kingsbury, et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self-eliminating spacer. p-Aminobenzyl self-eliminating spacers (e.g., PAB) are particularly suitable for use with Phe-Lys, Val-Ala or Val-Cit cleavable dipeptide units (PAB is positioned between the dipeptide and the camptothecin derivative (Z)).

[0194] A "non-self-eliminating" spacer unit is one in which some or all of the spacer unit remains attached to the moiety Z after enzymatic (e.g., proteolytic) cleavage of the antibody-moiety of interest conjugate. Examples of non-self-eliminating spacer units suitable for use as a spacer between a Gly-Gly-Phe-Gly amino acid unit and an exatecan molecule include, but are not limited to, -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2-C(=O)- and -CH 2 CH 2 -O-CH 2 -C(=O)-(e.g., GGFG-CH 2 CH 2 -O-CH 2 -C(=O)-exatecan unit). The use of such a spacer between the GGFG amino acid unit and exatecan results in the release of a molecule having the structure of compound 13. Other examples of non-self-eliminating spacer units include, but are not limited to, glycine spacer units and glycine-glycine spacer units. Other known combinations of peptide spacers susceptible to sequence-specific enzymatic cleavage can be used as well. For example, enzymatic cleavage of an antibody-target moiety conjugate containing a glycine-glycine spacer unit by tumor cell-associated proteases can result in the release of the glycine-glycine-drug moiety from the remainder of the antibody-target moiety conjugate. In one such embodiment, the glycine-glycine-drug moiety is then subjected to a separate hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[0195] Exemplary linker-cytotoxic agent moieties (XZ) can include any of the structures shown in Formulae III-IV below. [ka] [ka] [ka]

[0196] The spacers (Y) and (Y') are optionally linear or branched C 1 -C 20 Alkylene group, C 2 -C 20 Alkenylene group, C 2 -C 20 Alkynylene group, C 3-C 20 Cycloalkylene group, C 5 -C 20 Cycloalkenylene group, C 8 -C 20 Cycloalkynylene group, C 7 -C 20 Alkyl arylene group, C 7 -C 20 Aryl alkylene group, C 8 -C 20 Arylalkenylene groups and C 9 -C 20 and arylalkynylene groups, wherein the alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene, and arylalkynylene groups are independently selected from the group consisting of O, S, and NR 1 and R 1 is hydrogen, C 1 -C 24 Alkyl group, C 2 -C 24 Alkenyl group, C 2 -C 24 Alkynyl groups and C 3 -C 24 cycloalkyl groups, wherein said alkyl groups, alkenyl groups, alkynyl groups and cycloalkyl groups are optionally substituted.

[0197] The spacers (Y) and (Y') are optionally 1 -C 10 Alkylene-, -C 1 -C 10 Heteroalkylene-, -C 3 -C 8 Carbocyclo-, -O-(C 1 -C 8 Alkyl)-, -arylene-, -C 1 -C 10 Alkylene-arylene-, -arylene-C 1 -C10 Alkylene-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-, -C 3 -C 8 Heterocyclo-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-, -C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Heteroalkylene-C(=O)-, -C 3 -C 8 Carbocyclo-C(=O)-, -O-(C 1 -C 8 Alkyl)-C(=O)-, -arylene-C(=O)-, -C 1 -C 10 Alkylene-arylene-C(=O)-, -arylene-C 1 -C 10 Alkylene-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-C(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-C(=O)-, -C 3 -C 8 Heterocyclo-C(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-C(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10Alkylene-C(=O)-, -C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Heteroalkylene-NH-, -C 3 -C 8 Carbocyclo-NH-, -O-(C 1 -C 8 Alkyl)-NH-, -Arylene-NH-, -C 1 -C 10 Alkylene-arylene-NH-, -arylene-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-NH-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-NH-, -C 3 -C 8 Heterocyclo-NH-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-NH-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-NH-, -C 1 -C 10 Alkylene-S-, -C 1 -C 10 Heteroalkylene-S-, -C 3 -C 8 Carbocyclo-S-, -O-(C 1 -C 8 Alkyl)-)-S-, -Arylene-S-, -C 1 -C 10 Alkylene-arylene-S-, -arylene-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-S-, -(C 3 -C8 Carbocyclo)-C 1 -C 10 Alkylene-S-, -C 3 -C 8 Heterocyclo-S-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-S-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 Alkylene-S-, -C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Carbocyclo-OC(=O)-, -O-(C 1 -C 8 Alkyl)-OC(=O)-, -Arylene-OC(=O)-, -C 1 -C 10 Alkylene-arylene-OC(=O)-, -arylene-C 1 -C 10 Alkylene-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Carbocyclo)-OC(=O)-, -(C 3 -C 8 Carbocyclo)-C 1 -C 10 Alkylene-OC(=O)-, -C 3 -C 8 Heterocyclo-OC(=O)-, -C 1 -C 10 Alkylene-(C 3 -C 8 Heterocyclo)-OC(=O)-, -(C 3 -C 8 Heterocyclo)-C 1 -C 10 It can be defined as being or including alkylene -OC(=O)-, in each case -X, -R', -O, -OR', =O, -SR', -S - , -NR' 2 , -NR' 3+ , =NR', -CX 3 , -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO 2 , =N 2 , -N 3 , -NR'C(=O)R', -C(=O)R', -C(=O)NR' 2 , -SO 3 - , -SO 3 H, -S(=O) 2 R', -OS(=O) 2 OR', -S(=O) 2 NR', -S(=O)R', -OP(=O)(OR') 2 , -P(=O)(OR') 2 , -PO 3 , -PO 3 H 2 , -C(=O)X, -C(=S)R', -CO 2 R', -CO 2 , -C(=S)OR', C(=O)SR', C(=S)SR', C(=O)NR' 2 , C(=S)NR' 2 and C(=NR')NR' 2 wherein each X is independently a halogen, i.e., -F, -Cl, -Br, or -I, and each R' is independently -H, -C 1 -C 20 Alkyl, -C 6 -C 20 Aryl, or -C 3 -C 14 It is a heterocycle.

[0198] In one embodiment, the spacer (Y) can be defined as optionally including a reactive group (R) at, for example, one end of the chain, which is reactive with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody, or with a carbohydrate. In one aspect, the spacer Y is an R group -(succinimid-3-yl-N)-(CH 2 )n 3 -C(=O) [where n 3is an integer of 2 to 8, and -(succinimid-3-yl-N) is represented by the following formula F: [ka] Position 3 of the above structure can be the site of attachment to an antibody. Attachment to an antibody at position 3 is characterized by a bond involving thioether formation.

[0199] In one embodiment, the spacer (Y) can be defined as optionally comprising, for example, at one end of the chain, a reactive group (R) that is reactive with a complementary reactive group (R') that is attached to an amino acid of the antibody (e.g., via a free amino, hydroxyl, sulfhydryl or carboxyl group, or carbohydrate), or, after conjugation to the anti-Nectin-4 antibody, a residue for reaction of the reactive group (R) with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or a complementary reactive group (R') that is attached to an amino acid of the antibody. Exemplary pairs of reactive groups by R and R' include a variety of groups capable of biorthogonal reactions, preferably cycloadditions, such as Diels-Alder reactions or 1,3-dipolar cycloadditions between azides and cyclooctynes ​​(copper-free click chemistry), nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, and tetrazine ligation (see also WO 2013 / 092983 or US 2017 / 0072068 A1, the disclosures of which are incorporated herein by reference). For example, R may be an alkyne and R' may be an azide, or R may be an azide and R' may be an alkyne. Thus, the resulting linker and functionalized antibody, or Y element thereof, may in any embodiment include a group (RR') resulting from the reaction of R and R', for example, RR' may be or may include a triazole resulting from the reaction of an alkyne with an azide.

[0200] In one embodiment, the reactive groups R and R' are complementary reagents (i.e., click chemistry reagents or reactive groups) that can undergo a "click" reaction together. For example, 1,3-dipole functional compounds can react with alkynes in a cyclization reaction to form heterocyclic compounds, preferably in the substantial absence of an addition catalyst (e.g., Cu(I)). A variety of compounds with at least one 1,3-dipole group attached (having a three-atom pi-electron system containing four electrons delocalized to three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyldiazo groups.

[0201] Examples include O-phosphene aromatic esters, azides, fulminates, alkynes (including any constrained cycloalkynes), cyanides, anthracene, 1,2,4,5-tetrazine, or norbornene (or other strained cycloalkenes).

[0202] In one embodiment, R is a moiety bearing a terminal alkyne or azide, such moieties being described, for example, in U.S. Patent No. 7,763,736, the disclosure of which is incorporated herein by reference. Suitable reaction conditions for the use of copper (and other metal salts) as a catalyst for the click reaction between terminal alkynes and azides are provided in U.S. Patent No. 7,763,736.

[0203] In one embodiment, R is a substituted or unsubstituted cycloalkyne.Cycloalkyne containing compounds are described, for example, in U.S. Patent No. 7,807,619, the disclosure of which is incorporated herein by reference.

[0204] In one embodiment, the cycloalkyne has formula A: [ka] [In the formula, R1 is selected from carbonyl, alkyl ester, aryl ester, substituted aryl ester, aldehyde, amide, aryl amide, alkyl halide, thioester, sulfonyl ester, alkyl ketone, aryl ketone, substituted aryl ketone, and halosulfonyl; R 1 may be present at any position in the cyclooctyne group except the two carbons joined by the triple bond. The compound may be:

[0205] In some embodiments, the modified cycloalkyne is a cycloalkyne of formula A, in which one or more of the carbon atoms in the cyclooctyne ring other than the two carbon atoms joined by the triple bond are substituted with one or more electron-withdrawing groups, such as halo (bromo, chloro, fluoro, iodo), nitro, cyano, sulfone, or sulfonic acid groups. Thus, for example, in some embodiments, the subject modified cycloalkyne is of formula B: [ka] [In the formula, R 2 and R 3 each of which is independently selected from (a) H; (b) a halogen atom (e.g., bromo, chloro, fluoro, iodo); (c) -W-(CH 2 ) n -Z, where n is an integer from 1 to 4 (e.g., n=1, 2, 3, or 4), W, if present, is O, N, or S, and Z is nitro, cyano, sulfonic acid, or halogen; (d) -(CH 2 ) n -W-(CH 2 ) m -R 4 wherein n and m are each independently 1 or 2; W is O, N, S, or sulfonyl; and when W is O, N, or S, R 4 is nitro, cyano, or halogen; when W is sulfonyl, R4 is H; or (e) -CH 2 )nR 4 [wherein n is an integer from 1 to 4 (e.g., n=1, 2, 3, or 4), and R 4 is nitro, cyano, sulfonic acid, or halogen; and R 1 is selected from carbonyl, alkyl ester, aryl ester, substituted aryl ester, aldehyde, amide, aryl amide, alkyl halide, thioester, sulfonyl ester, alkyl ketone, aryl ketone, substituted aryl ketone, and halosulfonyl. 1 may be present at any position in the cyclooctyne group except the two carbons connected by the triple bond. is a cycloalkyne.

[0206] In one embodiment, R is a substituted or unsubstituted heterocyclic constrained alkyne. Cycloalkyne containing compounds are described, for example, in U.S. Pat. No. 8,133,515, the disclosure of which is incorporated herein by reference. In one embodiment, the alkyne is of formula C: [ka] [In the formula, Each R 1 is hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and C 1 -C 10 independently selected from the group consisting of alkyl and heteroalkyl; Each R 2 is hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and C 1 -C 10 X is independently selected from the group consisting of organic groups, 3 R 4 , N.H.-R. 4 , CH-N-OR 4 , CN-NR 3 R 4 , CHOR 4, or CHNHR 4 Each R 3 represents hydrogen or an organic group, R 4 represents the linking portion C of the linker. In one embodiment, R or R' is the following DBCO (dibenzocyclooctyl) group. [ka]

[0207] Alkynes, such as those described herein above, can be reacted with at least one 1,3-dipole functional compound in a cyclization reaction to form a heterocyclic compound, preferably in the substantial absence of an addition catalyst (e.g., Cu(I)). A wide variety of compounds having at least one 1,3-dipole group attached (having a three-atom pi-electron system containing four electrons delocalized to three atoms) can be used to react with the alkynes disclosed herein. Exemplary 1,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azoxy groups, and acyldiazo groups.

[0208] In the formulas herein, Y' is optionally absent or a spacer, optionally a self-eliminating spacer or a non-self-eliminating spacer, for example, including a p-aminobenzyl unit. Optionally, Y' is or includes a substituted or unsubstituted alkyl or heteroalkyl chain, Y' has a chain length of 2-40 atoms, optionally 2-30, 2-20, 4-40, 4-30, or 4-20 atoms, optionally one or more atoms may be other than carbon, for example oxygen, sulfur, nitrogen or other atoms, and optionally any carbon of the chain is substituted with alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide, or alkylamide.

[0209] An exemplary linker-cytotoxic agent molecule (e.g., the XZ moiety of Formulas I-XI) that can be conjugated to an anti-Nectin-4 antibody optionally has the formula V: (R)-(Y)-(Pep)-(Y')-(Z) Formula (V) [In the formula, R is a group reactive with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody, or a group reactive with a complementary reactive group (R') attached to an amino acid of the antibody, or after conjugation to the anti-Nectin-4 binding protein, R is the residue of a reactive group (R) with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody, or a complementary reactive group (R') attached to an amino acid of the antibody; Y is optionally absent or a spacer; Pep is or contains a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, e.g., a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide; Y' is optionally absent or a spacer, optionally a self-eliminating spacer or a non-self-eliminating spacer; Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exatecan, Dxd or SN-38 molecule. It can be shown by:

[0210] The resulting Nectin-4 binding immunoconjugate of the present invention has, for example, the formula (VI): Ab-(Y)-(Pep)-(Y')-(Z) Formula (VI) [In the formula, Ab is anti-nectin-4 antibody, Y is optionally absent or a spacer. Optionally, formula VI may include a residue of a reactive group (e.g., maleimide, primary amine) between (Ab) and (Y) and a side chain of an amino acid of the anti-Nectin-4 antigen binding protein (Ab) or a carbohydrate. Alternatively, the residue of a reactive group (e.g., maleimide, primary amine) between a side chain of an amino acid of the anti-Nectin-4 antigen binding protein (Ab) and a carbohydrate may be defined as being included in Y, Pep is or includes an amino acid unit (e.g., a peptidyl linker) that is cleaved by an intracellular peptidase or protease enzyme [e.g., (Pep) is a protease-cleavable di-, tri-, tetra-, or pentapeptide, e.g., a valine-citrulline, valine-alanine, or phenylalanine-lysine unit]; Y' is optionally absent or a spacer, optionally a self-eliminating spacer or a non-self-eliminating spacer; Z is a cytotoxic agent, optionally a camptothecin derivative, optionally an exatecan, Dxd or SN-38 molecule. It can be shown by:

[0211] Optionally, the formula can be defined as including or including a residue (RR') for reaction of a reactive group (R) with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or a complementary reactive group (R') attached to an amino acid of the antibody [e.g., between (Ab) and the terminus of Y (or (Pep or X) if Y is absent)].

[0212] In one example, where (RR') is a residue of a reactive group (R) and a complementary reactive group (R') attached to an antibody (e.g., R' is attached to an amino acid side chain or a glycan of the antibody), the Nectin-4-binding immunoconjugate of the present invention can be, for example, represented by the formula (VI bis ): Ab-(RR')-(Y)-(Pep)-(Y')-(Z) Formula (VI bis ) where Ab, Y, Pep, Y' and Z are as defined in formula VI, and RR' is the result of a biorthogonal reaction, preferably a cycloaddition, such as a Diels-Alder reaction or a 1,3-dipolar cycloaddition. In one embodiment, RR' is the result of the following: [ka] [In the formula, X 8 is O or NH, and X 9 is selected from H, methyl and pyridyl, and has the structure (RR' C ) and (RR' d ) [ka] Bonds indicate either single or double bonds.] The compound has a structure selected from the group consisting of:

[0213] In either embodiment, the exatecan molecule (or other hexacyclic camptothecin) can be defined as being linked to Y' [or (Pep) if Y' is absent)] via the amine at position 1 of exatecan.

[0214] In either embodiment, the SN-38 molecule (or other pentacyclic camptothecin) can be defined as being linked to Y' [or (Pep) if Y' is absent)] via the amine at position 9 of SN-38.

[0215] Cytotoxic agents, also referred to as (Z) moieties, include, for example, anti-neoplastic cytotoxic agents. Examples of cytotoxic agents are known in the art. For example, Z can be an alkylating agent, preferably a DNA alkylating agent. An alkylating agent is a compound that can replace a hydrogen atom with an alkyl group under physiological conditions (e.g., pH 7.4, 37°C, aqueous solution). Alkylation reactions are typically described in terms of the substitution reaction of N, O and S heteroatom nucleophiles with electrophilic alkylating agents, although Michael addition reactions are also important. Examples of alkylating agents include nitrogen and sulfur mustards, ethylenimines, methanosulfonates, CC-1065 and duocarmycins, nitrosoureas, platinum-containing agents, agents that result in topoisomerase II-mediated site-dependent alkylation of DNA (e.g., psorospermin and related bisfuranoxanthones), ecteinascidins and other or related DNA minor groove alkylating agents.

[0216] In one embodiment, Z is a chelated metal, e.g., a chelate of a positive divalent or positive trivalent metal having a coordination number of 2 to 8 (end points included). Specific examples of such metals include technetium (TC), rhenium (RE), cobalt (CO), copper (Cu), gold (Au), silver (Ag), lead (Pb), bismuth (Bi), indium (In), gallium (Ga), yttrium (Y), terbium (Tb), gadolinium (Gd), and scandium (SC). In general, the metal is preferably a radionuclide. Specific radionuclides include: 99 M.T.C., 186 R.E. 188 R.E. 58 Co, 60 Co, 67 Cu, 195 Au, 199 Au, 110 Ag, 203 Pb, 206 Bi, 207 Bi, 111 In, 67 Ga, 68 Ga, 88 Y, 90 Y,160 T.B., 153 Gd and 47 The chelated metal can be, for example, one of the above types of metals chelated with any suitable multidentate chelating agent, such as acyclic or cyclic polyamines, polyethers (e.g., crown ethers and their derivatives); polyamides; porphyrins; and carbocyclic derivatives.

[0217] Anti-nectin-4 antibodies or antibody fragments can also be used in diagnostic methods, for example, to detect nectin-4 tumor cells. In such embodiments, antibodies or antibody fragments can be conjugated to effector molecules, such as detectable substances useful in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, radionuclides, positron-emitting metals (for use in positron emission tomography), and non-radioactive paramagnetic metal ions. For a summary of metal ions that can be conjugated to antibodies for use as diagnostic methods, see U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin, and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin, and aequorin; and suitable radionuclides include 125 I, 131 I, 111 In and 99 Examples include Tc.

[0218] In one embodiment, the cytotoxic agent (Z) is a DNA minor groove binding and / or alkylating agent, such as a pyrrolobenzodiazepine, a duocarmycin, or a derivative thereof.

[0219] In further embodiments, the cytotoxic agent is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mitomycins, combretastatins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamicins, duocarmycins, tubulysins, dolastatins and auristatins, enediynes, amatoxins, pyrrolobenzodiazepines, ethylenimines, radioisotopes, therapeutic proteins and peptides, and toxins or fragments thereof.

[0220] In further embodiments, the cytotoxic agent is cyclophosphamide, ifosfamide, chlorambucil, 4-(bis(2-chloroethyl)amino)phenol, 4-(bis(2-fluoroethyl)amino)phenol, N,N-bis(2-chloroethyl)-p-phenylenediamine, N,N-bis(2-fluoro-ethyl)-p-phenylenediamine, carmustine, lomustine, treosulfan, dacarbazine, cisplatin, carboplatin, vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, irinotecan, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin, crisnatol, mitomycin C, mitomycin A, methotrexate, trimetrexate, mycophenolic acid, Triazofurin, ribavirin, hydroxyurea, deferoxamine, 5-fluorouracil, floxuridine, doxifluridine, raltitrexed, cytarabine, cytosine arabinoside, fludarabine, 6-mercaptopurine, thioguanine, raloxifene, megestrol, goserelin, leuprolide acetate, flutamide, bicalutamide, vertoporphin, phthalocyanine, photosensitizer PC4, demethoxyhypocreli interferon A, interferon-alpha, interferon-gamma, tumor necrosis factor, lovastatin, staurosporine, actinomycin D, bleomycin A2, bleomycin B2, peplomycin, daunorubicin, doxorubicin, N-(5,5-diacetoxypentyl)doxorubicin, morpholinodoxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, thapsigargin, N 8 -Acetylspermidine, tallysomycin, esperamicin, butyric acid, retinoic acid, 1,8-dihydroxybicyclo[7.3.1]tridec-4-ene-2,6-diyn-13-one, anguidine, podophyllotoxin, combretastatin A-4, pancratistatin, tubulysin A, tubulysin D, carminomycin, streptonigrin, erythromycin acetate, maytansine, maytansinol, calicheamicin, mertansine (DM1), N-acetylglucosamine, ... 1I -Calicheamicin, calicheamicin-gamma 1 I , calicheamicin-α 2 I , calicheamicin-α 3 I , duocarmycin SA, duocarmycin A, CC-1065, CBI-TMI, duocarmycin C2, duocarmycin B2, centanamycin, dolastatin, auristatin E, monomethylauristatin E (MMAE), monomethylauristatin F (MMAF), α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanine, amaninamide, amanulin, and amanuric acid, and derivatives thereof.

[0221] Exemplary auristatin embodiments have the following formulae (a1) and (a2): [ka] wherein the wavy lines in (a1) and (a2) indicate the sites of covalent attachment to a linker (e.g., linker X, or moiety Y, Pep, or Y′), independently at each position: R 2 H and C 1 -C 8 alkyl; R 3 , H, C 1 -C 8 Alkyl, C 1 -C 8 Carbocyclic, Aryl, C 1 -C 8 Alkyl-aryl, C 1 -C 8 Alkyl-(C 3 -C 8 carbocycle), C 3 -C 8 Heterocycles and C 1 -C 8 Alkyl-(C 3 -C 8 heterocycle); R 4 , H, C1 -C 8 Alkyl, C 3 -C 8 Carbocyclic, Aryl, C 1 -C 8 Alkyl-aryl, C 1 -C 8 Alkyl-(C 3 -C 8 carbocycle), C 3 -C 8 Heterocycles and C 1 -C 8 Alkyl-(C 3 -C 8 heterocycle); R 5 is selected from H and methyl; or R 4 and R 5 taken together form a carbocyclic ring of the formula -(CR a R b ) n (In the formula, R a and R b , H, C 1 -C 8 Alkyl and C 3 -C 8 carbocycle, and n is selected from 2, 3, 4, 5, and 6; R 6 H and C 1 -C 8 alkyl; R 7 , H, C 1 -C 8 Alkyl, C 3 -C 8 Carbocyclic, Aryl, C 1 -C 8 Alkyl-aryl, C 1 -C 8 Alkyl-(C 3 -C 8 carbocycle), C 3 -C 8 Heterocycles and C 1 -C 8 Alkyl-(C 3 -C 8 heterocycle); Each R8 is H, OH, C 1 -C 8 Alkyl, C 3 -C 8 Carbocyclic and O-(C 1 -C 8 alkyl); R 9 H and C 1 -C 8 alkyl; R 10 is aryl or C 3 -C 8 heterocycle; Z is O, S, NH, or NR 12 and R 12 is C 1 -C 8 is alkyl; R 11 , H, C 1 -C 20 Alkyl, aryl, C 3 -C 8 Heterocycle, -(R 13 O) m -R 14 , or -(R 13 O) m -CH(R 15 ) 2 m is an integer ranging from 1 to 1000; R 13 is C 2 -C 8 is alkyl; R 14 is H or C 1 -C 8 is alkyl; R 15 are each independently H, COOH, -(CH 2 ) n -N(R 16 ) 2 , -(CH 2 ) n -SO 3 -C 1 -C 8 is alkyl; R 16 are each independently H, C 1-C 8 Alkyl or -(CH 2 ) n -COOH; R 18 is -C(R 8 ) 2 -C(R 8 ) 2 -aryl, -C(R 8 ) 2 -C(R 8 ) 2 -(C 3 -C 8 heterocycle), and -C(R 8 ) 2 -C(R 8 ) 2 -(C 3 -C 8 carbocyclic ring); and n is an integer ranging from 0 to 6. The compound includes an N-terminally linked monomethyl auristatin drug moiety comprising any of the structures:

[0222] In one embodiment, R 3 , R 4 and R 7 is independently isopropyl or sec-butyl; R 5 is -H or methyl. In an exemplary embodiment, R 3 and R 4 are isopropyl, and R 5 is -H, R 7 is sec-butyl.

[0223] In yet another embodiment, R 2 and R 6 are methyl, and R 9 is -H.

[0224] In yet another embodiment, R 8 Each occurrence of -OCH 3 It is.

[0225] In an exemplary embodiment, R 3 and R 4are isopropyl, and R 2 and R 6 are methyl, and R 5 is -H, R 7 is sec-butyl, R 8 Each occurrence of -OCH 3 and R 9 is -H.

[0226] In one embodiment, Z is -O- or -NH-.

[0227] In one embodiment, R 10 is aryl.

[0228] In an exemplary embodiment, R 10 is -phenyl.

[0229] In an exemplary embodiment, when Z is -0-, R 11 is -H, methyl or t-butyl.

[0230] In one embodiment, when Z is -NH, R 11 -CH(R 15 ) 2 where R 15 Ha-(CH 2 ) n -N(R 16 ) 2 and R 16 -C 1 -C 8 Alkyl or -(CH 2 ) n -COOH.

[0231] In another embodiment, when Z is -NH, R 11 -CH(R 15 ) 2 where R 15 Ha-(CH 2 ) n -SO 3 It's H.

[0232] One exemplary auristatin embodiment of formula (a1) is MMAE: [ka] where the wavy line indicates a covalent bond to the linker.

[0233] Certain exemplary auristatin embodiments of formula (a2) include MMAF: [ka] where the wavy line indicates the covalent attachment of the antibody-drug conjugate to the linker (L) (see U.S. Patent Application Publication No. 2005 / 0238649 and Doronina et al. (2006) Bioconjugate Cfiem. 17: 1 14-124).

[0234] Other exemplary Z embodiments include monomethylvaline compounds having a phenylalanine carboxy modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848), and monomethylvaline compounds having a phenylalanine side chain modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0235] Other drug moieties include the following MMAF derivatives: [ka] [ka] [ka] [wherein the wavy line indicates a covalent bond to the linker] Examples include:

[0236] An example of a linker containing a primary amine-containing spacer (Y), valine-citrulline as the (Pep) moiety, and PAB as the (Y') moiety, together with MMAF as the (Z) moiety, is shown below. [ka]

[0237] In one embodiment, the Z moiety is a DNA minor groove binder, and Z may comprise a pyrrolobenzodiazepine (PBD). In one embodiment, Z is a pyrrolobenzodiazepine monomer. In one embodiment, Z is a pyrrolobenzodiazepine dimer comprising two pyrrolobenzodiazepine units. In one embodiment, Z is a pyrrolobenzodiazepine trimer comprising three pyrrolobenzodiazepine units. In one embodiment, Z is a pyrrolobenzodiazepine multimer comprising four or more pyrrolobenzodiazepine units. The structure of PBDs, as well as formulas and methods for producing them, are described, for example, in PCT Application Nos. WO2013 / 177481, WO2011 / 130616, WO2004 / 043880, WO2005 / 085251, WO2012 / 112687, and WO2011 / 023883, the disclosures of each of which are incorporated herein by reference.

[0238] Pyrrolo[2,1-C][1,4]benzodiazepines are a family of sequence-selective minor groove binding DNA interacting agents that covalently bind to guanine residues. It has been reported that (S)-chirality at the C11a position of the PBD gives the PBD the proper three-dimensional conformation that fits perfectly into the DNA minor groove. PBDs can have different effects and mechanisms of action. PBDs can be DNA binders or DNA alkylating agents that do not cause DNA cross-linking, or PBDs can be DNA cross-linking agents.

[0239] A pyrrolobenzodiazepine unit or monomer has the following general structure: [ka] [wherein PBD may vary in the number, type and position of substituents in both the aromatic A ring and the pyrrolo C ring, and may vary in the degree of saturation of the C ring. In the B ring, N, the electrophilic center responsible for DNA alkylation, 10 -C 11 At the N=C position, there is either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)). may have:

[0240] The biological activity of a PBD can be increased by linking two PBD monomers or units together via a flexible alkylene linker, typically via the C8 / C8'-hydroxyl functionality.

[0241] In one aspect of any of the embodiments herein, the pyrrolobenzodiazepine monomer or unit is a pyrrolo[2,1-C][1,4]benzodiazepine. In one aspect of any of the embodiments herein, the pyrrolobenzodiazepine dimer is a C8 / C8'-linked pyrrolo[2,1-C][1,4]benzodiazepine dimer.

[0242] The PBD can be attached to the linker through any suitable position, for example, the PBD can be linked to the linker through any position in the PBD unit shown below. [ka]

[0243] In one embodiment, a PBD dimer has the following general formula, where exemplary points of attachment to other substituents or functional groups in the compound are indicated by arrows: [ka] [In the formula, R 12 and R 12 ' and / or R 2 and R 2' are taken together to form a double bond =CH 2 or =CH-CH 3 or R 2 ' and R 12 ' does not exist, R 2 and R 12 teeth, (iia)C 1-5 Saturated aliphatic alkyl, (iib)C 3-6 saturated cycloalkyl, (iic)R 12 When the total number of carbon atoms in the group is 5 or less, [ka] (In the formula, R 21 , R 22 and R 23 Each of the 1-3 Saturated alkyl, C 2-3 Alkenyl, C 2-3 alkynyl and cyclopropyl), (iid) [ka] (In the formula, R 25a and R 25b One of them is H and the other is phenyl, optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (No) [ka] (In the formula, R 24 is H;C 1-3 Saturated alkyl;C 2-3 Alkenyl; C 2-3 alkynyl; cyclopropyl; phenyl optionally substituted with a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl. are independently selected from; R6 and R 9 are H, R, OH, OR, SH, SR, NH 2 , NHR, NRR', Nitro, Me 3 Sn and halo, where R and R' are optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclyl and C 5-20 aryl groups; R 7 are H, R, OH, OR, SH, SR, NH 2 , NHR, NHRR', Nitro, Me 3 Selected from Sn and Halo; (a)R 10 is H and R 11 OH, OR A where R A is alkyl or (b)R 10 and R 11 form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached, or (c)R 10 is H and R 11 SO z M, where z is 2 or 3, and M is a pharma- ceutically acceptable monovalent cation. Either; R” is C 3-12 It is an alkylene group, the chain of which may contain one or more heteroatoms, e.g., O, S, NR N2 (where R N2 is H or C 1-4 alkyl), and / or may be interrupted by aromatic rings, e.g., benzene or pyridine; Y and Y' are selected from O, S, or NH; R 6 ', R 7 ', R 9 ' are R 6 , R 7 and R 9 is selected from the same group as R 10 ' and R 11' is R 10 and R 11 where R 11 and R 11 SO z When M, M may represent a pharma- ceutically acceptable divalent cation. Includes the structure of.

[0244] In another example, the PBD dimer has the general formula: [ka] [In the formula, R 6 , R 7 , R 9 , R 6 ', R 7 ', R 9 ', R 10 , R 11 , R 10 ' and R 11 ' is as defined above, and the 'K' ring is a substituted or unsubstituted aromatic or non-aromatic ring, optionally 6-membered, optionally phenyl. Includes the structure of.

[0245] In one embodiment, the cytotoxic agent (Z) is a camptothecin, e.g., the cytotoxic agent (Z) has or includes the structure of camptothecin or a camptothecin analog. Camptothecin is well known and there are a variety of camptothecin analogs that share the core ring system with various substitutions, but preferably have modifications or substitutions in rings A and / or B of the basic camptothecin structure below. [ka]

[0246] Many camptothecin analogs have been reported, including topotecan, irinotecan, exatecan, Dxd, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, lurtotecan, camptothecin, gimatecan, belotecan, and rubitecan. Additional camptothecin analogs are disclosed in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392, Jpn. J. Cancer Res. 86: 776-782, and Takiguchi et al. 1997 Jpn. J. Cancer Res. 88:760-769, the disclosures of which are incorporated herein by reference. Four analogs, topotecan, irinotecan, belotecan, and Dxd (as part of trastuzumab deruxtecan), have been approved by the FDA. In one embodiment, the camptothecin analog is a pentacyclic compound (eg, camptothecin lacks an F ring). In one embodiment, the camptothecin analog is a hexacyclic compound, for example, including an F ring.

[0247] Some examples, such as the basic camptothecin structure, the SN-38 molecule (7-ethyl-10-hydroxycamptothecin; the active metabolite of irinotecan), and camptothecin analogs disclosed in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-139, have five rings (A, B, C, D and E rings) and can be attached to a linker (e.g., spacer Y or Y', or linker X) via, for example, a substituent on the B ring. SN-38: [ka] Compounds from Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-139: [ka]

[0248] Optionally, the camptothecin analogue may be a hexacyclic compound (with an additional F ring), where the compound is linked to the linker via a substituent on the F ring. Examples of such hexacyclic compounds include, but are not limited to, Dxd (CAS number: 1599440-33-1) and exatecan.

[0249] Thus, camptothecin analogs include exatecan, SN-38, and any of a variety of molecules containing such moieties, e.g., exatecan can be unsubstituted or substituted at the amine at position 1, e.g., the substituents are -C(=O)-, -OC(=O)-, -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2 -C(=O)-, -CH 2 CH 2 -O-CH 2 is or includes a -C(=O)- group or other groups set forth in US Pat. No. 6,835,807, the disclosure of which is incorporated herein by reference.

[0250] In one embodiment, the antibody of the present disclosure releases an exatecan molecule having the structure of Compound 1 (e.g., by enzymatic cleavage of the cleavable moiety followed by self-elimination of spacer Y') in vivo or in vitro in the presence of Nectin-4-expressing tumor cells.

[0251] Exatecan, a camptothecin derivative or analog, is described in Mitsui et al. 1995 Jpn. J. Cancer Res. 86: 776-782 and Takiguchi et al. 1997 Jpn. J. Cancer Res. 88: 760-769, the disclosures of which are incorporated herein by reference. The structure of exatecan is shown below as compound 1a. [ka]

[0252] Exatecan can be attached to the linker via the nitrogen atom of the amino group at position 1, and therefore, when the exatecan moiety is attached to the linker or is present in a linker-exatecan molecule ((XZ) molecule), for example, when conjugated to an antibody, exatecan can have the structure of compound 1b. [ka]

[0253] Thus, when the exatecan of compound 1a is attached to a linker via the amine at position 1 (and, for example, the linker is attached to an antibody), the exatecan is the group modified at position 1 (i.e., the NH 2It will be appreciated that the linker may be understood as a linker having a cleavable oligopeptide, with the aryl group being replaced by NH, or alternatively OH or O groups. For example, exatecan can be coupled to an antibody via a linker comprising a cleavable oligopeptide. Examples include di-, tri-, tetra- and pentapeptides such as glycine and phenylalanine-containing peptides as shown in U.S. Pat. No. 6,835,807, the disclosure of which is incorporated herein by reference, or the dipeptides valine-citrulline or valine-alanine, linked to a PAB molecule. A variety of suitable linker-Z structures are known that can liberate active exatecan or exatecan derivatives at the amino at the 1-position. Exatecan can be linked to a cleavable oligopeptide via a p-aminobenzyloxycarbonyl (PAB) group attached to the amine at the 1-position, as shown in formulas VII, VIII and IX, or the (PEG(8U)-Val-Ala-PAB-exatecan) linker in Example 7, which upon cleavage results in the liberation of exatecan having the structure of compound 1a. In one example, exatecan is attached to the amine at position 1 (CH 2 The NH or NH at position 1 of exatecan in compound 1 can be linked to a cleavable oligopeptide via the -C(=O) group, which upon cleavage results in the release of exatecan-containing compound 13 (Dxd). 2 Examples of the substituents in are -C(=O)-, -OC(=O)-, or -O-CH 2 -C(=O)-, HO-O-CH 2 -C(=O)-, -CH 2 CH 2 -C(=O)-, -CH 2 CH 2 CH 2 -C(=O)-, -CH 2 -O-CH 2 -C(=O)- and -CH 2 CH 2 -O-CH 2 -C(=O)- and other groups (CH 2 -C(=O)).

[0254] In one embodiment, a substituted exatecan derivative (eg, derivatized at the 1-position) has the structure of compound 13.

[0255] The structure of Dxd is shown below. [ka]

[0256] Dxd can be coupled to the linker via an oxygen atom at the end of the carbon chain, and therefore when the Dxd moiety is attached to the linker or present in a linker-Dxd molecule ((XZ) molecule), the carbon chain terminal OH can be replaced with O when conjugated, for example, to an antibody.

[0257] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in Formula VII below, where (Y) is a spacer comprising (e.g., at its terminus) a residue of reaction between a reactive group (R) and an amino acid residue, such as a free amino, hydroxyl, sulfhydryl, or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the S atom of one or more cysteinyl residues).Anti-Nectin-4 binding proteins functionalized with a linker comprising the structure of Formula VII or Compound 3 or 4 can release or provide (e.g., intracellularly in the presence of Nectin-4-expressing tumor cells) a compound having the structure of Compound 1a. [ka]

[0258] An exemplary linker having a maleimide as the R group may have the structure of Compound 3 below. Such a linker may be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond has been reduced with a reducing agent, for example, tris(2-carboxyethyl)phosphine hydrochloride. [ka]

[0259] The resulting antibody-drug conjugate comprises an antibody containing one or more cysteine ​​residues functionalized with a compound having the structure of Compound 3 (wherein Compound 3 is attached via the S atom of the cysteine ​​residue).

[0260] In another embodiment, the linker can have a primary amine as an R group, which when reacted with an antibody in the presence of a transglutaminase enzyme can result in an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (XZ) or an antibody functionalized therewith has or includes the structure shown below as compound 4. [ka]

[0261] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in formula VIII below, where (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) and a residue of reaction with an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl or carboxyl group on an antibody (e.g., an epsilon amino group of one or more lysine residues, a free carboxylic acid group of one or more glutamic or aspartic acid residues, or an S atom of one or more cysteinyl residues), or, e.g., a glycan structure of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of an antibody).Anti-nectin-4 binding proteins functionalized with a linker comprising the structure of formula VIII or compound 5, 6 or 7 can release or provide (e.g., intracellularly in the presence of nectin-4-expressing tumor cells) a compound having the structure of compound Ia. [ka]

[0262] Exemplary linkers having a maleimide as the R group may have the structure of compounds 5 or 6 below. Such linkers may be conjugated to antibodies via cysteine ​​residues in the antibody after the interchain disulfide bonds are reduced with a reducing agent. [ka] [ka]

[0263] In another embodiment, the linker can have a primary amine as an R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can result in an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (XZ) or an antibody functionalized therewith has or includes the structure shown below as compound 7. [ka]

[0264] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in formula IX below, where (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) and a residue of reaction with an amino acid residue, such as a free amino, hydroxyl, sulfhydryl or carboxyl group on an antibody (e.g., an epsilon amino group of one or more lysine residues, a free carboxylic acid group of one or more glutamic or aspartic acid residues, or an S atom of one or more cysteinyl residues), or a glycan structure, such as a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). An anti-nectin-4 binding protein functionalized with a linker comprising the structure of formula IX can release a compound having the structure of compound Ia (e.g., intracellularly in the presence of nectin-4-expressing tumor cells). [ka]

[0265] An exemplary linker having a maleimide as the R group may have the structure of compound 8 below. Such a linker may be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. [ka]

[0266] In another embodiment, the linker can have a primary amine as an R group and, when reacted with an antibody in the presence of a transglutaminase enzyme, can result in an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (XZ) or an antibody functionalized therewith has or includes the structure shown below as compound 9. [ka]

[0267] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in formula X below, where (Y) is a spacer that comprises (e.g., at its terminus) a residue of reaction between a reactive group (R) and an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure, e.g., of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). [ka]

[0268] An exemplary linker having a maleimide as the R group can have the structure of compound 10 below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond has been reduced with a reducing agent, for example, tris(2-carboxyethyl)phosphine hydrochloride. [ka]

[0269] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in formula XI below, where (Y) is a spacer that comprises (e.g., at its terminus) a residue of reaction between a reactive group (R) and an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure, e.g., of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). [ka]

[0270] An exemplary linker having a maleimide as the R group may have the structure of compound 11 below. Such a linker may be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. [ka]

[0271] In one embodiment, the linker moiety (XZ) is or includes the structure shown in formula VII below, where (Y) is a spacer that includes (e.g., at its terminus) a reactive group (R) and a residue of reaction with an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl, or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure, e.g., of a glycosylated amino acid residue (e.g., a native, cleaved, or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). The linker can include, e.g., 1-72, 1-16, 1-12, 1-8, 1-7, 1-6, 6-24, 6, 8, 16, 18, or 24 PEG units. In the following formula, n, which is the number of PEG units, can be, for example, 1 to 72, 5 to 23, 1 to 15, 1 to 16, 1 to 11, 1 to 12, 1 to 8, 1 to 7, 1 to 6, 7, 15, 17, or 23. [ka] Formula XII

[0272] In such embodiments, an immunoconjugate or antibody-drug conjugate comprising a linker moiety having a maleimide as an R group can have the structure of formula XII bis, where the maleimide moiety is coupled to a sulfur group (S) of an amino acid (i.e., cysteine) of an antibody (Ab) after the interchain disulfide bond is reduced with a reducing agent. [ka]

[0273] In the variant of formula XII where the intracellularly cleavable peptide is valine-citrulline, an immunoconjugate or antibody-drug conjugate including a linker moiety having a maleimide as the R group can have the structure of formula XIIter. [ka]

[0274] An exemplary linker having a maleimide as the R group may have the structure of compound 14a below. Such a linker may be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. In this example, the linker moiety contains eight PEG units. [ka]

[0275] According to this example, one immunoconjugate or antibody-drug conjugate containing such a linker moiety can have the structure of compound 14a bis below. [ka]

[0276] Another exemplary linker having a maleimide as the R group can have the structure of compound 14b below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. In this example, the linker moiety contains seven PEG units. [ka]

[0277] According to this example, an immunoconjugate or an antibody-drug conjugate containing such a linker moiety can have the structure of compound 14b bis below. [ka]

[0278] Another exemplary linker having a maleimide as the R group can have the structure of compound 14c below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. In this example, the linker moiety contains 16 PEG units. [ka]

[0279] According to this example, an immunoconjugate or antibody-drug conjugate containing such a linker moiety can have the structure of compound 14c bis below. [ka]

[0280] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in formula XIII below, where (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) and a residue of reaction with an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl or carboxyl group on an antibody (e.g., the epsilon amino group of one or more lysine residues, the free carboxylic acid group of one or more glutamic or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure, e.g., of a glycosylated amino acid residue (e.g., a native, cleaved or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). The linker can comprise, e.g., 1-72, 1-16, 1-12, 1-8, 1-7, 1-6, 6-24, 8, 16, 18 or 24 PEG units. In the following formula, n, which is the number of PEG units, can be, for example, 1 to 72, 5 to 23, 1 to 15, 1 to 16, 1 to 11, 1 to 12, 1 to 8, 1 to 7, 1 to 6, 7, 15, 17, or 23. [ka]

[0281] In such embodiments, an immunoconjugate or antibody-drug conjugate comprising a linker moiety having a maleimide as an R group can have the structure of Formula XIII bis, where the maleimide moiety is attached to a sulfur group (S) of an amino acid (i.e., cysteine) of an antibody (Ab) after the interchain disulfide bond has been reduced with a reducing agent. [ka]

[0282] An exemplary linker having a maleimide as the R group can have the structure of compound 15a below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. In this example, the linker moiety contains eight PEG units. [ka]

[0283] According to this example, an immunoconjugate or antibody-drug conjugate containing such a linker moiety can have the structure of compound 15a bis below. [ka]

[0284] Another exemplary linker having a maleimide as the R group can have the structure of compound 15b below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. In this example, the linker moiety comprises seven PEG units. [ka]

[0285] According to this example, an immunoconjugate or antibody-drug conjugate containing such a linker moiety can have the structure of compound 15b bis below. [ka]

[0286] In one embodiment, the linker moiety (XZ) is or includes the structure of formula XIV below, where (Y) is a spacer that includes (e.g., at its terminus) a reactive group (R) and a residue of reaction with an amino acid residue, e.g., a free amino, hydroxyl, sulfhydryl, or carboxyl group on an antibody (e.g., an epsilon amino group of one or more lysine residues, a free carboxylic acid group of one or more glutamic or aspartic acid residues, or an S atom of one or more cysteinyl residues), or a glycan structure, e.g., of a glycosylated amino acid residue (e.g., a native, cleaved, or otherwise modified N-glycan attached to Kabat residue N297 of an antibody). In the formula below, the number of PSAR units, n, can be, e.g., 1 to 72, 1 to 24, 1 to 16, 1 to 10, 1 to 12, 1 to 8, 1 to 6, 8, 16, 18, or 24. [ka]

[0287] An exemplary linker having a maleimide as the R group can have the structure of compound 16 below. Such a linker can be conjugated to an antibody via a cysteine ​​residue in the antibody after the interchain disulfide bond is reduced with a reducing agent. [ka]

[0288] According to this example, an immunoconjugate or antibody-drug conjugate containing such a linker moiety can have the structure of compound 16bis below. [ka]

[0289] In another embodiment, the linker can have a primary amine as an R group and can be reacted with an antibody in the presence of a transglutaminase enzyme to yield an antibody containing one or more acceptor glutamine residues functionalized with the linker. For example, the linker (XZ), or an antibody functionalized therewith, has or includes the structure shown as compound 12 below. [ka]

[0290] The anti-Nectin-4 binding protein functionalized with the oligopeptide-containing linker of formula XI or compounds 11 and 12 can be represented by the following structure: OH-CH 2 The -C(=O) substituent at the amine position results in release of the substituted exatecan (e.g., intracellularly in the presence of Nectin-4 expressing tumor cells). [ka]

[0291] When the exemplary linker of formula III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV is prepared as a structure having a primary amine, it can react with an antibody in the presence of a transglutaminase enzyme (e.g., bacterial transglutaminase, BTG), such that the transglutaminase enzyme catalyzes the conjugation of the linker to the acceptor glutamine residue within the primary structure of the antibody, for example, within the immunoglobulin constant domain or within the TGase recognition tag inserted or added (e.g., fused) to the constant region. Methods for BTG-mediated conjugation to antibodies and linkers for use in the conjugation are described in PCT Publication No. WO2014 / 202773, the disclosure of which is incorporated by reference. BTG-catalyzed conjugation allows for tight control of the average drug:antibody ratio in the composition. The term "transglutaminase" is used interchangeably with "TGase" or "TG" and refers to an enzyme that can crosslink proteins by acyl transfer reaction between the γ-carboxamide group of peptide-bound glutamine and a structurally related primary amine, such as the ε-amino group or aminopentyl group of lysine, resulting in an ε-(γ-glutamyl)lysine isopeptide bond. TGases include, in particular, bacterial transglutaminases (BTG), such as the enzyme with EC reference number EC2.3.2.13 (protein-glutamine-γ-glutamyltransferase). The term "acceptor glutamine" residue, when referring to a glutamine residue of an antibody, refers to a glutamine residue that is recognized by TGase and can be crosslinked by TGase via a reaction between glutamine and a structurally related primary amine, such as a lysine or aminopentyl group. Preferably, the acceptor glutamine residue is a surface-exposed glutamine residue.The term "TGase recognition tag" refers to a sequence of amino acids that includes an acceptor glutamine residue and that, when incorporated (e.g., added) into a polypeptide sequence under appropriate conditions, is recognized by a TGase and results in cross-linking by the TGase through a reaction between an amino acid side chain in the sequence of amino acids and a reactive partner. The recognition tag may be a peptide sequence that is not naturally present in a polypeptide that includes an enzyme recognition tag. Examples of TGase recognition tags include the amino acid sequences disclosed in WO2012 / 059882 and WO2014 / 072482.

[0292] As exemplified in WO 2013 / 092983 and WO 2020 / 188061, the disclosures of which are incorporated herein by reference, the linker-drug moiety (XZ) can be conjugated to a glutamine residue (acceptor glutamine) in an antibody in a two-step process comprising a first step of conjugating a moiety comprising a primary amine and a first reactive group (R) to the antibody in the presence of BTG, and then reacting the antibody-linker conjugate with a molecule comprising (i) a second reactive group (R') that is reactive with the first reactive group, and (ii) a cytotoxic agent (Z). Examples of reactive group pairs with R and R' include a variety of groups capable of biorthogonal reactions, such as between azides and cyclooctynes ​​(copper-free click chemistry), 1,3-dipolar cycloaddition between nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, and tetrazine ligation (see also WO 2013 / 092983). Thus, the resulting linker and functionalized antibody, or Y element thereof, can include an RR' group, e.g., a triazole, resulting from the reaction of R and R'.

[0293] The anti-nectin-4 immunoconjugate can be incorporated into the pharmaceutical formulation at a concentration of 1 mg / ml to 500 mg / ml, wherein the formulation has a pH of 2.0 to 10.0. The formulation may further comprise a buffer system, a preservative, an isotonicity agent, a chelating agent, a stabilizer and a surfactant. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation comprising water. Such formulations are typically solutions or suspensions. In a further embodiment, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation comprising at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution comprising at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension comprising at least 50% w / w water.

[0294] In another embodiment, the pharmaceutical formulation is a lyophilized formulation, whereto the physician or the patient adds solvents and / or diluents prior to use.

[0295] In another embodiment, the pharmaceutical formulation is a dried formulation (eg, a freeze-dried or spray-dried formulation) ready for use without prior dissolution.

[0296] In a further embodiment, a pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present in a concentration of 1 mg / ml or greater, and wherein the formulation has a pH of about 2.0 to about 10.0.

[0297] In another embodiment, the pH of the formulation is in the range selected from the list consisting of about 2.0 to about 10.0, about 3.0 to about 9.0, about 4.0 to about 8.5, about 5.0 to about 8.0, and about 5.5 to about 7.5.

[0298] In further embodiments, the buffering agent is selected from the group consisting of sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, bicine, tricine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffering agents constitutes an alternative embodiment of the invention.

[0299] In a further embodiment, the formulation further comprises a pharma- ceutically acceptable preservative. In a further embodiment, the formulation further comprises an isotonicity agent. In a further embodiment, the formulation also comprises a chelating agent. In a further embodiment of the invention, the formulation further comprises a stabilizer. In a further embodiment, the formulation further comprises a surfactant. For ease of explanation, see Remington: The Science and Practice of Pharmacy, 1999. th edition, 1995 is referred to.

[0300] Other ingredients may possibly be present in the pharmaceutical formulation of the present invention.Such additional ingredients may include wetting agents, emulsifiers, antioxidants, bulking agents, osmotic pressure regulators, chelating agents, metal ions, oil media, proteins (e.g., human serum albumin, gelatin or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine and histidine).Such additional ingredients should of course not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

[0301] Administration of the pharmaceutical compositions of the invention can be by several routes of administration, for example, intravenously. Appropriate antibody formulations can also be determined by examining the experience with other already developed therapeutic ADCs.

[0302] In any embodiment, a composition can be characterized as comprising a plurality of Nectin-4-binding immunoconjugates of the present disclosure, where at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in a sample have at least 4, 6 or 8 amino acid residues per antibody functionalized with a linker disclosed herein. In any embodiment, a composition can be characterized as comprising a plurality of Nectin-4-binding immunoconjugates of the present disclosure, where at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in a sample have at least 2, 4, 6 or 8 amino acid residues per antibody functionalized with a linker-camptothecin moiety, e.g., an (XZ) unit or (-(Y)-(Pep)-(Y')-(Z)) unit of the formulas herein. In any embodiment, a composition can be characterized as comprising a plurality of Nectin-4-binding immunoconjugates of the present disclosure, where at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in a sample have the same number of functionalized amino acids per antibody, which number can optionally be 4, 6, or 8.

[0303] Diagnostic methods, prognostic symptoms and treatment of malignant tumors In some aspects, methods and antibodies, antibody fragments and immunoconjugates useful for the diagnosis, prognosis, monitoring and treatment of cancers characterized by tumor cells expressing Nectin-4 on their surface are described. In the therapeutic methods, the treatment comprises administering an antibody of the present disclosure to a human subject or individual. In certain embodiments, improved methods are provided for improving the delivery of cytotoxic agents, particular camptothecin analogs, to Nectin-4 positive tumors. The improved delivery of cytotoxic agents may be the result of antibody-mediated inhibition of clustering and / or anchorage-independent growth of Nectin-4 expressing tumor cells, resulting in improved tumor infiltration of the cytotoxic agent and / or ADCs containing such agents. Treatment with the ADCs of the present disclosure is particularly advantageous for the treatment of diseases with lower or heterogeneous Nectin-4 expression, particularly for patients with resistant disease or for whom other ADCs are not suitable, and / or for use in combination with additional therapeutic agents (e.g., chemotherapeutic agents) that mediate toxicity as single agents.

[0304] In one embodiment, anti-Nectin-4 antibody or antibody fragment may be useful for sensitizing tumors to cytotoxic or chemotherapeutic agents. Anti-Nectin-4 antibody may be useful in enhancing or enhancing the toxicity of anti-cancer agents to cancers compared to the anti-cancer agents themselves by reducing the chemoresistance of cancerous cells. Cancer-sensitizing compositions comprising anti-Nectin-4 antibody or antibody fragment may be useful for sensitizing tumors (e.g., Pgp-expressing tumors) to anti-cancer agents, reducing chemoresistance, and improving the therapeutic effect of anti-cancer agents.

[0305] In one embodiment, the anti-Nectin-4 antibody or antibody fragment of the present disclosure can be specified for use in treating tumors in human individuals in combination with a chemotherapeutic agent, where the anti-Nectin-4 antibody, antibody fragment and chemotherapeutic agent are formulated for separate administration and administered simultaneously or sequentially. For example, the treatment can include administering to an individual an effective amount of each of (a) the anti-Nectin-4 antibody or antibody fragment of the present disclosure, and (b) an anti-cancer agent, optionally a chemotherapeutic agent, optionally a chemotherapeutic agent known to be capable of being transported by P-glycoprotein (Pgp), optionally an anthracycline, a vinca alkaloid, an etoposide, a taxane, a platinum compound or optionally a camptothecin.

[0306] In one embodiment, the antibody or antibody fragment is conjugated to a cytotoxic agent, such as a camptothecin, exatecan, or SN-38 molecule. The antibody or antibody fragment can typically be conjugated to multiple molecules of a cytotoxic agent, such as a camptothecin analog, exatecan. The cytotoxic agent, such as camptothecin or exatecan, can be conjugated to the antibody via a linker that includes a protease-cleavable oligopeptide linker (e.g., a linker-toxin of any of Formulas VII-XIV). An exemplary pharmaceutical composition can include, on average, 1-8 cytotoxic agent molecules (e.g., a camptothecin derivative, exatecan, a linker-toxin of any of Formulas VII-XIV) per antibody molecule, optionally 2-8, 4-8, 6-8 cytotoxic agent molecules per antibody molecule, or, for example, about 2, 4, 5, 6, 7, or 8 cytotoxic agent molecules per antibody molecule. When an antibody or antibody fragment is conjugated to exatecan (i.e., via a linker, e.g., a linker-toxin of any of Formulas VII-XIV), such immunoconjugates are particularly advantageous for treating Pgp-expressing tumors, e.g., tumors characterized by expression of Pgp (MDR1). Tumors characterized by expression of Pgp include tumors following treatment with chemotherapeutic agents and ADCs, e.g., treatment with ADCs containing an auristatin (MMAE) payload has been shown to induce Pgp expression in tumors. In addition, a variety of tumors are known that are characterized by native Pgp / MDR1 expression, for example, significant levels of MDR1 are expressed by renal carcinoma, adrenocortical carcinoma, bile duct carcinoma, liver cancer, rectal cancer, colon cancer, brain cancer, pancreatic cancer, prostate cancer, mesothelioma, gastric cancer, AML, thyroid cancer, DLBC, esophageal cancer, breast cancer, sarcoma, testicular cancer, uterine cancer, thymoma, cervical cancer, lung cancer, bladder cancer (e.g., urothelial carcinoma), and head and neck squamous cell carcinoma.

[0307] A Nectin-4 binding agent (e.g., an anti-Nectin-4 antibody or antibody fragment) conjugated to a cytotoxic agent can be advantageously used to treat individuals with Nectin-4-expressing cancers characterized by tumor cells expressing Nectin-4 (e.g., on the tumor cell membrane or cell surface). Examples of such cancers are urothelial cancer, breast cancer (e.g., triple-negative breast cancer, HER2-positive breast cancer), non-small cell lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, colorectal cancer (e.g., colon cancer), head and neck squamous cell carcinoma, and esophageal cancer.

[0308] Nectin-4 binding agents conjugated to cytotoxic agents can be used in Nectin-4 high expressing tumors.

[0309] Nectin-4 binding agents conjugated to cytotoxic agents can also be used in xenogeneic and / or low Nectin-4 expressing tumors, in which the immunoconjugates of the present disclosure can provide beneficial efficacy, optionally through circumvention of MDR1-mediated resistance and / or bystander antitumor effects.

[0310] A nectin-4 binding agent conjugated to a cytotoxic agent can be advantageously used to treat an individual regardless of the nectin-4 expression level, regardless of the heterogeneity of the nectin-4 expression level in the individual's tumor cells, and / or regardless of whether the individual has been previously treated with enfortumab vedotin. When the cytotoxic agent is exatecan, a nectin-4 binding agent conjugated to a cytotoxic agent can also be advantageously used to treat an individual regardless of the tumor Pgp expression and / or regardless of whether the individual has been previously treated with an ADC comprising a cytotoxic agent capable of being transported by Pgp (e.g., an ADC comprising an anti-nectin-4 antibody conjugated to a camptothecin analog other than exatecan, an ADC comprising an anti-nectin-4 antibody conjugated to Dxd (e.g., via a GGFG-containing linker), or an anti-nectin-4 antibody conjugated to an auristatin, an anthracycline, a vinca alkaloid, an etoposide, a taxane, or a platinum compound).

[0311] In one embodiment, the nectin-4 binding agent conjugated to a camptothecin derivative molecule can be advantageously used to treat an individual who has previously been treated with enfortumab vedotin. Such an individual may have a cancer characterized by heterogeneous and / or low nectin-4 expressing tumors, optionally after enfortumab vedotin treatment. Such an individual may have a cancer characterized by Pgp expressing tumors, optionally after enfortumab vedotin treatment. The individual may have a resistant cancer, a non-responsive cancer, a relapsed cancer and / or a progressed cancer despite (e.g., during or after) treatment with an antibody conjugated to an auristatin or MMAE molecule (e.g., enfortumab vedotin). For example, the individual may have a locally advanced or metastatic urothelial cancer and have previously been treated with an antibody conjugated to an auristatin or MMAE molecule (e.g., enfortumab vedotin).

[0312] In one embodiment, an immunoconjugate comprising a nectin-4 binding agent conjugated via a linker to exatecan is advantageously used to treat an individual who has previously been treated with an immunoconjugate comprising a nectin-4 binding agent conjugated via a linker to a cytotoxic agent capable of transport by Pgp. In one embodiment, an immunoconjugate comprising a nectin-4 binding agent conjugated via a linker to exatecan is advantageously used to treat an individual who has a cancer characterized by a xenogeneic and / or low nectin-4 expressing tumor after treatment with an immunoconjugate comprising a nectin-4 binding agent conjugated via a linker to a cytotoxic agent capable of transport by Pgp. In one embodiment, an immunoconjugate comprising a nectin-4 binding agent conjugated to exatecan via a linker is advantageously used to treat individuals with cancer that is resistant, unresponsive, relapsed and / or progressed despite (e.g., during or after) treatment with a nectin-4 binding agent conjugated to a cytotoxic agent capable of transport by Pgp via a linker. Optionally, the nectin-4 binding agent conjugated to a cytotoxic agent capable of transport by Pgp via a linker may comprise an anti-nectin-4 antibody conjugated to a camptothecin analogue via a linker to release a camptothecin analogue other than exatecan (e.g., Dxd or deruxtecan) upon cleavage of the linker. Optionally, the nectin-4 binding agent conjugated to a cytotoxic agent capable of transport by Pgp via a linker may comprise an anti-nectin-4 antibody conjugated to Dxd (e.g., via a GGFG-containing linker). Optionally, the Nectin-4 binding agent conjugated via a linker to a cytotoxic agent capable of being transported by Pgp may include an anti-Nectin-4 antibody conjugated to an auristatin, an anthracycline, a vinca alkaloid, an etoposide, a taxane or a platinum compound.Optionally, the nectin-4 binding agent conjugated via a linker to a cytotoxic agent capable of transport by Pgp may comprise an anti-nectin-4 antibody that binds to the IgV domain of nectin-4. Optionally, the nectin-4 binding agent conjugated via a linker to a cytotoxic agent capable of transport by Pgp may comprise an anti-nectin-4 antibody that binds to the VC1 cross-linking domain of nectin-4. In one embodiment, an immunoconjugate comprising a nectin-4 binding agent conjugated via a linker to exatecan comprises a linker having an enzymatically cleavable moiety (and optionally a self-eliminating spacer) that upon cleavage results in release of exatecan.

[0313] In advanced recurrent or metastatic urothelial carcinoma, a significant proportion of individuals express high levels of nectin-4 in tumor cells, e.g., H-scores of at least 290 (see EV-201 Clinical Trial Cohort 1 Nectin-4 Expression). However, a subset of patients has an H-score below 250, and some have an H-score below 200. A small number of patients have an H-score below 150, and some have an H-score below 100. In triple-negative breast cancer (TNBC), it has been reported that 62% of patients have high nectin-4 expression in tumor cells, and 38% have low nectin-4 expression in tumor cells (Rabat et al., 2017 Annals Onc. 28: 769-776). In other cancer types, the median H-scores of nectin-4 expression were typically lower than those observed in UC, particularly in non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer.

[0314] In one embodiment, the individual treated in accordance with the present disclosure has advanced recurrent or metastatic cancer, optionally advanced recurrent or metastatic urothelial carcinoma.

[0315] In one embodiment, an individual treated according to the present disclosure has a cancer (e.g., breast cancer) that tests positive for estrogen receptors and / or progesterone receptors and tests negative for epidermal growth factor receptor 2 (HER2) or excess HER2 protein, or the cancer may test positive for HER2 but express HER2 at low levels.

[0316] In one embodiment, the individual treated according to the present disclosure has triple negative breast cancer (TNBC), eg, breast cancer that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein.

[0317] In one embodiment, the individual treated according to the present disclosure has a cancer (e.g., breast cancer) that tests positive for HER2 protein, where optionally the cancer expresses excess HER2 protein (HER2 overexpression) or the cancer expresses low levels of HER2 protein (less than excess HER2 expression). In one embodiment, the individual is treated with an anti-Nectin-4 ADC in combination with an agent (e.g., an antibody) that binds to a HER2 polypeptide (e.g., trastuzumab, pertuzumab), where the agent that binds to HER2 may be an ADC, where the antibody that binds to HER2 may be conjugated to a cytotoxic agent, optionally an auristatin, a maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compound 1, 2 or 13), and where the antibody that binds to HER2 may be trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a; Enhertu®).

[0318] In one embodiment, the individual treated in accordance with the present disclosure has non-small cell lung cancer, optionally lung adenocarcinoma.

[0319] In one embodiment, the individual treated according to the present disclosure has pancreatic cancer.

[0320] In one embodiment, the individual treated according to the present disclosure has ovarian cancer.

[0321] In one embodiment, the individual treated according to the present disclosure has head and neck squamous cell carcinoma.

[0322] In one embodiment, the individual treated according to the present disclosure has esophageal cancer.

[0323] In one embodiment, the individual treated according to the present disclosure has colorectal cancer. Colorectal cancer (CRC), as used herein, refers to colon cancer, rectal cancer and colorectal cancer (cancer of both the colon and rectum).

[0324] In one embodiment, the individual treated according to the present disclosure has NSCLC or lung adenocarcinoma, gastric cancer, colorectal carcinoma, pancreatic cancer, urothelial carcinoma or bladder cancer that tests positive for HER2 protein, where the cancer may express too much HER2 protein (HER2 overexpression) or the cancer may express low levels of HER2 protein (less than overexpression). In one embodiment, an individual is treated with an anti-Nectin-4 ADC of the disclosure in combination with an agent (e.g., an antibody) that binds a HER2 polypeptide (e.g., an antibody comprising the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab), where the agent that binds HER2 can be an ADC, the antibody that binds HER2 can be conjugated to a cytotoxic agent, optionally an auristatin, a maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compounds 1, 2 or 13), and the antibody that binds HER2 can be trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a).

[0325] As shown herein, ADCs that release exatecan upon cleavage (e.g., upon cleavage of an intracellularly cleavable linker that contains a cytotoxic agent Z) are particularly advantageous for treating tumors that are resistant to ADCs having linkers that release a cytotoxic agent (other than exatecan) delivered by Pgp, e.g., auristatins or Dxd, upon cleavage. Thus, in one embodiment, when a HER2-positive cancer is treated with a combination of an anti-Nectin-4 binding agent (e.g., an anti-Nectin-4 ADC) and an anti-HER2 ADC, the anti-HER2 ADC can be designed to release exatecan upon cleavage (e.g., upon cleavage of an intracellularly cleavable linker that contains a cytotoxic agent Z). Anti-Nectin-4 binding agents (e.g., anti-Nectin-4 ADCs) can be designed to release exatecan upon cleavage (e.g., upon cleavage of an intracellularly cleavable linker that includes a cytotoxic agent Z) or can release any other cytotoxic agent (Z), where, for example, Z is a taxane, anthracycline, camptothecin, epothilone, mitomycin, combretastatin, vinca alkaloid, nitrogen mustard, maytansinoid, duocarmycin, tubulysin, dolastatin, auristatin, enediyne, pyrrolobenzodiazepine, amatoxine, or ethyleneimine.

[0326] Thus, in one aspect, the disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide for use in treating cancer (e.g., a HER2-positive Nectin-4-positive cancer), the immunoconjugate that binds to a human Nectin-4 polypeptide having the following formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4comprises a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; Z N4 contains cytotoxic agents] and the immunoconjugate that binds to the human Nectin-4 polypeptide is represented by the following formula: Ab HER2 -(X HER2 -(Z HER2 )) [In the formula, Ab HER2 is a polypeptide, peptide or antibody that specifically binds to a human HER2 polypeptide, and optionally, Ab HER2 may comprise the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab; X HER2 Ab HER2 and Z HER2 A molecule that connects X HER2 contains a moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z HER2 is exatecan] The immunoconjugate that binds to the human HER2 polypeptide releases exatecan upon cleavage of the cleavable moiety. N4 may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0327] Also, in one aspect, the disclosure provides an immunoconjugate that binds a human HER2 polypeptide for use in treating cancer (e.g., a HER2-positive Nectin-4-positive cancer), the immunoconjugate that binds a human HER2 polypeptide having the following formula: AbHER2 -(X HER2 -(Z HER2 )) [In the formula, Ab HER2 is a polypeptide, peptide or antibody that specifically binds to a human HER2 polypeptide, HER2 optionally comprises the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab; X HER2 Ab HER2 and Z HER2 A molecule that connects X HER2 contains a moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z HER2 is exatecan] and the immunoconjugate that binds to the human HER2 polypeptide is represented by the following formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z N4 contains cytotoxic agents] The immunoconjugate that binds to the human HER2 polypeptide releases exatecan upon cleavage of the cleavable moiety. N4may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0328] Thus, in one aspect, the disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide for use in treating cancer (e.g., a TROP-2 positive, Nectin-4 positive cancer), the immunoconjugate that binds to a human Nectin-4 polypeptide having the following formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z N4 contains cytotoxic agents] and the immunoconjugate that binds to the human Nectin-4 polypeptide is represented by the following formula: Ab TROP-2 -(X TROP-2 -(Z TROP-2 )) [In the formula, Ab TROP-2 is a polypeptide, peptide or antibody that specifically binds to a human TROP-2 polypeptide, HER2 optionally comprising the heavy and light chain CDRs or variable regions of datopotamab or sacituzumab; X TROP-2 Ab TROP-2 and Z TROP-2 A molecule that connects X TROP-2contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z TROP-2 is exatecan] The immunoconjugate that binds to the human TROP-2 polypeptide releases exatecan upon cleavage of the cleavable moiety. N4 may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0329] Also in one aspect, the disclosure provides an immunoconjugate that binds a human TROP-2 polypeptide for use in treating cancer (e.g., a TROP-2 positive Nectin-4 positive cancer), the immunoconjugate that binds a human TROP-2 polypeptide having the following formula: Ab TROP-2 -(X TROP-2 -(Z TROP-2 )) [In the formula, Ab TROP-2 is a polypeptide, peptide or antibody that specifically binds to a human TROP-2 polypeptide, TROP-2 optionally comprising the heavy and light chain CDRs or variable regions of datopotamab or sacituzumab; X TROP-2 Ab TROP-2 and Z TROP-2 A molecule that connects X TROP-2 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z TROP-2 is exatecan] and the immunoconjugate that binds to the human TROP-2 polypeptide is represented by the following formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z N4 contains cytotoxic agents] The immunoconjugate that binds to the human TROP-2 polypeptide releases exatecan upon cleavage of the cleavable moiety. N4 may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0330] Thus, in one aspect, the disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide for use in treating cancer (e.g., a B7H3-positive Nectin-4-positive cancer), the immunoconjugate that binds to a human Nectin-4 polypeptide having the formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z N4 contains cytotoxic agents] and the immunoconjugate that binds to the human Nectin-4 polypeptide is represented by the following formula: Ab B7H3 -(X B7H3 -(Z B7H3 )) [In the formula, Ab B7H3 is a polypeptide, peptide or antibody that specifically binds to a human B7H3 polypeptide, HER2 may optionally comprise the heavy and light chain CDRs or variable regions of enoblitutamab, ifinatamab, mirzotamab, obrindatamab, omburtamab, or vovlamitamab; X B7H3 Ab B7H3 and Z B7H3 A molecule that connects X B7H3 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z B7H3 is exatecan] The immunoconjugate that binds to the human B7H3 polypeptide releases exatecan upon cleavage of the cleavable moiety. N4 may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0331] Also, in one aspect, the disclosure provides an immunoconjugate that binds a human B7H3 polypeptide for use in treating cancer (e.g., a B7H3-positive Nectin-4-positive cancer), the immunoconjugate that binds a human B7H3 polypeptide having the following formula: Ab B7H3 -(X B7H3 -(Z B7H3 )) [In the formula, Ab B7H3 is a polypeptide, peptide or antibody that specifically binds to a human B7H3 polypeptide, B7H3 may optionally comprise the heavy and light chain CDRs or variable regions of enoblitutamab, ifinatamab, mirzotamab, obrindatamab, omburtamab, or vovlamitamab; X B7H3 Ab B7H3 and Z B7H3 A molecule that connects X B7H3 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or pentapeptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z B7H3 is exatecan] and an immunoconjugate that binds to the human B7H3 polypeptide is represented by the following formula: Ab N4 -(X N4 -(Z N4 )) [In the formula, Ab N4 is a polypeptide, peptide or antibody that specifically binds to a human Nectin-4 polypeptide; X N4 Ab N4 and Z N4 A molecule that connects X N4 contains a cleavable moiety (optionally a protease-cleavable di-, tri-, tetra- or penta-peptide), e.g., under physiological conditions, optionally under intracellular conditions; and Z N4 contains cytotoxic agents] The immunoconjugate that binds to the human Nectin-4 polypeptide is represented by the formula: N4 may be exatecan, and the immunoconjugate that binds to the human nectin-4 polypeptide may release exatecan upon cleavage of the cleavable moiety.

[0332] In one embodiment, the therapeutic method of the present disclosure does not depend on the assessment or detection of Nectin-4 expression in tumor tissue and / or does not depend on the expression level of Nectin-4 in tumor cells and / or the frequency or number of Nectin-4-expressing tumor cells in a tissue sample from said individual. In one embodiment, the therapeutic method of the present disclosure does not depend on the assessment or detection of Pgp(MDR1) expression in tumors.

[0333] In one aspect, the present invention provides a method of treating cancer and / or inducing an anti-tumor immune response in an individual in need thereof, wherein the individual has advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC), and wherein the method does not require prior determination of whether the individual has tumor tissue comprising cells (e.g., tumor cells) that express Nectin-4.

[0334] In one aspect, the present invention provides a method of treating cancer and / or killing tumor cells in an individual in need thereof, wherein the individual has advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC), and the method does not require prior determination of whether the individual has tumor tissue that contains cells (e.g., tumor cells) that express high levels of Nectin-4, e.g., as defined by immunohistochemical assessment (e.g., H-score or other suitable IHC scoring method).

[0335] In one embodiment, the method of treating cancer and / or killing tumor cells in an individual does not require pre-determining the level of Nectin-4 expression in the tumor cells.

[0336] In one embodiment, a method of treating cancer in an individual, optionally advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC, HER2-positive cancer), comprises treating an individual having a cancer characterized by an H-score of less than or equal to 290, 250, 200, 150 or 100 for Nectin-4 expression.

[0337] In any embodiment relating to treating or preventing cancer in an individual, the method can be defined as comprising (i) identifying an individual whose tumor cells express Nectin-4 (e.g., as determined by immunohistochemistry), and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody-drug conjugate of the present disclosure.

[0338] In any of the embodiments relating to treating or preventing cancer in an individual, the method includes the steps of (i) identifying an individual whose tumor cells express (a) Nectin-4 (e.g., as determined by immunohistochemistry) and (b) HER2 (wherein the tumor cells may express low levels of HER2) (e.g., as determined by immunohistochemistry; as determined by Herceptest™), and (ii) administering an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure, optionally in combination with an agent (e.g., an antibody) that binds to a Her2 polypeptide (e.g., an antibody). For example, the method can be defined as comprising administering to an individual in combination with an antibody that binds HER2, optionally an ADC, optionally conjugated to a cytotoxic agent, optionally an auristatin, a maytansinoid (e.g., DM1) or a camptothecin derivative (e.g., compounds 1, 2 or 13), and optionally trastuzumab emtansine or trastuzumab deruxtecan (DS-8201A), wherein the antibody that binds Her2 may optionally be HER2-binding mAb or trastuzumab deruxtecan (DS-8201A).

[0339] In any embodiment relating to treating or preventing cancer in an individual, the method can be defined as comprising the steps of (i) identifying an individual whose tumor cells have low or moderate levels of Nectin-4 expression (e.g., as determined by immunohistochemistry), and (ii) administering an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure to the individual identified in step (i).

[0340] In any embodiment relating to treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method can be defined as comprising (i) identifying an individual whose cancer is characterized by a low level of Nectin-4 expression (e.g., as determined by immunohistochemistry), and (ii) administering an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure to the individual identified in step (i). In one embodiment, the individual has a cancer characterized by an H-score of less than or equal to 150 or 100 for Nectin-4 expression.

[0341] In any embodiment relating to treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method can be defined as comprising (i) identifying an individual whose cancer is characterized by a moderate level of tumor Nectin-4 expression (e.g., as determined by immunohistochemistry), and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure. In one embodiment, the individual has a cancer characterized by an H-score for Nectin-4 expression of less than or equal to 290, 250, 200, 150, or less, and further, the cancer may be characterized by an H-score for Nectin-4 expression of at least 100.

[0342] In a further embodiment, a method for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual is provided, comprising (i) identifying an individual whose cancer is characterized by an H-score of less than or equal to 290, 250, 200, 150, 120 or 100 for tumor Nectin-4 expression, and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure. Optionally, step (i) may be defined as comprising evaluating Nectin-4 expression in tumor cells by histochemistry (e.g., IHC).

[0343] In a further embodiment, there is provided a method for treating or preventing cancer (e.g., Nectin-4 positive cancer, breast cancer) in an individual, comprising (i) identifying an individual whose cancer is characterized by a QS score of less than or equal to 200, 150, 120 or 100 for tumor Nectin-4 expression, and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure. Optionally, step (i) may be defined as comprising evaluating Nectin-4 expression in tumor cells by histochemistry (e.g., IHC).

[0344] A biological sample from an individual (e.g., from a biopsy) can be collected and evaluated. The sample can be stored as a formaldehyde (e.g., formalin) fixed paraffin embedded (FFPE) sample. After deparaffinization, the slide can be subjected to a method for detecting the expression of Nectin-4 (and / or HER2, TROP-2, B7H3).

[0345] The expression of Nectin-4, TROP-2, B7H3 and / or HER2 in tumor cells can be determined by any method known in the art.In certain embodiments, assays include immunohistochemistry (IHC) assays, fluorescence-activated cell sorting (FACS) assays, such as quantitative FACS, ELISA, immunoblotting (e.g., Western blotting, dot blotting or in-cell Western blotting), and other immunoassays.

[0346] IHC staining of tissue sections has been shown to be a reliable method of evaluating or detecting the presence of proteins in a sample. Immunohistochemistry techniques generally utilize antibodies to probe and visualize cellular antigens in situ by chromogenic or fluorescent methods. Thus, antibodies or antisera specific to each marker are used to detect expression, in some embodiments polyclonal antisera, and in some embodiments monoclonal antibodies. Antibodies can be detected by direct labeling of the antibody itself, for example, with radioactive labels, fluorescent labels, hapten labels such as biotin, or enzymes such as horseradish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibodies are used with labeled secondary antibodies, including antisera, polyclonal antisera, or monoclonal antibodies specific to the primary antibodies. Immunohistochemistry protocols and kits are well known in the art and are commercially available.

[0347] In some embodiments, the IHC assay is a direct assay that directly determines the binding of an antibody to a target antigen. This direct assay uses a labeling reagent that can be visualized without another antibody interaction, such as a fluorescent tag or an enzyme-labeled primary antibody. In some embodiments, the IHC assay is an indirect assay. In a typical indirect assay, an unconjugated primary antibody binds to an antigen, and then a labeled secondary antibody binds to the primary antibody. If the secondary antibody is conjugated to an enzyme label, a chromogenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification is performed because some secondary antibodies may react with different epitopes of the primary antibody. The primary and / or secondary antibodies used for immunohistochemistry are usually labeled with a detectable molecule. Numerous labels are available, including radioisotopes, colloidal gold particles, fluorescent labels, and enzyme-substrate labels.

[0348] Strong staining, moderate staining, and weak staining are well-known descriptions to those skilled in the art. In some embodiments, strong staining, moderate staining, and weak staining are calibrated levels of staining, where a range is established and the intensity of the staining is binned within the range. In some embodiments, strong staining is staining above 75% of the intensity range, moderate staining is staining between 25-75% of the intensity range, and low staining is staining below 25% of the intensity range. In some embodiments, those skilled in the art and familiar with a particular staining technique will adjust the bin size to define the staining category.

[0349] Control cell lines (e.g., centrifuged into pellets, formalin-fixed paraffin-embedded, prepared, e.g., as tissue microarrays, and stained, e.g., with anti-Nectin-4 antibodies) with various staining intensities (e.g., when stained with anti-Nectin-4 antibodies) can be utilized as controls for IHC analysis. Those skilled in the art will appreciate that other control cell pellets with negative, weak, moderate, and high C-MET staining intensities can be readily identified using the teachings of the present application and methods well known in the art and disclosed herein.

[0350] In certain embodiments, a cancer or tumor is considered to be a Nectin-4-expressing cancer tumor if it is Nectin-4 positive (eg, determined to be Nectin-4 positive using an IHC assay).

[0351] In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 5% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 10% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 20% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 30% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 40% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 50% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 60% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 70% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity). In certain embodiments, an individual's cancer or tumor is Nectin-4 positive if 80% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity).In certain embodiments, an individual's cancer or tumor is Nectin-4 positive if 90% or more of the tumor cells in a sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity).

[0352] In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 5% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 10% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 20% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 30% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 40% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 50% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 60% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 70% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 80% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity. In some embodiments, an individual's cancer or tumor is Nectin-4 positive when 90% or more of the tumor cells in a sample express Nectin-4 protein at moderate and / or strong staining intensity.

[0353] Evaluating immunohistochemical assays to determine whether an individual's cancer or tumor is characterized by high Nectin-4 expression (e.g., low or intermediate Nectin-4 expression) may generally involve application of known scoring methods.

[0354] Low, medium and high tumor Nectin-4 expression can be determined based on the "H score" as described in US Patent Application Publication No. 2013 / 0005678. The H score is obtained by the formula: (3×percentage of strongly stained cells)+(2×percentage of moderately stained cells)+(percentage of weakly stained cells) and ranges from 0 to 300. The H score has been used especially in UC.

[0355] In certain embodiments of any of the methods herein, low or moderate Nectin-4 expression (e.g., a tumor or tumor cell having a low or moderate level of Nectin-4 expression) corresponds to an H-score of about 250 or less, about 220 or less, about 200 or less, about 180 or less, about 160 or less, about 150 or less, about 140 or less, about 130 or less, about 120 or less, about 110 or less, or about 100 or less.

[0356] In certain embodiments of any of the methods herein, low Nectin-4 expression (e.g., a tumor or tumor cells having a low level of Nectin-4 expression) corresponds to an H-score of 200 or less, about 180 or less, about 160 or less, about 150 or less, about 140 or less, about 130 or less, about 120 or less, about 110 or less, or about 100 or less.

[0357] In certain embodiments of any of the methods herein, high Nectin-4 expression (eg, tumors or tumor cells with high levels of Nectin-4 expression) corresponds to an H-score of about 290 or greater.

[0358] In another example, Nectin-4 staining can be scored by Quick Score (QS) using the following formula: QS=P (percentage of positive cells)×I (intensity), with a maximum score of 300. QS has been used, for example, in breast cancer. For example, in TNBC, some research groups have defined low Nectin-4 expression groups as QS≦100. In certain embodiments of any of the methods herein, low or medium Nectin-4 expression (e.g., tumors or tumor cells with low or medium levels of Nectin-4 expression) corresponds to a QS score of about 200 or less, about 180 or less, about 160 or less, about 150 or less, about 140 or less, about 130 or less, about 120 or less, about 110 or less, or about 100 or less.

[0359] Assays for evaluating tumor cell expression of HER2 are well known in the art. For example, assays such as SPoT-Light HER2 CISH, which is approved by FDA, can be used to detect HER2 overexpression. Chromogenic in situ hybridization (CISH) detects HER2 gene amplification. This technique, also called Subtraction Probe Technology Chromogenic In Situ Hybridization, is a test used to confirm whether breast cancer cells overexpress HER2 receptor protein on the cell surface.

[0360] Another widely used assay for HER2 is HercepTest™ (Dako North America, Inc.), a semi-quantitative immunohistochemistry assay used to determine HER2 protein overexpression in formalin-fixed, paraffin-embedded cancer tissues. For example, tumors expressing low levels of HER2 can be identified by HercepTest™ with a score of +1 to +2.

[0361] In one embodiment, the treatment is used in individuals with pre-existing neuropathy, diabetes or hyperglycemia, heart failure, eye conditions, etc. Such conditions may make the individual unsuitable for treatment with an anti-Nectin-4 ADC, such as enfortumab vedotin, which has higher toxicity or a narrower therapeutic window than the anti-Nectin-4 antibody drug conjugates of the present disclosure.

[0362] In any embodiment, the treatment method may optionally include (a) assessing the stage of cancer and / or disease progression in the individual, and (b) if the individual has recurrent, metastatic and / or advanced cancer, administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure.

[0363] In certain embodiments, the present invention includes a method of treating a tumor in an individual having urothelial carcinoma, comprising: (a) assessing the stage of cancer and / or disease progression in the individual; and (b) if the individual has recurrent, metastatic and / or advanced cancer, administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure.

[0364] Optionally, an individual to be treated with the anti-Nectin-4 antibody drug conjugate of the present disclosure may have a cancer that is resistant, has not responded, or has relapsed and / or progressed despite (e.g., during or after) surgery and / or treatment with a therapeutic agent, such as a chemotherapeutic agent, an antibody, an ADC or radiation therapy (e.g., urothelial cancer, breast cancer (e.g., triple-negative breast cancer, HER2-positive cancer), non-small cell lung cancer, pancreatic cancer, ovarian cancer, gastric cancer, colorectal cancer, head and neck squamous cell carcinoma, or esophageal cancer).

[0365] In any of the embodiments herein, therapeutic response can be defined and / or evaluated according to well-known criteria, e.g., RECIST (Response Evaluation Criteria In Solid Tumors), e.g., version 1.1 (Eisenhauer et al. (2009) Eur. J. Cancer 45:228-247), or Immune-Related Response Criteria (irRC) (see Wolchock et al. (2009) Clinical Cancer Research 15:7412-7420).

[0366] Optionally, an individual to be treated with the anti-Nectin-4 antibody drug conjugate of the present disclosure may have a tumor or cancer that is resistant to, unresponsive to, or has progressed following treatment with chemotherapeutic agents known to be capable of transport by P-glycoprotein (Pgp), such as anthracyclines (doxorubicin, daunorubicin, taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vindesine), and etoposides. Compounds recognized by Pgp are typically characterized as being moderately hydrophobic (octanol-water partition coefficient, logP>1), often contain titratable protons with a net positive charge under physiological conditions, and are "natural products" with predominantly aromatic moieties.

[0367] In certain embodiments, ADCs comprising anti-Nectin-4 antibodies, antibody fragments are used or administered in the absence of concomitant administration of a chemotherapeutic agent.

[0368] In other embodiments, anti-Nectin-4 antibodies, antibody fragments, or ADCs comprising such may be used or administered in combination with a chemotherapeutic agent. Exemplary chemotherapeutic agents include, but are not limited to, amsacrine, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil, gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan, leucovorin, ribavir ... Liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin, tegafur-uracil, temozolomide, teniposide, thiotepa, thioguanine, topotecan, treosulfan, vinblastine, vincristine, vindesine, vinorelbine, or combinations thereof. In one embodiment, the anti-Nectin-4 antibody, antibody fragment (or ADC comprising such) and chemotherapeutic agent are formulated for separate administration and are administered simultaneously or sequentially.

[0369] Optionally, the individual can be characterized as having cancer that is progressing, relapsing, or not responsive to prior treatment with prior therapy, and further, the prior therapy can include administration of enfortumab vedotin and / or administration of a PD-1 neutralizing agent (e.g., pembrolizumab, atezolizumab, nivolumab), and the prior therapy can be a chemotherapeutic agent.

[0370] Optionally, in any embodiment, the individual can be characterized as being ineligible for treatment with enfortumab vedotin and / or as having a cancer that is not suitable or appropriate for treatment with enfortumab vedotin.

[0371] An exemplary treatment protocol for treating a human with an anti-Nectin-4 antibody conjugated to a camptothecin derivative molecule includes, for example, administering to a patient an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure, the method including at least one administration cycle in which at least one dose of an anti-Nectin-4 antibody conjugated to a camptothecin derivative molecule is administered at a dose of 0.1-10 mg / kg body weight, 0.1-5 mg / kg body weight, 0.1-1 mg / kg body weight, 1-10 mg / kg body weight, or 1-5 mg / kg body weight. In one embodiment, multiple doses are administered, for example at least 2, 3, 4, 5, 6, 8, 10 doses. In one embodiment, administration of multiple doses is separated by at least 2, 3, or 4 weeks. In one embodiment, administration is weekly, biweekly, triweekly, or quadruple weekly.

[0372] In one embodiment, the anti-Nectin-4 antibody drug conjugate of the present disclosure is administered by iv. EXAMPLES

[0373] Example 1: Human tumor cells co-expressing HER2, TROP-2, B7H3 and Nectin-4 A study of HER2 and Nectin-4 gene expression was performed using The Cancer Genome Atlas (a collaboration between the National Cancer Institute and the National Human Genome Research Institute), which is based on a multidimensional map of significant genomic alterations in different types of cancer. Significant correlations of HER2 and Nectin-4 expression were observed, especially in samples from pancreatic cancer, lung adenocarcinoma patients, breast cancer and bladder cancer patients. The highest correlation was observed in pancreatic cancer, with correlation values ​​of 0.71 by Spearman and 0.78 by Pearson.

[0374] HER2 and Nectin-4 expression was determined by flow cytometry in SUM185 and SUM190 human breast cancer tumor cell lines (Biovit Inc.). SUM185 originated from a pleural effusion of a patient with ER-negative, PR-negative, HER2-positive undifferentiated breast carcinoma. This cell line overexpresses HER2. SUM190 originated from a primary tumor from a patient with ER-negative, PR-negative, HER2-positive (amplified) breast cancer. Tumor cells were stained with anti-Nectin-4 (ASG-22ME modified as human IgG1 isotype with N297Q mutation to reduce binding to Fc gamma receptor), anti-TROP-2, anti-B7H3 or anti-Her2 (Trastuzumab modified as human IgG1 isotype with N297Q mutation to reduce binding to Fc gamma receptor) at 10 μg / ml (4° C.) and isotype controls, followed by staining with PE-conjugated polyclonal goat anti-human antibody at a dilution of 1:200. Samples were analyzed by cytofluorimetry using a Canto II (HTS).

[0375] Representative results for HER2 and Nectin-4 are shown in Figure 1 for SUM190 human breast cancer tumor cells and Figure 2 for SUM185 human breast cancer tumor cells. MFI: median fluorescence intensity. SUM190 tumor cells expressed HER2 at low to moderate levels (median fluorescence units 1777) and Nectin-4 at lower levels (median fluorescence units 991). SUM185 cells expressed HER2 at moderate to high levels (median fluorescence units 2880) and also expressed Nectin-4 at low levels (median fluorescence units 991). In addition, SUM185 cells expressed TROP-2 and B7H3 at high levels (median fluorescence units 17327 and 11481, respectively). Expression data are shown in Table 1 below. [Table 1]

[0376] Example 2: Efficacy of anti-nectin-4 camptothecin derivative ADC in combination with anti-HER2 ADC Anti-Nectin-4 antibody-drug conjugates were prepared and compared with Trastuzumab antibody-drug conjugates for efficacy against HER2+Nectin-4+ human tumor cells.Anti-Nectin-4 antibody-drug conjugates having VH and VL of SEQ ID NOs: 6 and 7 as human IgG1 isotype were prepared. [Table 2]

[0377] Anti-HER2 antibody-drug conjugates with trastuzumab heavy and light chains (human IgG1 isotype) were prepared. Both anti-nectin-4 and anti-HER2 antibodies were stochastically conjugated with linker-camptothecin derivatives through cysteine ​​residues of the antibodies, respectively, after partial reduction of the interchain disulfides. The reducing agent tris(2-carboxyethyl)phosphine hydrochloride ranging from 2 to 10 molar equivalents was incubated with the antibody (3 mg / ml) for 2 hours under stirring (350-400 rpm, +37°C) to reduce the disulfides. Linker-toxin conjugation was carried out by addition of a molar excess of linker-toxin of 9.2 or 12 molar equivalents, incubated overnight at +37°C on a stirring wheel. The resulting ADCs had an average drug loading (drug:antibody ratio) of approximately 8. In a further example, the same method was used to conjugate the anti-Nectin-4 antibody to a second camptothecin (SN-38) containing a linker. The ADCs used in this example are as follows:

[0378] N4 ADC1: Structure shown below: [ka] anti-nectin-4 conjugated to a linker having the formula: Her2 ADC1: Structure shown below: [ka] an anti-HER2 conjugated to a linker having the formula: N4 ADC2: Structure shown below: [ka] Anti-Nectin-4 conjugated to a linker having the formula:

[0379] The resulting ADCs were tested for their ability to induce cell death of Nectin-4 / HER2 expressing SUM190 when used in combination with MCF-7 tumor cells from Example 1. Briefly, cells were seeded in 96-well plates (V=80 μl). N4 ADC1 and HER2 ADC1 or human IgG1-isotype control (IC)-linker-toxin or media (5x concentration) were tested in 1:2 serial dilutions starting from (530 nM to 30 nM) and 1:5 serial dilutions (7 nM to 7x10) for N4 ADC1 and isotype control. -2 N4 ADC2 and isotype controls were tested at 530 nM to 5.3 10 -2 ADCs were tested in serial dilutions of 1:10 starting from 0.1%. The ability of the ADCs to induce cell death was determined by assessing confluence using an Incucyte S3-2 instrument, and viability at 6 days post-treatment was determined using a Cell Titer Glo® (CTG) assay with an Enspire2 instrument. The IC50 value of each ADC was determined using luminescent cell viability of the 6 day data by GraphPad Prism8. Experiments were repeated twice.

[0380] The results showed that the combination of anti-Nectin-4 ADC and anti-Her2 ADC showed improved efficacy (lower IC50) in killing Nectin-4+Her2+ tumor cells compared with either ADC used alone.

[0381] Example 3: Modeling and generation of a first set of anti-huNectin-4 antibodies with human framework sequences Human VH and VK templates were identified to introduce the CDRs of antibody 5E7. Each VH, VJ, VK and JK framework was analyzed individually. A parent antibody having the VH and VL amino acid sequences of SEQ ID NOs: 19 and 20, respectively, was cloned into the human subgroup IGHV1-46. * The heavy chain framework (FR1, FR2, FR3) derived from IGHJ4 * 01 (FR4) in VH and human subgroup IGKV2-28 * The light chain framework (FR1, FR2, FR3) derived from IGKJ4 * 01(FR4) into the VL.

[0382] The parental chimeric antibody Fab (HPLP) was modeled using the following heavy and light chain sequences: [Table 3]

[0383] The humanized antibody Fab H0L0 was modeled using the following heavy and light chain sequences: [Table 4]

[0384] To design the humanized variants of the light and heavy chains, we superimposed 3D models of the HPLP and H0L0 of 5E7 and probed every amino acid difference one by one. We also evaluated the intra- and extra-chain bonds between residues and ensured that critical low energy bonds were not broken by backmutating certain chains.

[0385] Starting with the raw CDR grafting, residues that differ in the framework between human and mouse were examined to identify potential backmutations.

[0386] Heavy Chain Design: To investigate the role of residue 72 (residue 71 by Kabat numbering), a backmutation was made at this residue to retain the leucine present in the parent antibody, which has the sequence TLD, whereas the H0L0 antibody has the sequence TRD. This backmutation needs to be combined with a mutation at residue 79 to avoid steric hindrance.

[0387] To investigate the role of the residue at position 79, this residue was backmutated to retain the threonine present in the parent antibody. The parent antibody has the sequence TTY, whereas the H0L0 antibody has the sequence TVY. The threonine at position 79 faces the inside of the VH domain and does not contact any other residues. The valine at position 79 was well superimposed with the threonine, but this residue forms many bonds with R72, M34, C22, and I51. The rotational position of I51 is explained by the contact with V79 (V78 in Kabat numbering). Backmutation to residue 72 (R72L; residue 71 in Kabat numbering) creates steric hindrance between I51 and L72 when V79 is present.

[0388] To investigate the role of residue 74, this residue was backmutated to retain the lysine present in the parent antibody. The parent antibody has the sequence DKS, whereas the H0L0 antibody has the sequence DTS. The lysine residue at position 74 is expressed on the surface of the molecule and is in a critical position, although not close to a potential binding site.

[0389] To investigate the role of residue 28, this residue was backmutated to retain the isoleucine present in the parent antibody. The parent antibody has the sequence YIF, whereas the H0L0 antibody has the sequence YTF. The isoleucine and threonine at position 28 were well superimposed. They are exposed on the surface of the molecule and are located near the paratope at the top of the antibody. A possible involvement of this residue in binding cannot be excluded. Moreover, this residue is included in CDR-H1 according to the IMGT definition.

[0390] The first heavy chain variant (H1) having the amino acid sequence set forth in SEQ ID NO: 39 had the substitutions R71L and V78T. The second heavy chain variant (H2) having the amino acid sequence set forth in SEQ ID NO: 41 had the substitutions R71L, T73K and V78T. The third heavy chain variant (H3) having the amino acid sequence set forth in SEQ ID NO: 43 had the substitutions T28I, R71L, T73K and V78T. The numbering of the substitutions is according to Kabat.

[0391] Light Chain Design: To investigate the role of the residue at position 2, this residue was backmutated to retain the valine residue of the parent antibody, which has the sequence DVV in the parent antibody, whereas the H0L0 antibody has the sequence DIV. The V2 residue interacts with residue K27 in CDR-L1 and further with residue E98 in CDR-L3.

[0392] To investigate the role of residue 69, this residue was backmutated, retaining the serine residue of the parent antibody. The parent antibody has the sequence SSS, whereas the H0L0 antibody has SGS. The S69 residue points towards the inside of the VL domain and forms multiple bonds with the adjacent residues W40 and M56 (located in CDR-L2), while G69 (G64 according to Kabat numbering) forms an h bond with M56. All of these residues overlap well.

[0393] To investigate the role of the residue at position 11, this residue was backmutated to retain the asparagine residue of the parent antibody. The parent antibody has a sequence SNP, whereas the H0L0 antibody has an SLP. The L11 residue is thought to interact with the P8 residue to strengthen the P8 β-strand.

[0394] To investigate the role of the residue at position 8, this residue was backmutated, retaining the alanine residue of the parent antibody. The parent antibody has the sequence SAL, whereas the H0L0 antibody has SPL. As mentioned above, residue P8 interacts with residue L11, and this interaction is believed to stiffen the P8 β-strand.

[0395] The first light chain variant (L1) having the amino acid sequence of SEQ ID NO: 61 had I2V and G64S substitutions. The second light chain variant (L2) having the amino acid sequence of SEQ ID NO: 63 had I2V, L11N and G64S substitutions. The third light chain variant (L3) having the amino acid sequence of SEQ ID NO: 65 had I2V, P8A, L11N and G64S substitutions. The numbering of the substitutions is according to Kabat.

[0396] The amino acid sequences of the variable regions of each of the heavy chains ("H" chains in Table 2) and light chains ("L" chains in Table 2) are shown in Table 2 below. [Table 5]

[0397] Antibodies were generated having the heavy and light chains shown in Table 3 below. [Table 6]

[0398] Example 4: Characterization of binding to Nectin-4 by SPR The antibodies in Table 3 of Example 3 were cloned and generated as human IgG1 isotype antibodies, purified, and then tested for binding to human Nectin-4. The affinity of 16 humanized variants, as well as their association and dissociation constants, were evaluated by SPR analysis. Table 4 summarizes all calculated constants (affinity constant KD(nM), association constant ka(1 / Ms) and dissociation constant kd(1 / s)).

[0399] Completely human IGKHV-46 * 01 and IGHJ4 * 01 heavy chain framework as well as the fully human IGKV-28 * 01 and IGKJ4 * The H0L0 antibody, which has a 01 light chain framework, had a kD value of 80.2 nM. As shown in Table 4, the other variants showed lower KDs than H0L0.

[0400] The heavy chain H3 was restored to a KD value close to that of the parent antibody. Therefore, the back mutations introduced into H3 are important for stabilizing the humanized antibody. A stepwise change in the dissociation constant value is observed from H0 to H3. [Table 7]

[0401] Example 5: Modeling and generation of a second set of anti-huNectin-4 antibodies with human framework sequences Based on the SPR data of the humanized antibody prepared in Example 3, a novel antibody was designed.

[0402] Heavy Chain Design: To investigate the role of the residue at position 38, this residue was backmutated, retaining the lysine residue of the parent antibody. The parent antibody has the sequence VKQ, whereas the H0L0 antibody has the sequence VRQ. K38 likely exists as part of a salt bridge with residue E46.

[0403] To investigate the role of the residue at position 40, this residue was backmutated, retaining the arginine residue of the parent antibody. The parent antibody has the sequence QRP, whereas the H0L0 antibody has the sequence QAP. Residue R40 contacts Q43, which in turn contacts Q39 and forms two hydrogen bonds with residue Q43 of the light chain. In such a backmutation, residue Q43 of the heavy chain is in a more distant position, but still maintains contact with Q39, and also maintains the two hydrogen bonds with L0-Q43.

[0404] To investigate the role of the residue at position 48, this residue was backmutated to retain the isoleucine residue of the parent antibody. The parent antibody has the sequence WIG, whereas the H0L0 antibody has the sequence WMG. Residue I48 interacts with A68, M81 and F64 (located in CDR-H2).

[0405] To investigate the role of the residue at position 70, this residue was backmutated to retain the leucine residue of the parent antibody. The parent antibody has the sequence TLT, whereas the H0L0 antibody has the sequence TMT. Residue L70 interacts with residues M81, I51 (located in CDR-H2), Y60 (located in CDR-H2) and W36. Residue M70 (M69 according to Kabat numbering) interacts with residues I51 (located in CDR-H2), Y60 (located in CDR-H2) and W36. Except for residue I51, which is in a different rotational position, all other residues are completely overlapping and the two networks are nearly equivalent.

[0406] The fourth heavy chain variant (H4) with the amino acid sequence shown in SEQ ID NO: 45 had a R38K substitution. The fifth heavy chain variant (H5) with the amino acid sequence of SEQ ID NO: 47 had a R38K and A40R substitution. The sixth heavy chain variant (H6) with the amino acid sequence of SEQ ID NO: 49 had a R38K, A40R and M48I substitution. The seventh heavy chain variant (H7) with the amino acid sequence of SEQ ID NO: 51 had a R38K, A40R, M48I and M69L substitution. Furthermore, the eighth (H8; SEQ ID NO: 53), ninth (H9; SEQ ID NO: 55) and tenth (H10; SEQ ID NO: 57) chains were designed with different combinations of substitutions. The numbering of the amino acid residues is according to Kabat.

[0407] The amino acid sequences of each heavy chain ("H" chain in Table 5) and light chain ("L" chain in Table 5) variable region are shown in Table 5 below. [Table 8]

[0408] Antibodies were generated having the heavy and light chains shown in Table 6 below. [Table 9]

[0409] Example 6: Characterization of binding to Nectin-4 by SPR (Second set) The antibodies in Table 6 of Example 5 were cloned and produced as human IgG1 isotype antibodies, purified, and then tested for binding to human Nectin-4. The affinity of 16 humanized variants, as well as their association and dissociation constants, were evaluated by SPR analysis. Table 7 summarizes all calculated constants (affinity constant KD(nM), association constant ka(1 / Ms) and dissociation constant kd(1 / s)).

[0410] Completely human IGKHV-46 * 01 and IGHJ4 * 01 heavy chain framework as well as the fully human IGKV-28 * 01 and IGKJ4 * The kD of the H0L0 antibody with the 01 light chain framework was 80.2 nM. As shown in Table 7, the other variants showed lower KDs than H0L0. In particular, variants H4L1, H5L3 and H8L1 showed KDs close to that of the chimeric parent antibody (KDs of 5.9 nM ± 1.2). [Table 10]

[0411] Example 7: Characterization of binding to Nectin-4 by flow cytometry assay The humanized antibodies in Table 3 of Example 3 and Table 6 of Example 5 were cloned, produced, purified, and then tested for binding to Nectin-4 expressing cells by flow cytometry. Binding of humanized variants in human IgG1 format was determined on the SUM190 cell line, which expresses high levels of Nectin-4. The EC50 and MedFi showing the maximum MedFi at saturation stage are shown in Table 8 below.

[0412] All humanized variants with L0 or L1 chains have lower binding capacity (low plateau phase) than the others. In contrast, all humanized variants with L2 or L3 chains have similar binding capacity compared to the parent chimeric 5E7 antibody (ch5E7). Variant H5L3 showed the highest MedFi value. [Table 11]

[0413] Example 8: Cellular internalization assay The humanized variants of Table 3 in Example 3 and Table 6 in Example 5 were cloned, generated, purified and then tested for their ability to induce Nectin-4 internalization. The chimeric parental 5E7 antibody and isotype control were used as negative controls. The analysis was performed using the Fab-ZAP Human Internalization Kit assay and the Cell Titer Glo® (CTG) assay was used as readout. The experiment was performed on two cell lines expressing different levels of Nectin-4. MDA-MB-468 has lower expression of Nectin-4 than SUM190.

[0414] Internalization assay in SUM190 cell line The results of the internalization assay in SUM190 cell line are shown in Table 9 below, which shows the internalization efficiency (normalized to the chimeric parent antibody internalization efficiency in SUM190 cell line). The experiment was performed twice (two independent experiments). [Table 12]

[0415] The internalization efficiency measured in experiments performed on the SUM190 cell line shows that the humanized variants H3L0, H4L2, H5L2, H5L3, H6L3 and H7L2 induce more than 75% internalization compared to the parent antibody (5E7), indicating interesting internalization potential. The H8L1 and H7L1 variants were more potent than the chimeric antibody 5E7 in the first experiment, although this result was not confirmed in the second experiment.

[0416] Internalization assay in MDA-M-468 cell line The results of the internalization assay in SUM190 cell line are shown in Table 10 below, which shows the internalization efficiency (normalized to the internalization efficiency of the chimeric parent antibody in MDA-M-468 cell line). The experiment was performed twice (two independent experiments).

[0417] [Table 13]

[0418] The results showed that only the H5L3 humanized variant consistently achieved an efficiency of 75% or more than the parent chimeric 5E7 antibody, whereas all other variants had lower efficiencies and many of them showed consistent results between experiments.

[0419] Example 9: In vitro cytotoxicity of antibodies as ADCs against tumor cell lines Nectin-4 low / SUM190 breast cancer model The ability of the 5E7 antibody conjugated with a camptothecin analog (Dxd or exatecan) to kill SUM190 cells was evaluated. In this experiment, the 5E7 antibody was tested alongside the anti-Ig-like V-domain antibodies enfortumab and N41, as well as a control antibody conjugated with the same toxin at an equivalent drug-antibody ratio. The antibody was designated ggfg-Dxd, with the structure shown below: [ka] The first ADC was prepared in which a camptothecin analog (Dxd) was conjugated (to a cysteine ​​residue) at eight toxins per antibody (DAR=8) via a linker comprising an intracellularly cleavable tetrapeptide linker (GGFG) having the formula:

[0420] The antibody has the following structure, called PEG(8U)-Val-Ala-PAB-Exatecan: [ka] A second ADC was prepared in which the compound was conjugated to another camptothecin analog (exatecan) at eight toxins per antibody (DAR=8) via a cleavable linker having the following structure:

[0421] Briefly, a dose range of each Ab tested (starting at 150 nM, 3 points at dilution 2, then 5 points at dilution 5) was incubated with cells for 5 days, after which cell viability was measured by addition of CTG substrate. Luminescence versus Ab concentration was plotted graphically.

[0422] For each ADC, a range of concentrations of the ADC were incubated with Nectin-4 expressing cells. After incubation, CTG substrate was added at a 1 / 1 ratio and the luminescence signal was read using a plate reader (Enspire). This allowed us to quantify the ATP present (an indicator of metabolically active cells) in proportion to cell viability.

[0423] The results are shown in Figure 3A, which shows the killing of human breast cancer cells by 5E7 antibody conjugated to camptothecin analog Dxd (via a ggfg-Dxd linker) or exatecan [via a PEG(8U)-Val-Ala-PAB-exatecan linker], along with an isotype control antibody (IC), all of which are at an equal drug-antibody ratio (DAR=8), compared to Enhertu® (trastuzumab deruxtecan (anti-HER2)), which binds to the V domain.

[0424] All Nectin-4 targeted ADCs reduced cell viability more efficiently than the non-targeted ADC (IC-GGFG-camptothecin).

[0425] SUM185, MDA-MB-468, MC38 and B16F10 cell lines Antibody 5E7 conjugated to exatecan (via PEG(8U)-Val-Ala-PAb-exatecan linker) was further tested for cell killing against other cancer cell lines. Figure 3B shows the efficacy of "5E7-exatecan" [5E7 conjugated to exatecan linker (PEG(8U)-Val-Ala-PAb-exatecan] in causing death of HER-2 and nectin-4 expressing SUM185 and SUM190 and MDA-MB-468 (TNBC) human tumor cells, as well as human nectin-4 expressing MC38 (colon cancer) and B16F10 (melanoma) mouse tumor cells. EC of cell viability 50 The values ​​are shown in Table 11 below. [Table 14]

[0426] Example 10: In vivo efficacy of ADCs in a mouse model of human breast cancer (Nectin-4 low / SUM190 model) The efficacy of the 5E7 antibody as a camptothecin ADC was compared to enfortumab and N41 in a mouse model of human breast cancer. In this experiment, the 5E7 antibody was tested with enfortumab and N41 conjugated to the ggfg-Dxd linker and a control Ab at a drug-antibody ratio equivalent to eight toxins per antibody (DAR=8).

[0427] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 with PBS. Tumors were 195–250 mm 3 Once tumor volume reached 150 mm, mice were randomized into groups of 9 mice for intravenous treatment with a single injection of 3 mg / kg camptothecin ADC. Tumor growth was followed twice weekly. Kaplan-Meier survival curves were established using GraphPad Prism V7 software according to the following criteria: tumor volume 150 mm 3Mice were euthanized and considered dead on the day of sacrifice (D) when tumors showed signs of necrosis (indicated by a red star in the individual tumor growth graphs).

[0428] The results showed that at a dose of 10 mg / kg, all ADCs were similarly effective in preventing tumor volume growth. However, at lower doses of ADC (3 mg / kg), antibody 5E7 showed a strong ability to block tumor growth, while both N41 and Enfortumab no longer showed the ability to control tumor growth. The results of the 3 mg / kg dose are shown in Figure 4.

[0429] Example 11: Comparison of in vitro efficacy of ADCs in drug-resistant breast cancer models (human breast cancer, HER2 / Nectin-4 high / SUM185 model) Next, the ability of 5E7 Ab conjugated with camptothecin analogues to kill SUM185 cells was evaluated in comparison with PADCEV™ (Enfortumab vedotin) and ENHERTU®. This setting is used as a model of anti-HER2 resistance. SUM185 cells express Nectin-4 at relatively high levels, and in these cells, Nectin-4 expression levels are approximately twice as high as HER2 (see Example 1). In this experiment, 5E7 antibody was tested as an ADC with DAR=8, together with PADCEV™ (DAR=4, FDA approved specifications for PADCEV®) and a control antibody conjugated with the same toxin, all at equivalent drug-antibody ratios. 5E7 was conjugated with camptothecin analogue Dxd via ggfg-Dxd linker.

[0430] Briefly, a dose range of each Ab tested (starting at 150 nM, 3 points at dilution 2, then 5 points at dilution 5) was incubated on cells for 5 days, after which cell viability was measured by addition of CTG substrate and luminescence versus Ab concentration was plotted graphically.

[0431] For each ADC, a range of concentrations of the ADC were incubated with Nectin-4 expressing cells. After incubation, CTG substrate was added in a 1:1 ratio and the luminescence signal was read on a plate reader (Enspire) allowing the quantification of ATP present (an indicator of metabolically active cells) in proportion to cell viability.

[0432] The results are shown in Figure 5. 5E7-ggfg-Dxd and PADCEV™ were able to kill SUM185 more efficiently than ENHERTU™. In each case, the Nectin-4 targeted ADC reduced cell viability more efficiently than the non-targeted ADC (IC) counterpart.

[0433] Example 12: Characterization of species cross-reactivity of anti-Nectin-4 antibodies Anti-Nectin-4 antibodies were tested by flow cytometry for binding to different CHO cell lines engineered to express mouse, cynomolgus monkey and rat Nectin-4 proteins, respectively (including an N-terminal V5 tag, not shown in the sequence below).

[0434] The mature amino acid sequence of the protein expressed by the cells was as follows: Mouse Nectin-4: ELETSDVVTVVLGQDAKLPCFYRGDPDEQVGQVAWARVDPNEGIRELALLHSKYGLHVNPAYEDRVEQPPPPRDPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARMRLRVLVPPLP SLNPGPPLEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTQSSRSFTHPRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDRRITHTLQVAFLAEASVRGLEDQNLWQVGREGATLKC LSEGQPPPKYNWTRLDGPLPSGVRVKGDTLGFPPLTTEHSGVYVCHVSNELSSRDSQVTVEVLDPEDPGKQVDLVSASVIIVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHSDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEDDDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 12) Rat Nectin-4: MPLSLGAEMWGPEAWLLLLFLASFTGRYSAGELETSDLVTVVLGQDAKLPCFYRGDPDEQVGQVAWARVDPNEGTRELALLHSKYGLHVSPAYEDRVEQPPPPRDPLDGSILLRNAVQADEGEYECR VSTFPAGSQARMRLRVLVPPLPSLNPGPPLEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTQSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHTLQVAFLAEASVRGLED QNLWHVGREGATLKCLSEGQPPPKYNWTRLDGPLPSGVRVKGDTLGFPPLTTEHSGVYVCHVSNELSSRASQVTVEVLDPEDPGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEEDDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 13) Cynomolgus Nectin-4: GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARADAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPL PSLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHVGREGAMLKC LSEGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 14)

[0435] Antibody 5E7 bound to human, cynomolgus and rat nectin-4 proteins, but not to mouse nectin-4 protein. Figures 6A and 6B show binding of anti-nectin-4 antibodies to rat and cynomolgus nectin-4 expressing CHO cell lines.

[0436] Example 13: Characterization of nectin family cross-reactivity Anti-nectin-4 antibodies were tested by flow cytometry for binding to different CHO cell lines engineered to express human nectin-1, nectin-2, nectin-3 and PVR proteins, respectively. Expression of each cell line was controlled and verified with known anti-human nectin-1, anti-human nectin-2, anti-human nectin-3 and anti-human PVR antibodies, respectively. The mature amino acid sequences of the proteins expressed by the cells were as follows: Nectin-1: MGLAGAAGRWWGLALGLTAFFLPGVHSQVVQVNDSMYGFIGTDVVLHCSFANPLPSVKITQVTWQKSTNGSKQNVAIYNPSMGVSVLAPYRERVEFLRPSFTDGTIRLSRLELEDEGVYICEFATFPT GNRESQLNLTVMAKPTNWIEGTQAVLRAKKGQDDKVLVATCTSANGKPPSVVSWETRLKGEAEYQEIRNPNGTVISRYRLVPSREAHQQSLACIVNYHMDRFKESLTLNVQYEPEVTIEGFDGNWYL QRMDVKLTCKADANPPATEYHWTTLNGSLPKGVEAQNRTLFFKGPINYSLAGTYICEATNPIGTRSGQVEVNITEFPYTPSPPEHGRRAGPVPTAIIGGVAGSILLVLIVVGGIVVALRRRRHTFKGDYSTKKHVYGNGYSKAGIPQHHPPMAQNLQYPDDSDDEKKAGPLGGSSYEEEEEEEEGGGGGERKVGGPHPKYDEDAKRPYFTVDEAEARQDGYGDRTLGYQYDPEQLDLAENMVSQNDGSFISKKEWYV (SEQ ID NO: 15) Nectin-2: MARAAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVED EGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQDPTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVTVTCKVEHESFEEPALIPVTLSVRYPPEVSISGYD DNWYLGRTDATLSCDVRSNPEPTGYDWSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAGAGATGGIIGGIIAAIIATAVAATGILICRQQRKEQTLQGAEEDEDLEGPPSYKPPTPKAKLEAQEMPSQLFTLGASEHSPLKTPYFDAGASCTEQEMPRYHELPTLEERSGPLHPGATSLGSPIPVPPGPPAVEDVSLDLEDEEGEEEEYLDKINPIYDALSYSSPSDSYQGKGFVMSRAMYV (SEQ ID NO: 16) Nectin-3: MARTLRPSPLCPGGGKAQLSSASLLGAGLLLQPPTPPPLLLLLFPLLLFSRLCGALAGPIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHN IGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETVAAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIISQYKLFPTRFARGRRITCVVKHPALEKDIRYSFILDIQYAPEVSVT GYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPDGLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADIEDLATEPKKLPFPLSTLATIKDDTIATIIASVVGGALFIVLVSVLAGIFCYRRRRTFRGDYFAKNYIPPSDMQKESQIDVLQQDELDSYPDSVKKENKNPVNNLIRKDYLEEPEKTQWNNVENLNRFERPMDYYEDLKMGMKFVSDEHYDENEDDLVSHVDGSVISRREWYV (SEQ ID NO: 17) PVR (Nectin-5): DVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGISRNAIIFLVLGILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQDPQTEGTR (SEQ ID NO: 18)

[0437] The results showed that there was no cross-reactivity of anti-nectin-4 antibodies to members of the human nectin family. The figure shows the flow cytometry results of anti-nectin-4 antibodies against human nectin 1, human nectin 2, human nectin 3, and human PVR-expressing CHO cells. None of the antibodies shown binds to each cell line.

[0438] Example 14: Competition-based epitope mapping for binding to nectin-4 by SPR Chimeric antibody 5E7 was tested together with previously reported antibodies N4.1 (N41), 14A5 and Enfortumab for their ability to compete with each other for binding to wild-type human Nectin-4 protein by SPR (surface plasmon resonance) with OCTET analysis using Ni-NTA (NTA) biosensor (Fortebio). Briefly, human Nectin4-His-BirA protein diluted to 5 μg / mL in kinetic buffer 10X was captured on the biosensor. The first antibody was diluted to 10 μg / mL in kinetic buffer 10X and injected, followed by a second injection of the first antibody diluted to 10 μg / mL in kinetic buffer 10X to saturate the signal. The second antibody was diluted to 10 μg / mL in kinetic buffer 10X and injected.

[0439] The results are shown in Table 12 below. Black boxes indicate substantial or direct competition between the first and second antibodies (first antibody blocks binding of the second antibody / resulting in loss of binding), boxes with crosses indicate potential partial competition (first antibody results in a potential reduction in binding of the second antibody but not loss of binding), and open boxes indicate no competition. Antibodies 5E7 competed with each other for binding to Nectin-4. [Table 15]

[0440] Example 15: Epitope mapping of antibodies using point mutants of Nectin-4 Point mutants of cell surface-expressed human nectin-4 The binding profile of the anti-nectin-4 antibodies to full-length and Ig-like V domain deleted proteins, together with the species binding profile of the antibodies (binding to human, cynomolgus monkey and rat nectin-4 but not to mouse nectin-4) and the inter-species differences within the non-human nectin-4 proteins, allowed the identification of residues at the junction of the Ig-like V domain and the Ig-like C2 type 1 domain (also called "C1"). In combination with the published structures of nectin-4 domains, mutations of nectin-4 at surface exposed amino acid residues were designed. The nectin-4 domain structure was modeled based on Protein Data Bank reference: 4FRW (domains V and C1). The human nectin-4 used was NCBI reference sequence: NP_112178.2, the mouse nectin-4 used was NCBI reference sequence: AAL79833.1, the cynomolgus nectin-4 used was NCBI reference sequence: XP_005541277.1, and the rat nectin-4 used was NCBI reference sequence: NP_001102546.1. Cells expressing nectin-4 variants were then used to test anti-nectin-4 antibodies for loss of binding to different nectin-4 variants to identify antibodies that bind to the same site on nectin-4. In particular, variants with C1-V junction substitutions at residues K197T and / or S199A, or variants 7, 7bis and 9 with additional and / or adjacent substitutions at the junction between domains C1 and V, can identify antibodies with advantageous applications as immunoconjugates. Mutant 7 had substitutions S195A / K197T / S199A. Mutant 7bis had substitutions A72P / G73N / K197T / S199A. Mutant 9 contained the key residue Q234 substitution and had substitutions L150S / S152A / Q234R / I236S. Figures 7A and 7B show molecular models of human nectin-4 protein and indicate the location of substituted residues in mutant 7 (7A) and mutant 7bis (7B), which are present in the C1 domain and identify two sites at the junction of domains C1 and V domains on opposite faces of the nectin-4 protein.8A and 8B show different views of the molecular model of human Nectin-4 protein and indicate the positions of the substituted residues in mutants 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0441] Nectin-4 mutants were generated by PCR. Amplified sequences were run on an agarose gel and purified using a Macherey Nagel PCR Clean-Up Gel extraction kit. The purified PCR products generated for each mutant were then ligated into an expression vector using the ClonTech InFusion system. Vectors containing mutant sequences were prepared as minipreps and sequenced. After sequencing, vectors containing mutant sequences were prepared as midipreps using the Promega PureYield® Plasmid Midiprep System. HEK293T cells were cultured in DMEM medium (Invitrogen), transfected with vectors using Invitrogen's Lipofectamine 2000, and incubated at 37°C for 48 hours in CO 2 After incubation in an incubator, the expression of the transgene was tested. Hek-293T cells were transfected with the mutants as shown in the table below. The targeted amino acid mutations are shown in Table 13 below, and also indicate the residue / position of the residue present in wild-type nectin-4 / residue present in mutant nectin-4, where the position reference is relative to the nectin-4 protein having the leader peptide shown in SEQ ID NO:1. [Table 16]

[0442] The results of the association of the antibodies with the Nectin-4 variants are shown in Table 14 below. (+) means that the antibody associates with Nectin-4. (-) means that the antibody does not associate with Nectin-4. The results related to variant 5 are irrelevant due to the absence of expression of said protein. [Table 17]

[0443] Thus, antibody 5E7 encompasses the C1 domain residues mutated in variants 7 and 7bis in its binding site on nectin-4 (S195A / K197T / S199A and A72P / G73N / K197T / S199A), and thus binds to a different epitope on nectin-4 than enfortumab, N41, and 14A5 (it binds to an epitope on the V domain of nectin-4).

[0444] Example 16: In vivo efficacy of exatecan ADC The human breast cancer cell line SUM190PT was cultured in cell culture medium containing the following: Ham's F12, FBS 1 g / L, HEPES 10 mM, ethanolamine 5 mM, insulin 5 μg / mL, hydrocortisone 1 μg / mL, apotransferrin 5 μg / mL, triiodothyronine (T3) 6.7 ng / mL, sodium selenite 8.7 ng / mL.

[0445] Immunocompromised CB17-SCID mice were used at 7-8 weeks of age. Anti-nectin-4 antibodies enfortumab, 5E7 and 6A7 with human Fc region were generated and conjugated to payloads Dxd or exatecan via intracellularly cleavable linkers ggfg-Dxd or PEG(8U)-Val-Ala-PAB-exatecan, respectively. Antibodies 6A7 and 5E7 share most CDRs, have comparable nectin-4 binding affinity, and bind to the same site on nectin-4 (see PCT / EP2021 / 082872, filed November 24, 2021, for the amino acid sequence of 6A7). The ggfg-Dxd linker releases Dxd upon cleavage, and the PEG(8U)-Val-Ala-PAB-exatecan linker releases exatecan upon cleavage.

[0446] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 with PBS. Tumors grew to 146.9 ± 63.2 mm depending on the experiment. 3 or 213.2 ± 77.5 mm 3 On day 21, when tumor volume reached an average of 1,000 mm, mice were randomized into groups of 8 or 9 mice depending on the experiment and treated intravenously with a single injection of 3 or 10 mg / kg body weight of ADC or PBS as control. Tumor growth was followed twice weekly. Kaplan-Meier survival curves were established according to the following criteria by using GraphPad Prism V7 software: tumor volume 1500 mm or more; 3 Mice were euthanized and considered dead on the day of sacrifice (D) if tumors reached 0.01%. Mice were euthanized and considered dead on the same day (D) if tumors were highly necrotic.

[0447] Results from the 3 mg / kg dose are shown in Figures 9A and 9B, and show that the anti-IgVC1 ADC 5E7-ggfg-Dxd, which was found to lose binding to Nectin-4 with the K197T / S199A mutation, showed the highest efficacy in suppressing tumor growth in mice. Furthermore, 5E7 conjugated with PEG(8U)-Val-Ala-PAB-exatecan, designed to release exatecan upon linker cleavage, showed higher efficacy in controlling tumor growth in mice compared to 5E7 conjugated with ggfg-Dxd, designed to release Dxd upon linker cleavage, as shown in Figure 9C at the 10 mg / kg dose.

[0448] Example 17: In vitro efficacy of ADCs in Pg-p-expressing cancer models MC-38 cells, which endogenously express MDR1 P-glycoprotein (Pgp), were genetically engineered to express Nectin-4 and cultured in DMEM + 10% FBS. Cells were treated in the presence of ADCs with either vehicle (DSMO) or cyclosporine A (5 μM, stock solution in DMSO), which is known to act as an inhibitor of Pgp. The ADCs tested were as follows: (a) PADCEV (trademark) (b) antibody 5E7 conjugated to deruxtecan (Dxd) via a ggfg-Dxd linker that releases Dxd upon cleavage (5E7-GGFG-DxD); and (c) Antibody 5E7 conjugated to exatecan via a PEG(8U)-Val-Ala-PAB-exatecan linker that releases exatecan upon cleavage (5E7-exatecan). .

[0449] ADCs and equivalent isotype control ADCs were incubated at 150–2.3 10 -3 A range of nM doses were used. After 5 days of co-incubation with cells, cell viability was measured by adding Cell Titer Glo™ (CTG) substrate. Luminescence was plotted against Ab concentration.

[0450] Figure 10A shows the luminescence (an indicator of cell viability) of cells treated with Padcev™ (enfortumab vedotin), antibody 5E7 conjugated with Dxd, or 5E7 conjugated with exatecan. The ADC with exatecan as the payload (5E7-exatecan) was so potent that it reduced cell viability in this setting of drug resistance. Figure 10B shows the tumor growth (area under the curve) of MC38 cells treated with 150 nM ADC of Padcev® (enfortumab vedotin), antibody 5E7 conjugated with Dxd, or 5E7 conjugated with exatecan, in the presence or absence of the Pgp inhibitor cyclosporine, normalized with control antibody. This result suggests that the antitumor activity of antibody 5E7 conjugated to Padcev® and Dxd is negatively affected by Pgp at concentrations at which 5E7 conjugated to exatecan is highly effective.

[0451] Example 18: In vivo efficacy of branched PEG-dipeptide-exacan ADCs An anti-VC domain anti-Nectin-4 antibody was conjugated to exatecan via different branched PEG linkers and the resulting ADC was evaluated in vivo in the SUM190 tumor model (Nectin-4 low / SUM190 breast cancer model).

[0452] An ADC was generated in which the anti-VC domain anti-Nectin-4 (N4) human IgG1 isotype antibody 6A7 was conjugated to exatecan (at a cysteine ​​residue) via different linkers with eight toxins per antibody (DAR=8). The linker-toxins were tested as follows: [ka] [ka] [ka] [ka]

[0453] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 with PBS. Tumors with a volume of 200–250 mm were cultured in vitro. 3 Upon reaching 100 mg / kg, mice were randomized into groups of 10 for intravenous treatment with a single injection of 3 mg / kg ADC. Tumor growth was followed twice weekly. Kaplan-Meier survival curves were established using GraphPad Prism V7 software according to the following criteria: tumor volume >1500 mm 3 Mice were euthanized and considered dead on the day of sacrifice (D) when tumors showed signs of necrosis (indicated by red asterisks in individual tumor growth graphs).

[0454] The results show that at a dose of 3 mg / kg, all ADCs were effective in blocking tumor volume growth (indicated by red stars in the individual tumor growth graphs). When various linker toxins were administered as free toxins (not conjugated to anti-Nectin-4 antibodies; denoted as "IC"), they were less effective in blocking tumor growth. The IC (free toxin) results are shown in Figure 11. The ADC results are shown in Figure 12.

[0455] Example 19: In vivo pharmacokinetics and efficacy of branched PEG-dipeptide-exatecan ADC The anti-VC domain anti-Nectin-4 antibody 6A7 was conjugated to exatecan (DAR=8) via a VA-PAB-exatecan-PEG(16 U) linker (structure shown below) and evaluated in vivo in the SUM190 tumor model (low Nectin-4 / SUM190 breast cancer model) at various low doses. Different dose regimens up to 1 mg / kg body weight were tested in this study to evaluate the correlation between antitumor efficacy and circulating ADC concentrations. [ka]

[0456] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl of Matrigel containing growth factors diluted 1 / 2 in PBS. Tumors with a volume of 150–250 mm were grown. 3 At that time, mice were randomized into groups of 20 mice and treated intravenously with PBS as a control or a single injection of ADC at either 0.11 mg / kg, 0.33 mg / kg, 0.66 mg / kg or 1 mg / kg body weight (2.2 μg, 6.6 μg, 13.2 μg and 20 μg doses, respectively). Tumor growth was measured and plasma samples were taken on the same day, plasma samples were taken 5 minutes, 5 hours and 24 hours after treatment, followed by both plasma samples and tumor growth measurements at 72 hours and 7 days, and weekly thereafter.

[0457] The results are shown in Figure 13. The top left panel of Figure 13 shows that PBS did not prevent tumor volume growth. The top right panel of Figure 13 shows the results for ADC at a dose of 1 mg / kg, which demonstrated potent antitumor efficacy. The bottom panel of Figure 13 shows the concentration of ADC in plasma over time, demonstrating that the ADC remained detectable throughout the period during which it demonstrated antitumor activity.

[0458] Example 20: In vivo safety of branched PEG-dipeptide-exatecan ADC A dose ranging study of DAR-8's anti-VC domain anti-Nectin-4 antibody conjugated with the VA-PAB-Exatecan-PEG(16U) linker was performed at doses ranging from 3 g / kg body weight to 30 mg / kg body weight.

[0459] In the first experiment, Sprague Dawley mice were injected intravenously with 3, 10 or 30 mg / kg body weight of the ADC as a bolus on days 1 and 22. Plasma samples were collected over the course of the study (n=3 males per time point for each dose level) and concentrations of total anti-Nectin-4 antibodies (total antibodies; TA) (DAR≧0) and ADC (DAR≧1) were measured by ELISA, and free exatecan (Exa) was measured by LC-MS.

[0460] Figure 14A shows the results for the 3 mg / kg dose (top) and the 10 mg / kg dose (bottom), and Figure 14B shows the results for the 30 mg / kg dose. For each analyte, concentration values ​​below the lower limit of quantification (LLOQ) are plotted as LLOQ / 2. The Y-axis represents the plasma concentration of the analyte in ng / mL, and the X-axis represents time in days. Symbols and bars indicate the mean and standard deviation for each group.

[0461] In a second experiment, Mauritius cynomolgus monkeys were injected intravenously with 3, 10 or 30 mg / kg body weight of the ADC as a bolus on days 1 and 22. Plasma samples were collected over the course of the study (n=1 male and n=1 female per dose level) and concentrations of total anti-Nectin-4 antibody (full antibody; TA) (DAR≧0) and ADC (DAR≧1) were measured by ELISA, and free exatecan (Exa) was measured by LC-MS.

[0462] Figure 15A shows the results for the 3 mg / kg dose (top) and the 10 mg / kg dose (bottom), and Figure 15B shows the results for the 30 mg / kg dose. For each analyte, concentration values ​​below the lower limit of quantification (LLOQ) are plotted as LLOQ / 2. The Y-axis represents the plasma concentration of the analyte in ng / mL, and the X-axis represents time in days. Symbols and bars indicate the mean and standard deviation for each group.

[0463] Results in both rats and non-human primates demonstrated that the ADC was safe and well tolerated at the highest dose tested (30 mg / kg body weight).

[0464] All references cited herein, including publications, patent applications, and patents, are incorporated by reference in their entirety herein to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein (to the maximum extent permitted by law), notwithstanding any separate provided incorporation of the particular reference made elsewhere in this specification.

[0465] Unless otherwise stated, all exact values ​​provided herein represent the corresponding approximations (e.g., all exemplary exact values ​​provided for a particular factor or measurement can also be considered to provide the corresponding approximate measured value, where appropriate modified by "about"). When "about" is used in connection with a numerical value, this can be specified to include values ​​corresponding to + / - 10% of the stated numerical value.

[0466] Description of any aspect or embodiment of the invention herein using terms such as "comprising," "having," "including," or "containing" in reference to a single or multiple elements is intended to provide support for similar aspects or embodiments of the invention that "consist," "consist essentially of," or "substantially comprise" that particular single or multiple elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood to also describe a composition consisting of that element, unless otherwise stated or clearly contradicted by context).

[0467] The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to better illustrate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0468]

Table 18

Table 19

Table 20

Table 21

Table 22

Table 23

Table 24

Claims

1. An antibody or antibody fragment that specifically binds to human nectin-4 polypeptide, wherein the antibody or antibody fragment comprises a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 47; and a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO:

65.

2. The antibody according to claim 1, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 77; and a light chain containing the amino acid sequence of SEQ ID NO:

78.

3. An antibody or antibody fragment is conjugated to a linker-payload portion (X-Z), and the antibody-linker-payload has the following structure: 【Chemistry 1】 (In the formula, n is 15) The antibody or antibody fragment according to claim 1 or 2, having the following characteristics.

4. Formula (II): Ab-(X-(Z) n ) m Formula (II) [In the formula, Ab is the antibody or antibody fragment according to claim 1; X is a molecule linking Ab and Z, and X contains a cleavable portion (a protease-cleavable di, tri, tetra, or pentapeptide, for example, under physiological conditions, and optionally under intracellular conditions); Z contains a cytotoxic agent (optionally a camptothecin analog, optionally a penta-ring camptothecin analog, optionally a hexa-ring camptothecin analog); n is 1 or 2; and m is between 1 and 8. An immunoconjugate that binds to human nectin-4 polypeptide, as shown by [the symbol].

5. The immunoconjugate has the following structure: 【Chemistry 2】 or 【Transformation 3】 or 【Chemistry 4】 (In the formula, n is 5 to 23; and Ab is an antibody or antibody fragment that specifically binds to human nectin-4 polypeptide. The immunoconjugate according to claim 4, including the immunoconjugate described in claim 4.

6. The immunoconjugate according to claim 5, wherein n is 15, and the immunoconjugate is characterized by a DAR of 6 to 8, wherein the DAR may be 6 or, optionally, 8.

7. A method for producing an antibody-drug conjugate (ADC), wherein the method comprises conjugating an anti-nectin-4 antibody (Ab) with a cytotoxic agent (Z) as described in claim 1, and a linker (X) links the anti-nectin-4 antibody (Ab) and the cytotoxic agent (Z).

8. The method provides the anti-nectin-4 antibody described in claim 1, and the anti-nectin-4 antibody is given by the following formula: 【Transformation 5】 The method according to claim 7, comprising contacting and / or reacting with a linker payload portion (X-Z) having a linker payload portion

9. A pharmaceutical composition for use in treating cancer in an individual, comprising an immunoconjugate according to any one of claims 4 to 6 or an antibody drug conjugate manufactured according to the method of any one of claims 7 or 8.

10. The pharmaceutical composition according to claim 9, wherein the individual has a nectin-4 expressing cancer characterized by low or moderate expression of nectin-4 on tumor cells (which may optionally be determined by immunochemistry).

11. The pharmaceutical composition according to claim 9, wherein the individual has urothelial carcinoma, TNBC, non-small cell lung cancer, pancreatic cancer, ovarian cancer, head and neck squamous cell carcinoma, or esophageal cancer.

12. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to claim 1 or 2, an immunoconjugate according to any one of claims 4 to 6, or an antibody drug conjugate manufactured according to the method of any one of claims 7 to 8, and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising the antibody or antibody fragment described in Claim 3, and a pharmaceutically acceptable carrier.

14. A nucleic acid or set of nucleic acids encoding the heavy chain and / or light chain of an antibody or antibody fragment according to claim 1 or 2.

15. A hybridoma or recombinant host cell that produces the antibody or antibody fragment according to claim 1 or 2.

16. A method for producing an antibody or antibody fragment, comprising culturing the cells described in Claim 15 and recovering the antibody or antibody fragment.

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