Nectin-4 binding agents

Abstract

The present invention relates to antibodies and antibody fragments that bind to a Nectin-4 polypeptide. The present invention also relates to antibody-drug conjugates comprising the antibodies or antibody fragments, and to methods of preparing the conjugates, pharmaceutical compositions, and methods of using the same to diagnose, treat, or prevent diseases, such as cancers characterized by Nectin-4-expressing tumor cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 345,453, filed on May 25, 2022, which is incorporated herein by reference in its entirety; including any drawings.

[0003] Reference to a sequence listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a 97KB file named "Nectin-4-3 PCT" created on May 22, 2023. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0005] The present invention relates to antibodies and fragments thereof that bind to nectin-4 polypeptides. The present invention also relates to cells that produce such compounds; methods for preparing such compounds; conjugates and methods for preparing conjugates; pharmaceutical compositions comprising the compounds; and methods for using such compounds to diagnose, treat or prevent diseases (e.g., cancers characterized by nectin-4 expressing tumor cells). Background Art

[0006] Nectin-4 is a surface molecule of the nectin protein family that plays a key role in various biological processes during development and adult life, such as polarity, proliferation, differentiation and migration of epithelial cells, endothelial cells, immune cells and neuronal cells. Nectin-4 was originally cloned from human trachea by the Lopez group in 2001 (see Reymond et al. (2001) J. Biol. Chem. 276(46): 43205-15). Nectins are the main receptors for polio, herpes simplex and measles viruses, and are further involved in several human pathological processes. Nectin-4 is expressed at significantly higher levels in several tumors and is particularly overexpressed in breast cancer, including triple-negative breast cancer (TNBC) (see M-Rabet et al. Ann Oncol. 2017 Apr 1; ​​28(4): 769-776), pancreatic cancer and urothelial carcinoma. In addition, nectin-4 is also expressed in tumor samples of non-small cell lung cancer, ovarian cancer, head and neck squamous cell carcinoma, and esophageal cancer. Challita-Eid et al. (2016) Cancer Res. 76(10):3003-3013 reported moderate to strong staining by immunohistochemistry (H score ≥100) in bladder (60%) and breast (53%) tumor tissues. Zeindler et al. 2019 Frontiers in Medicine 6:200 reported high expression of nectin-4 in 86 of 148 TNBC cases (58%). In the serum of patients with these cancers, soluble forms of nectin-4 were detected and associated with poor prognosis. Serum nectin-4 levels increased during metastatic progression and decreased after treatment. These results suggest that nectin-4 may be a reliable target for cancer treatment.

[0007] 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 and is currently in clinical studies for the treatment of patients with solid tumors. Challita-Eid et al. (2016), supra, developed an anti-nectin-4 antibody conjugated to the highly potent microtubule disruptor MMAE based on the antibody AGS-22. This work led to the ADC candidate enfortumab vedotin (see U.S. Pat. No. 8,637,642 and PCT Publication No. WO2012 / 047724, Agensys Inc.), which has achieved promising results in human clinical trials for the treatment of patients with locally advanced or metastatic urothelial carcinoma who had previously received platinum-containing chemotherapy and PD-1 / PD-L1 checkpoint inhibitors in the neoadjuvant / adjuvant, locally advanced or metastatic setting. Several other groups have also proposed anti-Nectin-4 agents that are conjugated to a variety of toxic agents. PCT patent application WO2018 / 158398 (INSERM) reports several anti-Nectin-4 antibodies and proposes potential conjugation to a range of cytotoxic agents. Similarly, U.S. Patent No. 8,637,642 (Agensys) also provides anti-Nectin-4 antibodies and proposes potential conjugation to a range of cytotoxic agents. In addition, Bicycle Therapeutics reported the development of an anti-Nectin-4 targeting agent that comprises a Nectin-4 binding protein conjugated to a cytotoxic auristatin (MMAE) payload via a cleavable linker valine-citrulline (val-cit). To date, all anti-Nectin-4 antibodies that have been reported to be active when conjugated to chemotherapeutic agents target the Ig-like V-domain of Nectin-4, and it is therefore believed that the Ig-like V-domain provides the strongest internalization of anti-Nectin-4 ADCs.

[0008] Vitin-Enfortuzumab (anti-Nectin-4 ADC) binds to the Ig-like V-domain of Nectin-4, which is associated with highly internalizing anti-Nectin-4 antibodies. Vitin-Enfortuzumab showed an impressive treatment response in the EV-201 Phase 2 study (2019), with an ORR (objective response rate) of 44% and a CR (complete response rate) of 12% for UC, with approximately half of the patients discontinuing treatment. Most discontinuations were due to disease progression as assessed by RECIST (48%) or clinical symptoms (5%). In addition, 18% of the patients who discontinued experienced adverse events, particularly neuropathy. Therefore, ADCs targeting Nectin-4 have limitations, and the art needs improved benefits for patients with UC and other cancers. Summary of the invention

[0009] Provided herein are variable heavy (VH) domains and variable light (VL) domains for 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 a heavy chain comprising an 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 a light chain comprising an amino acid sequence of SEQ ID NO: 59, 61, 63 or 65.

[0010] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has 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: 69, and the light chain variable region has at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) with the amino acid sequence of SEQ ID NO: 70.

[0011] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided, which comprises: a heavy chain variable region (VH), which comprises CDR1, CDR2 and CDR3 having the amino acid sequences of SEQ ID NOs: 21, 22 and 23, respectively, and the framework FR1, FR2 and FR3 amino acid sequences from the human IGHV1-46*01 gene (and optionally an additional framework FR4 amino acid sequence from the human IGHJ4*01 gene); and a light chain variable region (VL) CDR1, CDR2 and CDR3, which comprises the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively, and the framework FR1, FR2 and FR3 amino acid sequences from the human IGKV2-28*01 gene (and optionally an additional framework FR4 amino acid sequence from the human IGKJ4*01 gene). The framework sequences from specific human IGHV, IGHJ, IGKV and IGKJ genes may contain one or more amino acid substitutions, as further described herein.

[0012] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided, comprising a heavy chain variable region comprising one, two, three, four, five, six, seven or eight amino acid substitutions at a Kabat position selected from 28, 38, 40, 48, 69, 71, 73, 78. Optionally, the threonine residue at position 28 is substituted with an isoleucine residue. Optionally, the arginine residue at position 38 is substituted with a lysine residue. Optionally, the alanine residue at position 40 is substituted with an arginine residue. Optionally, the methionine residue at position 48 is substituted with an isoleucine residue. Optionally, the methionine residue at position 69 is substituted with a leucine residue. Optionally, the arginine residue at position 71 is substituted with a leucine residue. Optionally, the threonine residue at position 73 is substituted with a lysine residue. Optionally, the valine residue at position 78 is substituted with a threonine residue.

[0013] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided, comprising a light chain variable region comprising one, two, three or four amino acid substitutions at kabat positions selected from 2, 8, 11, 64. Optionally, the isoleucine residue at position 2 is substituted with a valine residue. Optionally, the proline residue at position 8 is substituted with an alanine residue. Optionally, the leucine residue at position 11 is substituted with asparagine. Optionally, the glycine residue at position 64 is substituted with a serine residue.

[0014] 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) with 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) with a light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0015] In one embodiment, an anti-nectin-4 antibody or antibody fragment is provided, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has 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: 47, and the light chain variable region has at least about 80% sequence identity (e.g., at least about 85%, 90%, 95%, 97%, 98% or 99% identity, or 100% identity) with the amino acid sequence of SEQ ID NO: 65.

[0016] In one embodiment, a nectin-4 binding protein is provided, which comprises an antibody or antibody fragment of the present disclosure, and optionally further comprises an additional antigen binding domain.

[0017] In any embodiment, the antibody or antibody fragment can optionally be characterized as a monoclonal form, a humanized form, and / or an isolated form.

[0018] In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to the VC1 bridging domain of the nectin-4 polypeptide.

[0019] In any aspect, the antibodies or antibody fragments of the present disclosure are used to prepare antibody drug conjugates (ADCs). In any aspect, the antibodies of the present disclosure are used to reduce intercellular adhesion between nectin-4 expressing tumor cells, inhibit nectin-4: nectin-1 interactions, inhibit nectin-4: nectin-4 interactions, reduce the growth of nectin-4 expressing tumor cells and / or reduce cluster formation of nectin-4 expressing tumor cells (e.g., for use in the above methods).

[0020] According to one of the present inventions, an antibody drug conjugate is provided, which comprises an anti-Nectin-4 antibody or antibody fragment conjugated 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 linker-toxin comprises any one of Formulae III to XIV.

[0021] In one embodiment, the antibody drug conjugate (ADC) comprises an anti-nectin-4 antibody or antibody fragment linked to at least one cytotoxic drug 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.

[0022] In one embodiment, the antibody drug conjugate (ADC) comprises: (a) at least one antigen binding domain that binds to a nectin-4 polypeptide, the antigen binding domain comprising (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 drug portion.

[0023] 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.

[0024] In some aspects, antibodies (e.g., full-length antibodies, antibody fragments) that bind to human Nectin-4 polypeptides are capable of reducing intercellular adhesion between Nectin-4 expressing tumor cells and / or reducing growth and / or cluster formation of Nectin-4 expressing tumor cells (e.g., as assessed using 3-dimensional or non-adherent tumor cell cultures; tumor spheroid assays) and are used to prepare antibody drug conjugates.

[0025] In some aspects, the antibody (e.g., full length antibody, 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 wherein the cell is a tumor cell).

[0026] In any aspect herein, the anti-Nectin-4 antibody or antibody fragment or antibody-drug conjugate comprising such antibody or fragment is capable of undergoing intracellular internalization upon binding to Nectin-4 on the surface of a tumor cell.

[0027] In some aspects, a method of preparing an antibody drug conjugate is provided, the method comprising conjugating an anti-Nectin-4 antibody or antibody fragment (eg, an antibody or antibody fragment according to the disclosure) to a cytotoxic agent (eg, a linker or linker-toxin according to the disclosure).

[0028] In some aspects, preparation of the antibody drug conjugate includes the step of conjugating the antibody to the cytotoxic agent (eg, via a linker moiety that further comprises an intracellularly cleavable moiety).

[0029] In one aspect, 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., dipeptide, tripeptide, tetrapeptide, or pentapeptide). In one aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a cytotoxic agent (e.g., a camptothecin derivative) via an intracellularly cleavable (e.g., protease-cleavable) dipeptide, tripeptide, tetrapeptide, or pentapeptide and a self-eliminating spacer. In one aspect, the anti-Nectin-4 antibody or antibody fragment is conjugated to a cytotoxic agent (e.g., a camptothecin derivative) via an intracellularly cleavable (e.g., protease-cleavable) tetrapeptide or pentapeptide and a self-eliminating or non-self-eliminating spacer.

[0030] In one aspect, the cytotoxic agent is a highly potent chemotherapeutic agent, optionally a cytotoxic agent selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mytomycins, combretastatins, vincaalkaloids, nitrogen mustards, The cytotoxic agents include, for example, mustards, maytansinoids, duocarmycins, tubulysins, dolastatins and auristatins, enediynes (e.g., calicheamicins, esperamicins, shishijimicins and namenamicins), pyrrolobenzodiazepines (e.g., pyrrolobenzodiazepine dimers and indole-pyrrolobenzodiazepine dimers), amatoxins and ethyleneimines. In one embodiment, the cytotoxic agent is a DNA damaging agent, including, for example, a DNA intercalator, such as an agent that inserts itself into the DNA structure of a cell and binds to the DNA, thereby causing DNA damage (e.g., daunorubicin). The compound includes a topoisomerase inhibitor, i.e., a chemical compound that blocks the action of topoisomerases (topoisomerases I and II). Such compounds are used for a wide range of solid tumors and hematological malignancies, particularly lymphomas. Topoisomerase I inhibitors include camptothecins, such as irinotecan (approved for the treatment of colon cancer), topotecan (approved for the treatment of ovarian cancer and lung cancer), camptothecin, lamellarin D, indenoisoquinoline, indimitecan. Other camptothecins include silatecan, cositecan, ilotecan, lurtotecan, gimatecan, belotecan and rubitecan.Topoisomerase II inhibitors include, for example, etoposide (VP-16), teniposide, doxorubicin, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, and HU-331 (a quinolone synthesized from cannabidiol).

[0031] Optionally, the antibody or antibody fragment is functionalized (eg, conjugated, covalently bound) with a linker-toxin of any of Formulae III to XIV.

[0032] In one aspect, the cytotoxic agent is a camptothecin analog, such as exotecan, Dxd, or a SN-38 molecule.

[0033] In one aspect, the present invention provides a nectin-4 binding antibody or antibody fragment of the present disclosure, which is conjugated (e.g., covalently bound) to a camptothecin (e.g., a camptothecin analog, exotecan or an exotecan derivative, a Dxd molecule, or a SN-38 molecule).

[0034] In any of the embodiments herein, the nectin-4 binding antibodies or antibody fragments of the present disclosure conjugated (e.g., covalently bound) to a camptothecin analog (e.g., exotecan or SN-38 molecule) 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 residues, non-natural amino acid residues) that are functionalized via a linker with a molecule comprising the structure of Compound 1 or 2. In any of the embodiments herein, the nectin-4 antibody or antibody fragment can be characterized as being functionalized with a linker-camptothecin molecule having the structure of Formula III, IV, V, VI, VII, VII, IX, X, XI, XII, XIII, or XIV, or functionalized with any one of Compounds 3 to 16.

[0035] In one embodiment, the nectin-4 antibody or antibody fragment conjugated to the camptothecin derivative is a nectin-4 antibody or antibody fragment conjugated to an exotecan molecule, such as a molecule having the structure of compound 1 (1a or 1b). In any embodiment, the cleavable linker or immunoconjugate or ADC comprising the linker can be characterized as, for example, releasing an exotecan molecule, such as a molecule having the structure of compound 1 (1a or 1b), upon enzymatic cleavage of the linker. In one embodiment, the humanized nectin-4 antibody or antibody fragment conjugated to the camptothecin derivative is a nectin-4 antibody or antibody fragment conjugated to an SN-38 molecule (e.g., a molecule having the structure of compound 2). In one embodiment, a nectin-4 antibody or antibody fragment 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, or an unnatural amino acid residue) that is functionalized with a molecule having the following structure via a linker (e.g., a cleavable linker molecule with or without an additional spacer, such as a spacer (Y′) described herein):

[0036]

[0037]

[0038] In one embodiment, the nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent can be designated as an immunoconjugate represented by formula (I):

[0039] Ab-XZ Formula (I)

[0040] in,

[0041] 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 present disclosure, optionally the antibody binds to the VC1 bridging domain of human nectin-4 and / or exhibits reduced binding to a mutant human nectin-4 polypeptide comprising an amino acid substitution at residues K197 and / or S199 (see SEQ ID NO: 1) compared to binding to a wild-type human nectin-4 polypeptide);

[0042] X is a linker molecule connecting Ab and Z (e.g., covalently bound to each of Ab and Z), wherein X comprises a moiety cleavable, e.g., under physiological conditions, optionally under intracellular conditions, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, optionally wherein X further comprises a self-immolative or non-self-immolative spacer system (Y′) positioned between the cleavable moiety and Z, optionally wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety; and

[0043] Z is a cytotoxic agent, optionally wherein Z is a camptothecin analog, optionally wherein Z is exotecan, optionally wherein Z is exotecan and cleavage of the linker results in release of a compound having the structure of Compound 1 (exotecan).

[0044] In one embodiment, the nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent can be designated as an immunoconjugate represented by formula (I):

[0045] Ab-XZ Formula (I)

[0046] in,

[0047] Ab is an antibody or antibody fragment that specifically binds to a human Nectin-4 polypeptide according to the present disclosure;

[0048] X is a linker molecule connecting Ab and Z (e.g., covalently bound to each of Ab and Z), wherein X comprises a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide, wherein X further comprises a self-immolative or non-self-immolative spacer system (Y′) positioned between the cleavable moiety and Z, and wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety; and

[0049] Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exotecan molecule or a SN-38 molecule.

[0050] In one embodiment, a method of delivering or targeting a cytotoxic agent (optionally a camptothecin analog) to a tumor, a method of releasing a cytotoxic agent (optionally a camptothecin analog, Dxd, exotecan) in a tumor (e.g., in a subject suffering from cancer), or a method of sensitizing a tumor or cancer to a cytotoxic agent (optionally a camptothecin analog) is provided, the method comprising administering to a subject suffering from cancer an immunoconjugate represented by formula (I):

[0051] Ab-XZ Formula (I)

[0052] in,

[0053] Ab is an antibody or antibody fragment that specifically binds to human nectin-4 polypeptide;

[0054] X is a linker molecule connecting Ab and Z (e.g., covalently bound to each of Ab and Z), wherein X comprises a moiety cleavable, e.g., under physiological conditions, optionally under intracellular conditions, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, optionally wherein X further comprises a self-immolative or non-self-immolative spacer system (Y′) positioned between the cleavable moiety and Z, optionally wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety; and

[0055] Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exotecan molecule or an SN-38 molecule, optionally wherein Z is exotecan and wherein cleavage of the linker results in release of a compound having the structure of Compound 1 (exotecan).

[0056] In one embodiment, the antibody or antibody fragment conjugated to a cytotoxic agent (optionally a camptothecin analog) can be designated as an immunoconjugate represented by formula (II):

[0057] Ab-(X-(Z) n ) m Formula (II)

[0058] in,

[0059] Ab is an antibody or antibody fragment that specifically binds to a human Nectin-4 polypeptide according to the present disclosure;

[0060] X is a linker molecule connecting Ab and Z, wherein X comprises a moiety cleavable, e.g., under physiological conditions, optionally under intracellular conditions, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, optionally wherein X further comprises a self-eliminating or non-self-eliminating spacer system (Y′) positioned between the cleavable moiety and Z, optionally wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety;

[0061] Z is a cytotoxic agent, optionally a camptothecin analog, optionally Z is a molecule comprising an exotecan molecule or an SN-38 molecule, such as a molecule having the structure of Compound 1 or 2;

[0062] n is 1; and

[0063] m is 4 to 8, or optionally m is an integer selected from 4, 5, 6, 7 or 8.

[0064] In one embodiment, the nectin-4 antibody or antibody fragment conjugated to a cytotoxic agent (optionally a camptothecin analog) can be characterized as a composition of an immunoconjugate represented by formula (II):

[0065] Ab-(X-(Z) n )m Formula (II)

[0066] in,

[0067] Ab is an antibody or antibody fragment that specifically binds to a human Nectin-4 polypeptide according to the present disclosure;

[0068] X is a molecule linking Ab and Z, wherein X comprises a moiety cleavable, e.g., under physiological conditions, optionally under intracellular conditions, optionally a protease cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, optionally wherein X further comprises a self-eliminating or non-self-eliminating spacer system (Y′) positioned between the cleavable moiety and Z, optionally wherein X further comprises a spacer (Y) positioned between Ab and the cleavable moiety;

[0069] Z is a cytotoxic agent, optionally a camptothecin analog, optionally Z is a molecule comprising an exotecan molecule or an SN-38 molecule;

[0070] wherein 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 moieties) between 2 and 4, between 4 and 8, optionally between 6 and 8. Optionally at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the composition have m (the number of XZ moieties) of, or at least 4, 6, 7 or 8.

[0071] In Formula I or II, the (XZ) moiety can optionally be characterized as having a structure of any one of Formulas III to XI or any one of Compounds 3-12.

[0072] In Formula I or II, molecule X or spacer Y can optionally be designated as comprising a reactive group (R) or a reactive group (R) that reacts with an amino acid of an antigen binding protein (e.g., an antibody) or with a complementary reactive group (R′) of an amino acid attached to an antibody or antibody fragment.

[0073] In any of the embodiments herein, the exotecan molecule may be designated as being conjugated to the linker (X) via the amine at position 1 of the exotecan (when the exotecan molecule is part of the linker, NH replaces NH2 at position 1). In one embodiment, exotecan is conjugated to the linker (X), for example, to the carbonyl group of the p-aminobenzyloxycarbonyl (PAB) self-immolative spacer portion of X. In any of the embodiments herein, the SN-38 molecule may be designated as being conjugated to the linker (X) via the OH at position 9 (when the SN-38 molecule shown in compound 2 is part of the linker, O replaces OH at position 9).

[0074] In one embodiment, the nectin-4 binding agent conjugated to exotecan 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 ​​residues, glutamine residues) functionalized via a spacer (Y) with a linker-exotecan molecule comprising the following structure:

[0075]

[0076] In one embodiment, the humanized nectin-4 antibody or antibody fragment conjugated to exotecan 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 ​​residues, glutamine residues) functionalized with a linker-exotecan comprising the following structure via a spacer (Y):

[0077]

[0078] In one embodiment, the humanized nectin-4 antibody or antibody fragment conjugated to exotecan 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 ​​residues, glutamine residues) functionalized with a linker-exotecan molecule comprising the following structure via a spacer (Y):

[0079]

[0080] In one embodiment, the humanized nectin-4 antibody or antibody fragment conjugated to exotecan 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 ​​residues, glutamine residues) functionalized with a linker-exotecan molecule comprising the following structure via a spacer (Y):

[0081]

[0082] The spacer (Y) may be specified as or include a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-100 atoms, 2-40 atoms, optionally 2-30, 2-20, 4-40, 4-30 or 4-20 atoms, optionally wherein one or more atoms may not be carbon, such as oxygen, sulfur, nitrogen or other atoms, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide. For example, Y may include one or more ethylene oxide monomers, optionally Y includes a polyethylene oxide moiety, optionally Y includes the structure -(CH2CH2O) x -, wherein x is 1 to 24, optionally 1 to 12, optionally 1 to 8, optionally 1 to 6.

[0083] In some aspects, a linker-exitecan molecule having the following structure is provided:

[0084]

[0085] In certain embodiments, an immunoconjugate that binds to a human nectin-4 polypeptide is provided, wherein the immunoconjugate is represented by any one of the following formulae:

[0086]

[0087] wherein n is 1-15, 5-15, 5-23 (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

[0088] Wherein Ab is an antibody or an antibody fragment thereof that specifically binds to a human nectin-4 polypeptide, optionally wherein the 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 NO: 19, 47, 69 or 77; and 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 NO: 20, 65, 70 or 78. S may be designated as an atom of a cysteine ​​residue of the antibody (Ab).

[0089] In one embodiment, an immunoconjugate that binds to a human nectin-4 polypeptide is provided, wherein the immunoconjugate is represented by the following formula:

[0090]

[0091] 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 DAR is between 6 and 8, optionally wherein DAR is 6, optionally wherein DAR is 8; and wherein S is an atom of a cysteine ​​residue of the antibody (Ab). In one embodiment, the 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, optionally wherein 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 NOS: 78.

[0092] In one aspect, the treatment shows 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 conjugated to auristatin; Vitin-Enfortumab). In one aspect, a method of treating and / or preventing cancer and / or killing tumor cells in an individual in need thereof is provided, wherein the treatment comprises administering a Nectin-4 binder conjugated to a camptothecin derivative (e.g., exotecan or SN-38 molecule) 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times at a frequency of 1 to 2 times per month (e.g., once every two weeks, once every three weeks, or once every four weeks).

[0093] Also provided is a method for treating or preventing a disease (e.g., cancer) in an individual in need thereof using an antibody or fragment thereof optionally conjugated to a cytotoxic drug moiety. In one embodiment, the antibody or fragment optionally conjugated to a cytotoxic drug moiety is administered to an individual with cancer in an amount and frequency sufficient to kill Nectin-4 expressing tumor cells. In one embodiment, the individual suffers from a Nectin-4 expressing tumor, optionally wherein the tumor is a HER-2 expressing tumor or a HER2-negative tumor, such as urothelial carcinoma, head and neck squamous cell carcinoma, or esophageal cancer. In one embodiment, the cancer or tumor is a late recurrent or metastatic cancer, optionally a late recurrent or metastatic urothelial carcinoma. In one embodiment, the cancer or tumor is triple negative breast cancer (TNBC).

[0094] In one aspect, the treatment methods herein can be used in individuals with nectin-4 expressing cancers, regardless of the level of nectin-4 expression on the tumor cells.

[0095] In one aspect, the treatment methods herein may be advantageously used in individuals whose tumor cells express P-glycoprotein (Pgp).

[0096] In one aspect, the treatment methods disclosed herein can be advantageously used in patients who have received chemotherapy (e.g., chemotherapy agents transported by P-glycoprotein (Pgp), platinum agents (e.g., oxaliplatin, cisplatin, carboplatin, nedaplatin, phenoplatin, picoplatin, satraplatin), taxanes (e.g., paclitaxel (Taxol TM ) and docetaxel TM )) of individuals who have received prior treatment.

[0097] The increased anti-tumor efficacy and larger therapeutic window provided by the anti-nectin-4 antibodies of the present disclosure (e.g., particularly when conjugated to camptothecin derivatives) provide the possibility of improving treatment outcomes in individuals with tumors that are resistant, unresponsive, or have progressed after treatment with a composition comprising an anti-HER2 agent (e.g., trastuzumab, an ADC comprising trastuzumab) or with a composition comprising another anti-nectin-4 agent (e.g., an ADC comprising enfortuzumab; Vitin-enfortuzumab). The improved therapeutic window provides the possibility of combined treatment with other agents (particularly chemotherapeutic agents and / or anti-HER2 agents).

[0098] In one aspect, the treatment methods herein can be advantageously used in individuals whose tumors or cancers are resistant (i.e., have not responded 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 enfortuzumab; Vitin-enfortuzumab)).

[0099] In one aspect of the embodiments herein, the individual has received prior treatment with radiation therapy, surgery, chemotherapy, and / or treatment with a biologic agent.

[0100] In one aspect of the embodiments herein, the individual has received prior treatment with an anti-nectin-4 agent, optionally an anti-nectin-4 agent comprising a cytotoxic moiety in addition to a topoisomerase inhibitor (eg, Vidin-Enfortumab).

[0101] In one aspect of the embodiments herein, a method of treating cancer, killing tumor cells and / or delivering a cytotoxic agent to a tumor in an individual in need thereof is provided, the method comprising administering to an individual who has received prior treatment with a nectin-4 binding agent conjugated to an auristatin, optionally vestin-enfortumab, a therapeutically effective amount of an immunoconjugate that binds to a human nectin-4 polypeptide, wherein the immunoconjugate is represented by any one of the following formulae:

[0102]

[0103]

[0104] wherein n is 5-23, optionally n is 7-15, optionally n is 15, and wherein Ab is an antibody or antibody fragment thereof that specifically binds to a human nectin-4 polypeptide. Optionally, the immunoconjugate is characterized in that the DAR is between 6 and 8, optionally wherein the DAR is 6, optionally wherein the DAR is 8.

[0105] In one aspect, the invention provides therapeutic methods that can be used to mediate an anti-tumor effect in an individual at a dose that is lower or below the dose 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, less than 125 mg fixed dose.

[0106] In one aspect, the disclosed treatment methods can be used in individuals with existing neuropathy, diabetes or hyperglycemia, cardiac insufficiency, or ocular pathology.

[0107] In one aspect, the disclosed therapeutic methods can be used in individuals with a nectin-4 expressing cancer characterized by low or moderate levels of tumor cell expression of a nectin-4 polypeptide (eg, expression of a nectin-4 polypeptide at the tumor cell membrane).

[0108] In any of the embodiments herein, an 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 transported by P-glycoprotein (Pgp, the product of the human MDR1 gene), a platinum agent, a taxane), wherein the additional cytotoxic agent is administered separately from the anti-Nectin-4 antibody conjugated to the cytotoxic agent.

[0109] In another aspect of the present invention, pharmaceutical compositions and kits are provided, which contain an anti-nectin-4 antibody or antibody fragment, optionally conjugated to a cytotoxic drug portion, and one or more additional ingredients, which may generally be active ingredients or inactive ingredients (e.g., various carriers) that facilitate the formulation, delivery, stability or other properties of the composition.

[0110] These aspects will be more fully described, and additional aspects, features, and advantages will be apparent, in the description of the invention provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1Shown are 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 express HER2 at low to moderate levels (median fluorescence units of 1777) and Nectin-4 at low levels (median fluorescence units of 991).

[0112] Figure 2 Shown are 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 express HER2 at moderate to high levels (median fluorescence unit of 2880) and Nectin-4 at high levels (median fluorescence unit of 4326).

[0113] Figure 3A Killing of human breast cancer cells by 5E7 antibody, conjugated to camptothecin analog Dxd (via GGFG-Dxd linker as shown in Example 9) or exotecan (via (PEG(8U)-Val-Ala-PAB-exotecan) linker as shown in Example 9), all at equal drug / antibody ratio (DAR=8), and to V domain binding Enhertu TM (Trastuzumab delutec (anti-HER2)) Comparison. Figure 3B Shown is the efficacy of "5E7-Ixitecan" (5E7 conjugated to the Ixitecan linker (PEG(8U)-Val-Ala-PAB-Ixitecan) shown in Example 9) in causing the death of HER-2 and Nectin-4 expressing SUM185, SUM190, MDA-MB-468 (TNBC) human tumor cells and MC38 (colon cancer) and B16F10 (melanoma) mouse tumor cells, as well as the EC50 values ​​for the ability of 5E7-Ixitecan to cause cell death.

[0114] Figure 4 The efficacy of a single dose of 3 mg / kg of 5E7 antibody in the form of a camptothecin ADC is shown in a human breast cancer mouse model compared to the same doses of Enfortuzumab, N41, and an isotype control antibody (IC). Each antibody was conjugated to the same camptothecin analog (Dxd) and a tetrapeptide-containing linker (GGFG) at an equal drug / antibody ratio (DAR=8). Only 5E7 was able to effectively control tumor growth.

[0115] Figure 5 Shown with Padcev TM(Vitin-Enfortumab) (Enfortumab conjugated to the auristatin compound MMAE at DAR=4) and Enhertu TM In contrast, the 5E7 antibody killed SUM185 human breast cancer cells expressing nectin-4 at relatively high levels.This setting was used as a model for anti-HER2 resistance.

[0116] Fig. 6A and Figure 6B Shown is the binding of anti-Nectin-4 antibody 5E7 on rat and cynomolgus monkey Nectin-4 expressing CHO cell lines, respectively, as determined by flow cytometry.

[0117] Fig. 7A and Figure 7B The structure of the human nectin-4 protein is shown with substitutions shown in color; white regions corresponding to the C1 domain residues substituted in mutants 7 (7A) and 7bis (7B) are indicated.

[0118] Fig. 8A and Figure 8B Several views of the structure of the human nectin-4 protein are shown with substitutions shown in color; white areas corresponding to residues substituted in mutants 1, 2, 3, 4, 5, 6, 7, 8 and 9 are indicated.

[0119] Fig.9A , Fig. 9B and Fig. 9C Shown is the evolution of tumor growth over time (days after tumor implantation) in mice treated with ADC. Fig.9A and Fig. 9B Shown are treatments with different anti-nectin-4 antibodies (5E7 or 6A7) conjugated to the same linker-payload at the same DAR by intravenous injection at a dose of 3 mg / kg body weight. Fig. 9C Shown are treatments with a dose of 10 mg / kg body weight by intravenous injection of antibody 5E7 conjugated to linkers designed to release Dxd or exotecan upon linker cleavage.

[0120] Fig. 10A Shown with Padcev TM Luminescence (indicating cell viability) of cells treated with 5E7 (vitin-enfortumab), antibody 5E7 conjugated to Dxd, or 5E7 conjugated to exotecan. These cells are MC-38 cells that endogenously express MDR1 p-glycoprotein and are engineered to express nectin-4. ADCs with exotecan as the payload are highly effective in reducing cell viability in this drug resistance setting. Fig. 10BFigure 2 shows the effect of Padcev on ADC at 150 nM in the presence or absence of the Pgp inhibitor cyclosporine and normalized to control antibody. TM Tumor growth (area under the curve) of MC38 cells treated with 5E7 conjugated to Dxd or 5E7 conjugated to exotecan, indicating that Padcev TM The antitumor activity of antibody 5E7 conjugated to Dxd was negatively affected by Pgp.

[0121] Fig.11 and Fig.12 Results are shown for evaluation of free toxin and ADC in mice at a dose of 3 mg / kg. Fig.11 The results of IC (free toxin) are shown in . Fig.12 The results of ADC are shown in .

[0122] Fig.13 The results of the in vivo anti-tumor efficacy of anti-Nectin-4 ADC in mice and the plasma concentration of ADC over time are shown. The upper left figure shows that PBS did not prevent the increase in tumor volume. The upper right figure shows that the ADC at a dose of 1 mg / kg showed strong anti-tumor efficacy. The lower figure shows the concentration of ADC in plasma over time.

[0123] FIG. 14 shows in vivo plasma concentrations of ADC in rats. Fig.14A The 3 mg / kg dose (upper panel) and the 10 mg / kg dose (lower panel) are shown, and Fig. 14B Results are shown for the 30 mg / kg dose.

[0124] FIG. 15 shows in vivo plasma concentrations of ADC in non-human primates. Fig.15A The 3 mg / kg dose (upper panel) and the 10 mg / kg dose (lower panel) are shown, and Fig. 15B Results are shown for the 30 mg / kg dose.

[0125] Table 15 shows the nucleic acid and amino acid sequences disclosed in this application. DETAILED DESCRIPTION

[0126] definition

[0127] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used in conjunction 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.

[0128] When "comprising" is used, this may optionally be replaced by "consisting essentially of" or "consisting of.

[0129] "Nectin-4" and "Nectin-4 polypeptide" refer to proteins or polypeptides encoded by the NECTIN4 gene (see Uniprot Accession No. Q96NY8) or cDNA prepared from such gene. Any naturally occurring isoform, allele or variant is encompassed in the term Nectin-4 polypeptide (e.g., a Nectin-4 polypeptide that is 95%, 98% or 99% identical to SEQ ID NO: 1 or a contiguous sequence of at least 100, 200, 300, 400 or 500 amino acid residues thereof). The 510 amino acid residue sequence of a typical human Nectin-4 (isomer 1), including a 31 amino acid signal peptide, is shown below:

[0130]

[0131]

[0132] SEQ ID NO: 1 corresponds to UniProt KB identifier Q96NY8-1, the disclosure of which is incorporated herein by reference.

[0133] 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 (eg, non-human primates, cynomolgus monkeys).

[0134] The term "HER2" (also known as HER2 / neu and ErbB-2) stands for "human epidermal growth factor receptor 2." It encompasses variants and isoforms of HER2.

[0135] The terms "immunoconjugate" and "antibody conjugate" are used interchangeably and refer to an antigen-binding agent, such as an antibody-binding polypeptide or an antibody, conjugated to another molecule (e.g., a camptothecin derivative, an exotecan molecule, an SN-38 molecule). When the immunoconjugate comprises an antigen-binding agent conjugated to a therapeutic agent (e.g., a cytotoxic agent or an anticancer agent), the immunoconjugate may also be referred to as an "antibody drug conjugate" or "ADC". Examples of cytotoxic agents include camptothecins, taxanes, anthracyclines, camptothecins, epothilones, mitomycins, compretins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamycins, duocarmycins, tubulysins, pyraclostrobins and auristatins, enediynes, pyrrolobenzodiazepines, and ethyleneimines.

[0136] As used herein, "treatment" generally means obtaining the desired pharmacological and physiological effects. The effect may be preventive in terms of preventing or partially preventing a disease, its symptoms or conditions and / or may be therapeutic in terms of partial or complete cure of a disease, condition, symptom or side effect attributable to the disease. The term "treatment" as used herein encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease, such as preventive early asymptomatic intervention; (b) inhibiting the disease, such as preventing its development; or alleviating the disease, such as causing regression of the disease and / or its symptoms or symptoms, such as improvement or remediation of damage, for example, in a subject who has been diagnosed with the disease. Optionally, treatment may cause (e.g., may be characterized as a method of causing) a reduction in tumor burden, a reduction in the size and / or number of lesions, a reduction or delay in cancer progression (e.g., an increase in progression-free survival), a delay or prevention of cancer metastasis, and / or an increase in survival. Optionally, treatment can cause or provide (eg, can be characterized as a method of causing or providing) stable disease, a partial response, or a complete response in a subject, eg, according to standardized criteria, optionally RECIST criteria.

[0137] Whenever a nectin-4 binding agent (e.g., an antibody or antibody fragment, an immunoconjugate) is referred to as "cancer treatment", this includes:

[0138] (a) A method of treating cancer, said method comprising the steps of: administering a nectin-4 binding agent (for at least one treatment) to an individual, mammal, especially a human in need of such treatment, in a dose (therapeutically effective amount) allowing the treatment of cancer, optionally as specified herein;

[0139] (b) Use of nectin-4 binding agents in treating cancer;

[0140] (c) a nectin-4 binding agent for use in treating cancer (especially in humans);

[0141] (d) Use of a nectin-4 binding agent in the preparation of a pharmaceutical preparation for treating cancer;

[0142] (e) A method for preparing a pharmaceutical preparation for treating cancer using a nectin-4 binding agent, the method comprising mixing the nectin-4 binding agent with a pharmaceutically acceptable carrier;

[0143] (f) a pharmaceutical preparation comprising an effective amount of a nectin-4 binding agent suitable for treating cancer;

[0144] (g) Any combination of (a), (b), (c), (d), (e) and (f) that is patentable subject matter in the country in which this application is filed.

[0145] As used herein, the term "biopsy" is defined as the removal of tissue for the purpose of examination, such as to establish a diagnosis. Examples of biopsy types include - by applying suction, such as via a needle attached to a syringe; - by instrumental removal of tissue fragments; - by removal via an endoscope with appropriate instruments; - by surgical excision, such as surgical excision of the entire lesion; etc.

[0146] As used herein, the term "antibody" refers to polyclonal antibodies and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, antibodies are classified into one of five major categories: IgA, IgD, IgE, IgG and IgM. Several of these are further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Exemplary immunoglobulin (antibody) structural units include tetramers. Each tetramer is composed of two pairs of identical polypeptide chains, each pair having a "light" chain (about 25kDa) and a "heavy" chain (about 50-70kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids that are primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) refer to these light chains and heavy chains, respectively. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called "α", "δ", "ε", "γ" and "μ", respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known. IgG is an exemplary class of antibodies employed herein because they are the most commonly used antibodies under physiological conditions and because they are the easiest to prepare in a laboratory setting. Optionally, the antibody is a monoclonal antibody. Specific 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.

[0147] The amino acid residues of an antibody that are responsible for antigen binding may also be referred to as a hypervariable region. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (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 those residues from a "hypervariable loop" (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 an analogous system for defining essential amino acids responsible for antigen binding. Typically, the numbering of the amino acid residues in this region is performed by the method described in Kabat et al., supra. Phrases such as "Kabat position", "variable domain residue numbering as in Kabat" and "according to Kabat" refer to this numbering system for heavy chain variable domains or light chain variable domains in this article. Using the Kabat numbering system, the actual linear amino acid sequence of a peptide may include fewer or additional amino acids corresponding to the shortening of the FR or CDR of the variable domain or the insertion therein. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of CDR H2 (residue 52a according to Kabat) and a residue inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c, etc. according to Kabat). The Kabat numbering of the residues of a given antibody can be determined by comparing the sequence of the antibody with the "standard" Kabat numbering sequence at the homology region.

[0148] The term "specific binding" means that the antibody can preferably bind to a binding partner, such as Nectin-4, in a competitive binding assay, as assessed using a recombinant form of the protein, an epitope therein, or the 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 by radiolabeling, physical methods (such as mass spectrometry), or direct or indirect fluorescent labeling using, for example, cytofluorimetry (e.g., FACScan). An amount of binding greater than that seen with a control nonspecific agent indicates that the agent binds to the target.

[0149] 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 a recombinant molecule (e.g., Nectin-4) or a surface expressed molecule (e.g., Nectin-4). For example, if the test antibody reduces binding of an antibody having a heavy chain variable region of any of SEQ ID NO: 19 and a corresponding 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 antibody is said to "compete" with such an antibody, respectively.

[0150] The term "internalization" is used interchangeably with "intracellular internalization" and refers to molecular, biochemical and cellular events associated with the process of translocating a molecule from the extracellular surface of a cell to the intracellular surface of a cell. The processes responsible for the intracellular internalization of molecules are well known and may involve, among other things, the internalization of a complex of an extracellular molecule (such as a hormone, an antibody, and a small organic molecule); a membrane-associated molecule (such as a cell surface receptor); and a membrane-associated molecule bound to an extracellular molecule (e.g., a ligand that binds to a transmembrane receptor or an antibody that binds to a membrane-associated molecule). Thus, "inducing and / or increasing internalization" includes events in which intracellular internalization is initiated and / or the rate and / or extent of intracellular internalization is increased.

[0151] As used herein, the term "affinity" refers to the strength with which an antibody binds to an epitope. The affinity of an antibody is given 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 aDefined by 1 / Kd. Methods for determining the affinity of monoclonal antibodies can be found in the following references: Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); Coligan et al. (eds.), Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993) and Muller, Meth. Enzymol., Vol. 92: 589-601 (1983), all of which are incorporated herein by reference in their entirety. A standard method for determining the affinity of monoclonal antibodies, well known in the art, is to use surface plasmon resonance (SPR) screening (e.g., by using BIAcore TM SPR analysis device).

[0152] In the present context, a "determinant" refers to a site of interaction or binding on a polypeptide.

[0153] The term "epitope" refers to an antigenic determinant, and is a region or area on an antigen to which an antibody binds. A protein epitope may include amino acid residues that are directly involved in binding and amino acid residues that are effectively blocked by a specific antigen-binding antibody or peptide, i.e., amino acid residues within the "footprint" of an antibody. It is the simplest form or smallest structural region on a composite antigen molecule that can be combined with, for example, an antibody or a receptor. An epitope may be linear or conformational / structural. The term "linear epitope" is defined as an epitope (primary structure) consisting of continuous amino acid residues on a linear amino acid sequence. The term "conformational or structural epitope" is defined as an epitope (secondary, tertiary and / or quaternary structure) consisting of amino acid residues that are not all continuous and therefore represent separate parts of a linear amino acid sequence that are close to each other by molecular folding. Conformational epitopes depend on 3-dimensional structures. Therefore, the term "conformation" is usually used interchangeably with "structure".

[0154] The term "agent" is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract prepared from biological material. The term "therapeutic agent" refers to an agent that has biological activity.

[0155] The terms "Fc domain", "Fc portion" and "Fc region" refer to the C-terminal fragment of an antibody heavy chain, e.g., about amino acids (aa) 230 to about aa 450 from a human gamma (γ) heavy chain, or its corresponding sequence in other types of antibody heavy chains (e.g., α, δ, ε and μ of a human antibody), or its naturally occurring allotypes. Unless otherwise indicated, the generally accepted Kabat amino acid numbering of 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 Institutes of Health, Bethesda, MD).

[0156] As used herein, so-called "framework" or "FR" residues refer to regions of antibody variable domains other than those regions defined as CDRs. Each antibody variable domain framework can be further subdivided into continuous regions (FR1, FR2, FR3, and FR4) separated by these CDRs.

[0157] The terms "isolated," "purified," or "biologically pure" mean substantially or essentially free from components normally associated with it as found in its native state. Purity and homogeneity are generally determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein is substantially purified if it is the predominant species present in a preparation.

[0158] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

[0159] The term "recombinant", when used with 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 by 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 not found in the native (non-recombinant) form of the cell, or expresses native genes that are otherwise abnormally expressed, under-expressed, or not expressed at all.

[0160] Within the present context, the term antibody "binding" to a polypeptide or epitope designates an antibody that binds to said determinant with specificity and / or affinity.

[0161] The term "identity" or "identical" when used in the context of a relationship between sequences of two or more polypeptides refers to the degree of sequence relatedness between the polypeptides, as determined by the number of matches between chains of two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences with gapped alignments (if any) solved by a particular mathematical model or computer program (i.e., an "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 Primers, 1993. Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math., vol. 48, p. 1073 (1988).

[0162] The method for determining identity is designed to give the maximum match between the sequences tested. The method for determining identity is described in publicly available computer programs. The computer program method for determining the identity between two sequences includes the GCG program package, including GAP (Devereux et al., "Nucl. Acid. Res.", Vol. 12, p. 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wisconsin), BLASTP, BLASTN and FASTA (Altschul et al., "Journal of Molecular Biology (J. Mol. Biol., Vol. 215, p. 403-410 (1990)). The BLASTX program can be obtained from the National Center for Biotechnology Information (NCBI) and other sources ("BLAST Manual (BLAST Identity is determined by using the publicly available PCR method (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.

[0163] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain including a fully saturated (no double or triple bond) hydrocarbon group. Alkyl can have, for example, 1 to 20 carbon atoms (regardless of when it appears herein, numerical ranges such as "1 to 20" refer to each integer within a given range; for example, "1 to 20 carbon atoms" means that the alkyl can be composed of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, but this definition also encompasses the appearance of the term "alkyl" when no numerical range is specified). The alkyl group of the compound can be named "C1-C4 alkyl" or similar names. For example only, "C1-C4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include but are by no means limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Alkyl can be substituted or unsubstituted.

[0164] As used herein, the term "heteroalkyl" refers to a straight or branched chain alkyl group containing one or more heteroatoms, ie, elements other than carbon (including but not limited to oxygen, sulfur, nitrogen, phosphorus) replacing one or more carbon atoms.

[0165] Whenever a group is described as "substituted", the group is substituted with one or more of the specified substituents. If no substituents are specified, it means that the specified "substituted" group may be substituted with one or more groups individually and independently selected from the following: alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heteroalkyl, aryl, heteroaryl, heteroalicyclic, aralkyl, heteroaralkyl, (heteroalicyclic)alkyl, hydroxy, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, halogen, thiocarbonyl, carbamoyl, thiocarbamoyl, amido, sulfonylamino, sulfonylamino, carboxyl, isocyanate, thiocyanate, isothiocyanate, nitro, silyl, sulfinyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethylsulfonyl, trihalomethylsulfonamido, amino, mono-substituted amino and di-substituted amino, and protected derivatives thereof.

[0166] Where the number of substituents is not specified (e.g., haloalkyl), one or more substituents may be present. For example, "haloalkyl" may contain one or more of the same or different halogens. As another example, "C1-C3 alkoxyphenyl" may contain one or more of the same or different alkoxy groups containing one, two, or three atoms.

[0167] Unless the context clearly indicates otherwise, references to a "compound" or "formula" with a particular number (e.g., "Compound 1," "Compound 2," "Formula I," or "Formula II") refer to all compounds derived from the compound with the particular number or formula. Compound 1, for example, includes references to Compounds 1a and 1b.

[0168] Production of antibodies and antibody fragments

[0169] Hypervariable regions, heavy and light chain CDRs, heavy and light chain variable regions, and antibodies comprising them (e.g., full-length antibodies or antibody fragments, multispecific antigen-binding proteins, chimeric antigen receptors) will bind to human Nectin-4 expressed on the surface of cells (e.g., tumor cells). In one embodiment, the binding of the antibody to Nectin-4 is mediated by a single antigen-binding domain or by two identical antigen-binding domains, optionally wherein each antigen-binding domain comprises a VH and VL domain pair. In some embodiments, a single VH or VL variable domain is provided; such domains can, for example, be incorporated into polypeptides expressed and produced separately, and can then be combined to form a Nectin-4 binding domain.

[0170] When used in a therapy to eliminate nectin-4-expressing tumor cells, as an immunoconjugate or as an unconjugated ("naked") antibody (e.g., optionally in combination with a separately administered cytotoxic agent), a nectin-4 binding antibody (e.g., a full-length antibody, an antibody fragment) or a protein, conjugate or complex comprising such an antibody can advantageously inhibit cell-cell interactions mediated by nectin-4, for example, as determined by assessing cell cluster formation (e.g., in a three-dimensional cell culture system, by assessing tumor spheroid formation or growth) and / or anchorage-independent growth of nectin-4-expressing cells. When used in therapy to eliminate nectin-4 expressing tumor cells, the nectin-4 binding antibody may advantageously be capable of sensitizing the tumor to the cytotoxic agent, either as an immunoconjugate with a cytotoxic agent or as an unconjugated ("naked") antibody in combination with a cytotoxic agent administered separately, e.g., the antibody may enhance the ability of the cytotoxic agent to inhibit the proliferation of tumor cells or cause the death of tumor cells, e.g., the ability to inhibit the proliferation of tumor cells that are clustered (e.g., spheroids) or cause their death, or the ability to inhibit the formation or growth of tumor cell clusters (e.g., spheroids). When used as an immunoconjugate in therapy to eliminate nectin-4 expressing tumor cells, the anti-nectin-4 immunoconjugate may advantageously be capable of causing the death of nectin-4 expressing tumor cells when conjugated to a cytotoxic molecule as disclosed herein.

[0171] Tumor cells expressing Pgp may have reduced sensitivity to certain cytotoxic agents. Cells expressing Pgp (eg, tumor cells) may optionally be used to test antibodies and cytotoxic agents (whether or not the cytotoxic agent is conjugated to the antibody).

[0172] Tumor spheroids are generally less sensitive to chemotherapy, in part due to the protection provided by their structure and also due to their slower proliferation rate, and therefore tumor spheroids can be used to evaluate the anti-tumor effects of antibodies or immunoconjugates. The ability of antibodies or immunoconjugates to inhibit spheroid formation or disrupt cancer cell spheroid formation in a concentration-dependent manner can be tested. For example, real-time digital photography can be used to test the ability of antibodies or immunoconjugates to alter spheroid formation in different cancer cell lines.

[0173] The antibody or immunoconjugate can, for example, be characterized as being able to maintain cancer cells as single cells (or increase their maintenance), which cells can thus become more sensitive to cytotoxic agents (e.g., chemotherapy). Promoting the maintenance of cancer cells as single cells to increase 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, alone or in combination, in the presence of the antibody or immunoconjugate, optionally further compared to antibodies or immunoconjugates that bind only to the Ig-like V domain or the Ig-like C21 or type 2 domain. Maintaining cancer cells as single cells to increase 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 the chemotherapeutic agent or ADC will be lower when the antibody binds to the VC1 domain of Nectin-4, or for ADCs where the antibody binds to the VC1 domain of Nectin-4, the dose used to control tumor growth will be lower. Sensitivity to chemotherapy can be assessed as cell viability, cell proliferation or cytotoxicity, for example, monitored using different readouts such as CTG by luminescence, confluence using Incucyte, or caspase 3 / 7 or annexin V. In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to a wild-type human nectin-4 polypeptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 1, and to a modified human nectin-4 polypeptide having the amino acid sequence of SEQ ID NO: 10 (a nectin-4 protein containing Ig-like C21 and type 2 domains but lacking an Ig-like V-type domain). Optionally, the antibody retains partial binding to a polypeptide lacking an Ig-like V-type domain, optionally wherein the antibody retains 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 wherein the reduced level is between 5%-50%, optionally between 5%-30%, optionally between 5%-25%, optionally between 5%-15% of binding to wild-type Nectin-4. Optionally further, the anti-Nectin-4 antibody does not bind to a Nectin-4 polypeptide lacking both an Ig-like V-type domain and an Ig-like C21-type domain, for example a polypeptide having the amino acid sequence of SEQ ID NO: 11. Optionally, binding is assessed by flow cytometry using cells prepared to express a Nectin-4 polypeptide, and the level of fluorescence intensity (e.g., MFI) is determined.

[0174] 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. The corresponding Nectin or PVR protein may be specified to be expressed at the cell surface.

[0175] In one embodiment, the anti-Nectin-4 antibody or antibody fragment binds:

[0176] A nectin-4 polypeptide having an amino acid sequence of SEQ ID NO: 1, and

[0177] A nectin-4 polypeptide having the amino acid sequence of SEQ ID NO: 10 (optionally at a reduced level, optionally at a level between 5%-50% compared to binding to SEQ ID NO: 1), wherein the anti-nectin-4 antibody or antibody fragment does not bind to any of the following:

[0178] A polypeptide having the amino acid sequence of SEQ ID NO: 13,

[0179] A polypeptide having the amino acid sequence of SEQ ID NO: 16,

[0180] A polypeptide having the amino acid sequence of SEQ ID NO: 17, and

[0181] A polypeptide having the amino acid sequence of SEQ ID NO:18.

[0182] Optionally further, the anti-nectin-4 antibody or antibody fragment does not bind to the polypeptide having the amino acid sequence of SEQ ID NO:11.

[0183] Optionally further, the anti-nectin-4 antibody or antibody fragment binds to rat nectin-4 polypeptide (eg, the amino acid sequence of SEQ ID NO: 13).

[0184] In one embodiment, the anti-nectin-4 antibody or antibody fragment binds to the VC1 bridging domain, epitope or determinant of nectin-4.

[0185] In one embodiment, the anti-Nectin-4 antibody or antibody fragment binds to human Nectin-4 at 1, 2, 3, 4 or 5 or more residues selected from the group consisting of A72, G73, S195, K197, S199, L150, S152, Q234 and I236 (with reference to SEQ ID NO: 1). In one embodiment, the anti-Nectin-4 antigen binding protein or antibody binds to human Nectin-4 at 1, 2 or 3 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 binding of the antibody or antibody fragment to a mutant Nectin-4 polypeptide (eg, as expressed at the surface of a cell) in which the residue is substituted with a different residue has been reduced or lost.

[0186] In one embodiment, the anti-nectin-4 antibody or antibody fragment is capable of binding to both an Ig-like V-type domain and an Ig-like C21-type domain, for example, binding to a determinant or epitope on an Ig-like V-type domain and a determinant or epitope on an Ig-like C21-type domain.

[0187] In one embodiment, the anti-nectin-4 antibody or antibody fragment retains at least partial binding to a modified nectin-4 polypeptide comprising an Ig-like C21-type domain but lacking 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 binding at a reduced level (e.g., as assessed by flow cytometry) compared to binding to a wild-type human nectin-4 polypeptide having an amino acid sequence of residues of SEQ ID NO: 1. The nectin-4 polypeptide can be designated as expressed at the surface of a cell (e.g., a host cell prepared to express the polypeptide).

[0188] Antibodies can be produced by a variety of techniques known in the art. Typically, they are produced by immunizing non-human animals (preferably mice) 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 a human Nectin-4 polypeptide or a fragment or derivative thereof, typically an immunogenic fragment, i.e., a portion of a polypeptide comprising an epitope (e.g., an epitope recognized by the 5E7 antibody) exposed on the surface of a cell expressing the Nectin-4 polypeptide. The Nectin-4 polypeptide may be designated as comprising or consisting of a VC1 bridging domain or a fragment or subsequence thereof. Such fragments typically contain at least about 7 consecutive amino acids of the mature polypeptide sequence, and even more preferably at least about 10 consecutive amino acids thereof. The fragments are typically derived substantially 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 located on the cell surface. In a specific embodiment, the immunogen comprises intact cells, particularly intact human cells, which are optionally treated or lysed. In another preferred embodiment, the polypeptide is a recombinant Nectin-4 polypeptide. In specific embodiments, the immunogen comprises intact nectin-4 expressing cells.

[0189] The step of immunizing a non-human mammal with an antigen can be performed in any manner well 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, New York (1988), the entire disclosure of which is incorporated herein by reference).

[0190] Antibodies can also be generated by selecting combinatorial libraries of immunoglobulins, as disclosed, for example, in (Ward et al., Nature, 341 (1989), p. 544, the entire disclosure of which is incorporated herein by reference).

[0191] Provided herein are modified human receptor framework sequences into which antibody CDRs can be incorporated, such that the resulting anti-nectin-4 variable regions have high binding and internalization efficacy in nectin-4 expressing cells. The advantage of the antibodies is low or reduced immunogenicity in humans, such as low ability or likelihood to induce an undesirable anti-nectin-4 antibody directed immune response after administration to humans. Examples of such antibodies disclosed herein include antibodies comprising a H0, H1, H2, H3, H4, H5, H6, H7, H8, H9 or H10 VH domain in combination with a L0, L1, L2 or L3 VL domain. Examples of antibodies of the present disclosure include antibodies comprising a VH and VL domain pair 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.

[0192] In one aspect, the antibody or antibody fragment that binds to the human nectin-4 polypeptide comprises a VH and VL framework (e.g., FR1, FR2, FR3, and FR4) of human origin (e.g., derived from a human amino acid sequence). In one aspect, the antibody or antibody fragment comprises: HCDR1 (heavy chain CDR1), the HCDR1 comprising the amino acid sequence SYWMH as shown in SEQ ID NO: 21; and HCDR2 (heavy chain CDR2), the HCDR2 comprising the amino acid sequence EIDPSDSYTNYNQKFKG as shown in SEQ ID NO: 22; HCDR3 (heavy chain CDR3), the HCDR3 comprising the amino acid sequence GYGNYGDY as shown in SEQ ID NO: 23; LCDR1, the LCDR1 comprising the amino acid sequence RSSKSLLHSNGITYLY as shown in SEQ ID NO: 24; LCDR2, the LCDR2 comprising the amino acid sequence QMSNLAS as shown in SEQ ID NO: 25; LCDR3, the LCDR3 comprising the amino acid sequence AQNLELPWT as shown in SEQ ID NO: 26.

[0193] In one embodiment, the antibody comprises a heavy chain framework derived from human subgroup IGHV1-46 (optionally and IGHJ4, preferably IGHJ4*01), optionally IGHV1-46 is IGHV1-46*01. In one embodiment, the antibody comprises a light chain framework derived from human subgroup IGKV2-28 (optionally and IGKJ, preferably IGKJ4*01), optionally IGKV2-28 is IGKV2-28*01.

[0194] The antibody may further comprise one, two, three, four, five or more amino acid substitutions across the human heavy and / or light chain frameworks, for example to enhance affinity, stability or other properties of the antibody.

[0195] Optionally, in any embodiment, an antibody can be designated as an antibody other than a murine parent antibody (eg, a murine parent antibody having the corresponding VH and VL of SEQ ID NOs: 19 and 20).

[0196] Optionally, the human framework comprises one or more mutations (e.g., back mutations) to introduce residues present at specific positions in non-human mammals (e.g., mice). Optionally, the human framework of the heavy chain variable region of the antibody comprises one, two, three, four, five, six, seven or eight mutations at kabat positions selected from 28, 38, 40, 48, 69, 71, 73 or 78.

[0197] In one aspect of any of the embodiments herein, the threonine residue at position 28 of the Kabat heavy chain is substituted with a threonine residue.

[0198] In one aspect of any of the embodiments herein, the arginine residue at position 38 of the Kabat heavy chain is substituted with a lysine residue.

[0199] In one aspect of any of the embodiments herein, the alanine residue at position 40 of the Kabat heavy chain is substituted with an arginine residue.

[0200] In one aspect of any of the embodiments herein, the methionine residue at position 48 of the Kabat heavy chain is substituted with an isoleucine residue.

[0201] In one aspect of any of the embodiments herein, the methionine residue at position 69 of the Kabat heavy chain is substituted with a leucine residue.

[0202] In one aspect of any of the embodiments herein, the arginine residue at position 71 of the Kabat heavy chain is substituted with a leucine residue.

[0203] In one aspect of any of the embodiments herein, the threonine residue at position 73 of the Kabat heavy chain is substituted with a lysine residue.

[0204] In one aspect of any of the embodiments herein, the valine residue at position 78 of the Kabat heavy chain is substituted with a threonine residue.

[0205] Optionally, the human framework of the light chain variable region of the antibody comprises one, two, three or four mutations at kabat positions selected from 2, 8, 11 or 64.

[0206] In one aspect of any of the embodiments herein, the isoleucine residue at position 2 of the Kabat light chain is substituted with a valine residue.

[0207] In one aspect of any of the embodiments herein, the proline residue at position 8 of the Kabat light chain is substituted with an alanine residue.

[0208] In one aspect of any of the embodiments herein, the leucine residue at position 11 of the Kabat light chain is substituted with asparagine.

[0209] In one aspect of any of the embodiments herein, the glycine residue at position 64 of the Kabat light chain is substituted with a serine residue.

[0210] 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) is used to describe positions within the VH and VL domains herein.

[0211] In one aspect, 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 one 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 one of SEQ ID NOs: 59, 61, 63 or 65.

[0212] In one aspect, 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).

[0213] 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) with 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) with a light chain comprising the amino acid sequence of SEQ ID NO: 78.

[0214] DNA encoding the antibody can be prepared and placed in an appropriate expression vector for transfection into an appropriate host. The host is then used to recombinantly produce the antibody or its variant, such as a humanized form of the monoclonal antibody, an active fragment of an antibody, a chimeric antibody comprising the antigen recognition portion of the antibody, or a form comprising a detectable portion.

[0215] DNA encoding the monoclonal antibodies of the present disclosure can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). In one aspect, nucleic acids encoding the heavy or light chains of the anti-nectin-4 antibodies or antibody fragments of any embodiment herein are provided. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell (which does not originally produce immunoglobulins) to achieve the synthesis of the monoclonal antibody in a recombinant host cell. Such DNA sequences can be modified for any of a number of purposes, such as for humanizing antibodies, producing fragments or derivatives, or for modifying sequences of antibodies, such as in an antigen binding site, to optimize the binding specificity of the antibody. In one embodiment, isolated nucleic acid sequences encoding the light and / or heavy chains of an antibody are provided, as well as recombinant host cells comprising (e.g., in their genome) such nucleic acids. Recombinant expression in bacteria of DNA encoding antibodies is well known in the art (see, e.g., Skerra et al., Curr. Opinion in Immunol., Vol. 5, p. 256 (1993); and Pluckthun, Immunol., Vol. 130, p. 151 (1992)).

[0216] Fragments and derivatives of antibodies (which are covered by the terms "antibody" or "antibodies" as used in this application, unless otherwise specified or clearly contradictory from the context) can be produced by techniques known in the art. A "fragment" comprises a portion of an intact antibody, typically the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment, which is a polypeptide having a primary structure consisting of an uninterrupted sequence of one continuous amino acid residue (referred to herein as a "single-chain antibody fragment" or "single-chain polypeptide"); and multispecific (e.g., bispecific) antibodies formed from antibody fragments. For example, a multispecific protein may comprise the hypervariable regions (e.g., VH and VL) of an antibody of any embodiment 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).

[0217] Typically, the affinity of the anti-nectin-4 antibodies provided herein for nectin-4 polypeptides (e.g., nectin-4 polypeptides produced in the Examples herein) is about 10 4 to about 10 11 M -1 (For example, about 10 7 to about 10 10 M -1 ) is within the range of. For example, in certain aspects, the present disclosure provides an average dissociation constant (K D ) is less than 1×10 -7 M anti-nectin-4 antibodies, such as by surface plasmon resonance (SPR) screening (such as by using BIAcore TM In more specific exemplary aspects, the present disclosure provides a KD of about 1×10 -7 M is about 1×10 -10 Anti-nectin-4 antibody of M.

[0218] The antibody can be characterized, for example, by an average KD of no more than about (i.e., having a better affinity than) 200, 150, 100, 80, 70, 60, 40, 30, or 25 nanomolar. The KD can be determined, for example, by immobilizing a recombinantly produced human nectin-4 protein on a chip surface and then applying the test antibody in solution. In one embodiment, the method further comprises the step (d): selecting an antibody from (b) that is capable of competing with the control antibody for binding to nectin-4.

[0219] Anti-Nectin-4 antibodies may or may not have substantial binding to human Fcγ receptors. In one embodiment, the anti-Nectin-4 antibody comprises an Fc domain of the human IgG1 isotype that retains binding to human Fcγ receptors. In one embodiment, the anti-Nectin-4 antibody comprises an Fc domain of the human IgG1 isotype that has been modified (e.g., by introducing substitutions) to increase binding to human Fcγ receptors (e.g., CD16A). In another embodiment, the anti-Nectin-4 antibody may be prepared so that it does not substantially bind to human Fcγ receptors (e.g., any one or more of CD16A, CD16B, CD32A, CD32B, and / or CD64). Such antibodies may include constant regions of various heavy chains that are known to lack binding to Fcγ receptors or have low binding to them. Alternatively, antibody fragments that do not contain (or contain part of) a constant region (such as a F(ab′)2 fragment) can be used to avoid Fc receptor binding. Fc receptor binding can be assessed according to methods known in the art, including, for example, testing the binding of antibodies to Fc receptor proteins in a BIACORE assay. In addition, any antibody IgG isotype can generally be used, wherein the Fc portion is modified (e.g., by introducing 1, 2, 3, 4, 5 or more amino acid substitutions) to minimize or eliminate binding to Fc receptors (see, e.g., WO 03 / 101485, the disclosure of which is incorporated herein by reference). Assays such as evaluating Fc receptor binding are well known in the art and are described, e.g., in WO 03 / 101485. An example of a silent Fc IgG1 antibody is a LALA mutant comprising L234A and L235A mutations in the IgG1 Fc amino acid sequence. Another example of a Fc silent mutation is a mutation at residue D265 or D265 and P329, such as used as a DAPA (D265A, P329A) mutation in an IgG1 antibody (US 6,737,056). Another silent IgG1 antibody includes a mutation at residue N297 (eg, N297A, N297S mutation), which results in an aglycosylated / non-glycosylated antibody.Other silent mutations included substitutions at residues L234 and G237 (L234A / G237A); substitutions at residues S228, L235, and R409 (S228P / L235E / R409K, T, M, L); substitutions at residues H268, V309, A330, and A331 (H268Q / V309L / A330S / A331S); 1S); substitutions at residues C220, C226, C229, and P238 (C220S / C226S / C229S / P238S); substitutions at residues C226, C229, E233, L234, and L235 (C226S / C229S / E233P / L234V / L235A); substitutions at residues K322, L235, and L236 (C226S / C229S / E233P / L234V / L236A); at residues 233, 234, 235, 237, and 238 as defined by the EU numbering system comprise a sequence selected from the group consisting of PAAAP, PAAAS, and SAAAS (see WO2011 / 066501). In one embodiment, the antibody has a substitution in the heavy chain constant region at any one, two, three, four, five or more of the 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 (the numbering of the residues in the heavy chain constant region is according to EU numbering as in Kabat).

[0220] In the abbreviations used herein, the format is: wild-type residue:position in polypeptide:mutated residue, where the residue position is indicated according to EU numbering as in Kabat.

[0221] In a specific embodiment, an antibody is provided that substantially binds to the same epitope or determinant as monoclonal antibody 5E7; optionally, the antibody comprises a hypervariable region of antibody 5E7. In any embodiment herein, antibody 5E7 may be characterized by an amino acid sequence and / or a nucleic acid sequence encoding it. In one embodiment, the monoclonal antibody comprises a Fab or F(ab')2 portion of 5E7. An antibody or antibody fragment comprising the heavy chain variable region of 5E7 is also provided. According to one embodiment, the antibody or antibody fragment comprises three CDRs of the heavy chain variable region of 5E7. An antibody or antibody fragment further comprising one, two or three CDRs of the variable light chain variable region of 5E7 or the CDRs of the light chain variable region of 5E7 is also provided. Optionally, HCDR1, 2, 3 and LCDR1, 2, 3 sequences can be designated as all (or each independently) sequences of 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 chain or heavy chain CDRs may comprise one, two, three, four or five or more amino acid modifications (eg, substitutions, insertions or deletions).

[0222] In one aspect, the anti-nectin-4 antibody or antibody fragment comprises 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. In another aspect, 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 Structural domain.

[0223] The VH and VL comprise (e.g., are modified to incorporate) a human acceptor framework. In one embodiment, the anti-nectin-4 antibody comprises a VH CDR1, CDR2, and / or CDR3 (e.g., according to Kabat numbering) of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19. In one embodiment, the 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 an amino acid sequence of SEQ ID NO: 20. In one embodiment, the anti-nectin-4 antibody comprises a VH and a VL, the VH comprising a Kabat CDR1, CDR2, and / or CDR3 of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 19, and the VL comprising a Kabat CDR1, CDR2, and / or CDR3 of a light chain variable region having an amino acid sequence of SEQ ID NO: 20.

[0224] 5E7 VH:

[0225]

[0226] 5E7VL:

[0227]

[0228] 5E7CDR:

[0229]

[0230] In one embodiment, the anti-nectin-4 antibody may, for example, comprise 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. The anti-nectin-4 antibody may, for example, comprise: a HCDR1 having an amino acid sequence: SYWMH (SEQ ID NO: 21) or a sequence of at least 4 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be substituted with a different amino acid; a HCDR2 having an 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 wherein one or more of these amino acids may be substituted with a different amino acid; a HCDR3 having an amino acid sequence: GYGNYGDY (SEQ ID NO: 23) or a sequence of at least 4, 5 or 6 consecutive amino acids thereof, optionally wherein one or more of these amino acids may be substituted with a different amino acid; a LCDR1 having an 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 wherein one or more of these amino acids may be substituted with a different amino acid; a HCDR4 having an amino acid sequence: QMSNLAS (SEQ ID NO: 25) or a sequence of at least 4, 5, 6, 7, 8, 9 or 10 consecutive amino acids thereof IDNO: 25) LCDR2 region or a sequence of at least 4, 5 or 6 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be substituted with different amino acids; and / or having the amino acid sequence: AQNLELPWT (SEQ ID NO: 26) LCDR3 region or a sequence of at least 4, 5, 6, 7 or 8 consecutive amino acids thereof, optionally wherein one or more of these amino acids can be deleted or substituted with different amino acids. The CDR positions can be numbered according to Kabat.

[0231] Advantageously, the antibodies of the present disclosure can be used in a method for preparing an antibody-conjugate. In one embodiment, the method for preparing 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 designated as 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), for example, when conjugated, X is the residue of the linker after covalent attachment to one or both of Ab and Z.

[0232] In the embodiments herein, the method for preparing an antibody-drug conjugate includes the step of contacting and / or reacting an anti-Nectin-4 antibody or antibody fragment (Ab) of the present disclosure with a cytotoxic agent (Z). The contact may be performed under suitable conditions so that an antibody-drug conjugate of one aspect of the present disclosure is formed or obtained. Z may, for example, be contained in a compound comprising a cytotoxic agent (Z) and a linker (X) or a portion of a linker (X), such that the step includes 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 may optionally specify the following steps: isolating or recovering the formed antibody-drug conjugate, and optionally further processing the composition for use as a medicament, optionally formulating the antibody (e.g., with a pharmaceutical excipient) for administration to a human subject.

[0233] Optionally, the method of preparing the ADC comprises conjugating the antibody or antibody fragment to 2, 3, 4, 5, 6, 7 or 8 cytotoxic agent molecules. Optionally, the composition obtained is characterized in that the DAR is between 2 and 4, between 4 and 6, between 6 and 8. In one embodiment, the method comprises conjugating the antibody to 4 cytotoxic agent molecules. In one embodiment, the method comprises conjugating the antibody to 8 cytotoxic agent molecules. Optionally, the method further comprises assessing the DAR, and if the DAR corresponds to a predetermined specification (e.g., a DAR or DAR range disclosed herein, a DAR of about 2, 4, 6 or 8, etc.), the composition is further processed for use as a drug, and the antibody is optionally formulated (e.g., with a pharmaceutical excipient) for administration to a human subject.

[0234] In some embodiments, the linker (X)-(Z) element is prepared and isolated prior to contacting (and reacting) the compound comprising (X) and (Z) with (Ab), thereby forming an antibody drug conjugate.

[0235] In some embodiments, the method comprises:

[0236] (a) contacting and / or reacting a linker (X) or a portion of a linker (X) with (Ab) to form an Ab-X conjugate, and

[0237] (b) contacting and / or reacting the Ab-X of step (a) with a cytotoxic agent (Z) or a compound comprising a linker (X) and a second moiety of (Z), thereby forming an antibody drug conjugate.

[0238] X may, for example, 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, for example, be an oligopeptide (e.g., a dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide). The spacer Y may be positioned between Ab and the cleavable moiety, and the spacer system (Y') may be positioned between the cleavable moiety and Z.

[0239] In some embodiments, linker X or spacer Y can be optionally specified to include a reactive group (R) that is capable of reacting with an amino acid of the antibody or with a complementary reactive group (R') attached to an amino acid of the antibody (e.g., under suitable conditions, optionally after deprotection). Optionally, R is a group reactive with a free amino, hydroxyl, sulfhydryl, or carboxyl group on the antibody. For example, R can be a maleimide-N-group having the following structure, which reacts with a thiol group (also known as a sulfhydryl) on the antibody:

[0240]

[0241] In some embodiments, linker X or spacer Y may optionally be designated as a residue comprising a reactive group R that reacts with an amino acid of the antibody or with a complementary reactive group (R') attached to an amino acid of the antibody. Optionally, R is a residue that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody.

[0242] In any embodiment, the method includes a step of preparing, selecting or providing an anti-Nectin-4 antibody or antibody fragment 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). In one embodiment, the 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.

[0243] For example, an anti-nectin-4 antibody or antibody fragment may be tested for its ability to bind to the VC1 bridging domain of nectin-4. An antibody or antibody fragment determined to bind to the VC1 bridging 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 may be tested for its ability to bind to a mutant nectin-4 polypeptide (e.g., a mutant nectin-4 polypeptide comprising a substitution at residues 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)).

[0244] In another example, an anti-Nectin-4 antibody or antibody fragment can be tested for its ability to reduce cell-to-cell adhesion between Nectin-4 expressing tumor cells (e.g., adherent tumor cells) and / or its ability to reduce the growth of Nectin-4 expressing tumor cells (e.g., in three-dimensional cell culture, in a tumor spheroid formation assay). The antibody or antibody fragment determined to have the ability to reduce cell-to-cell adhesion between Nectin-4 expressing tumor cells and / or its ability to reduce the growth of Nectin-4 expressing tumor cells (e.g., adherent tumor cells) is then contacted and / or reacted with a compound (e.g., a linker (X) and / or a cytotoxic agent (Z)).

[0245] In another example, an anti-nectin-4 antibody or antibody fragment can be tested for its ability to sensitize a tumor to a cytotoxic agent (e.g., cytotoxic agent Z or a drug of the same class as Z, such as a camptothecin agent). The 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)).

[0246] 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)).

[0247] 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 a portion of a linker, followed by a reaction that conjugates the cytotoxic agent to the antibody-linker composition.

[0248] In one embodiment, a method for preparing an antibody-conjugate is provided, the method comprising:

[0249] (i) obtaining an anti-nectin-4 antibody by culturing a host cell transformed with an expression vector containing a polynucleotide encoding the antibody; collecting and purifying the antibody of interest from the culture obtained in the previous step;

[0250] (ii) contacting the anti-nectin-4 antibody or antibody fragment with a compound (L) comprising (a) a first reactive group capable of reacting with an amino acid (e.g., a side chain of an amino acid or a glycan, or a group attached to an amino acid or a glycan of an amino acid) of the antibody and (b) a second reactive group (R′) to obtain a modified antibody comprising one or more amino acids functionalized with the compound (L); and

[0251] (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 dipeptide, tripeptide, tetrapeptide, or pentapeptide) cleaved by an intracellular peptidase or protease; (c) an optional non-self-eliminating or self-eliminating spacer (Y'); and (d) a cytotoxic agent (Z). Optionally, the compound of step (ii) further comprises a spacer (Y) placed between R and the amino acid unit.

[0252] Optionally, the step of obtaining anti-nectin-4 antibodies may further include the following steps: preparing antibody-producing cells by immunizing animals with antigens, collecting blood, and measuring its antibody titer to determine when to remove the spleen; preparing myeloma cells; fusing the antibody-producing cells with myeloma; screening a group of hybridomas that produce the desired antibodies; dividing the hybridomas into single-cell clones (clones); culturing hybridomas or raising animals implanted with hybridomas to produce large amounts of monoclonal antibodies; and / or examining the biological activity and binding specificity of the monoclonal antibodies produced thereby, or measuring their properties as labeling agents; etc.

[0253] Optionally, the step of obtaining an anti-nectin-4 antibody may further include the step of preparing a cell library.

[0254] In one embodiment, R and R' are capable of a click reaction or a cycloaddition, optionally wherein R comprises or is an alkyne moiety and R' comprises or is an azide moiety, or wherein R' comprises or is an alkyne moiety and R comprises or is an azide moiety, and wherein the reaction of step (ii) is a 1,3-dipolar cycloaddition.

[0255] In one embodiment, the reaction of step (i) is carried out in the presence of a catalyst, optionally an enzyme (eg, transglutaminase).

[0256] In one embodiment, step (i) includes a step of modifying the anti-nectin-4 antibody or antibody fragment prior to contacting the anti-nectin-4 antibody or antibody fragment with compound (L). For example, the antibody or antibody fragment may be modified by reacting or contacting the antibody or antibody fragment with an enzyme capable of modifying antibody glycosylation (e.g., at Kabat residue N297). In one example, the modification includes deglycosylation of an antibody glycan having a core N-acetylglucosamine in the presence of an endoglycosidase to obtain an antibody comprising a core N-acetylglucosamine substituent, wherein the core N-acetylglucosamine and the core N-acetylglucosamine substituent are optionally fucosylated. Examples of endoglycosidases include EndoS, EndoA, EndoE, Endo18A, EndoF, EndoM, EndoD, EndoH, EndoT, and EndoSH and / or combinations thereof.

[0257] The antigen binding protein (e.g., antibody) molecule and the cytotoxic agent (e.g., camptothecin derivative molecule) are connected via a linker. In such embodiments, the immunoconjugate can be represented, for example, by formula (II):

[0258] Ab-(X-(Z) n ) m Formula (II)

[0259] in,

[0260] Ab is an anti-nectin 4 antibody or antibody fragment);

[0261] X is a linker connecting Ab and Z, e.g., the residue of a linker after being covalently linked to one or both of Ab and Z;

[0262] Z is a cytotoxic agent, such as a camptothecin analog, optionally Z comprises the structure of Compound 1 or 2 (exitecan or SN-38 molecule);

[0263] n is 1 or 2; and

[0264] When n is 1, m is 1 to 8, or optionally m is an integer selected from 1 to 8 or 1 to 6, optionally m is an integer selected from 1 to 4, optionally m is 2 or 4; optionally, m is 2, 3, 4, 5, 6, 7 or 8; and when n is 2, m is 1 to 4, or optionally m is an integer selected from 1 to 4 or 1 to 3, optionally m is an integer selected from 1 to 4, optionally m is 2 or 4; optionally, m is 1, 2, 4 or 4. Optionally, "n" can be designated to represent the degree of branching or polymerization. "n" and "m" can be designated to represent average values ​​in a composition comprising multiple antibodies.

[0265] In one embodiment, X represents a molecule comprising a cleavable portion, for example under physiological conditions, optionally under intracellular conditions. In one embodiment, X represents a molecule comprising (i) a spacer (Y), (ii) a cleavable portion and (iii) an optional self-eliminating or non-self-eliminating spacer system (Y'). The spacer Y can be positioned between Ab and the cleavable portion, and the spacer system (Y') can be positioned between the cleavable portion and Z. The molecule X or spacer Y can optionally be designated as comprising a reactive group (R) or a reactive group R that reacts with an amino acid of the antibody or with a complementary reactive group (R') of an amino acid attached to the antibody.

[0266] The variable m represents the -X-(Z) of each antibody molecule in the immunoconjugate. n In a composition comprising multiple anti-nectin-4 ADCs, the number of -XZ moieties "m" per antibody molecule can vary. Thus, in an exemplary composition comprising multiple immunoconjugates of the formulae herein, m is the number of -X-(Z) moieties per Ab. n The average number of moieties, in this case, m, may also be referred to as the average drug loading or drug:antibody ratio (DAR). The average drug loading or DAR may advantageously be between 1 and about 8 moieties per Ab (-X-(Z) n ) moiety. The number "n" of Z moieties attached to moiety X can be, for example, 1 or 2. Typically, n is 1. In some embodiments, n is 1, and m represents the average drug loading, m is between 2 and 8. In some embodiments, n is 1, and m represents the average drug loading, m is between 2 and 6. In some embodiments, n is 1, and m represents the average drug loading, m is between 4 and 8. In some embodiments, n is 1, and m represents the average drug loading, m is between 6 and 8, optionally about 6, 7, or 8. In some embodiments, n is 1, and m represents the average drug loading, m is between 4 and 6, optionally about 4, 5, or 6.

[0267] The number of (-XZ) moieties per Ab can be characterized by conventional methods, 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, the separation, purification, and characterization of homogeneous immunoconjugates in which m is a particular value can be achieved by means such as reverse phase HPLC or electrophoresis to distinguish from immunoconjugates with other drug loadings.

[0268] In one embodiment, the anti-nectin-4 composition for use in the therapeutic methods of the present disclosure is characterized by comprising a plurality of immunoconjugates represented by formula (I):

[0269] Ab-(X-(Z) n) m Formula (II)

[0270] in,

[0271] Ab is an anti-nectin-4 antigen binding protein (eg, an antibody or an antibody fragment);

[0272] X is a molecule that connects Ab and Z, e.g., the residue of a linker after being covalently linked to one or both of Ab and Z;

[0273] Z is a cytotoxic agent, optionally a topoisomerase inhibitor, optionally a camptothecin analog, optionally a camptothecin analog comprising exitecan or an SN-38 molecule (e.g., a molecule having the structure of Compound 1 or 2);

[0274] n is 1 or 2; and

[0275] wherein 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 wherein 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.

[0276] In one embodiment, the anti-nectin-4 composition for use in the therapeutic methods of the present disclosure is characterized by comprising a plurality of immunoconjugates represented by formula (I):

[0277] Ab-(X-(Z) n ) m Formula (II)

[0278] in,

[0279] Ab is an anti-nectin-4 antigen binding protein (eg, an antibody or an antibody fragment);

[0280] X is a molecule that connects Ab and Z, e.g., the residue of a linker after covalent attachment to one or both of Ab and Z;

[0281] Z is a camptothecin analog, which includes a molecule of exotecan or SN-38, such as a molecule including the structure of Compound 1 or 2;

[0282] n is 1; and

[0283] wherein 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.

[0284] It should be understood that a variety of methods can be used for the covalent attachment of the joint comprising a cytotoxic agent to an antibody or antigen-binding proteins, non-specifically or specifically attached to a specific amino acid residue.Joint (X) can, for example, under physiological conditions, optionally include a cleavable part under intracellular conditions as shown in the examples, so that the cutting of the joint releases a cytotoxic agent (e.g., compound 1, compound 2, compound 13, etc.) in the intracellular environment.Joints can be combined with chemically reactive groups on antibody molecules, such as free amino groups, imino groups, hydroxyls, thiols or carboxyl groups (e.g., attached to N-terminal or C-terminal, attached to the free carboxylic acid groups of the epsilon amino group of one or more lysine residues, one or more glutamic acid or aspartic acid residues, or attached to the sulfhydryl group of one or more cysteinyl residues), attached to carbohydrates, or generally attached to any reactive group introduced or engineered into an antibody. The site to which the linker is attached may be a natural residue in the amino acid sequence of the antibody molecule, or it may be introduced into the antibody molecule, for example by recombinant DNA technology (e.g., by introducing a cysteine ​​or protease cleavage site in the amino acid sequence, by introducing a non-natural amino acid residue) or by protein biochemistry (e.g., reduction, pH adjustment or proteolysis, by glycoengineering, enzymatic modification of amino acids and glycans).

[0285] In some embodiments, the linker (X) comprises peptide residues comprising an amino acid selected from the group consisting of phenylalanine, glycine, valine, alanine, lysine, citrulline, serine, glutamic acid and aspartic acid, optionally the linker (X) comprises a dipeptide, tripeptide or tetrapeptide residues.

[0286] In certain embodiments, the intermediate as the precursor of the joint (X) reacts with the cytotoxic agent (Z) under appropriate conditions. In certain embodiments, a reactive group is used on the cytotoxic agent and / or the intermediate. In some embodiments, the reaction product between the cytotoxic agent and the intermediate or the derived cytotoxic agent is subsequently reacted with the antibody molecule under appropriate conditions. In other embodiments, the precursor of the joint (X) first reacts with the antibody molecule under appropriate conditions to produce an antibody combined with the precursor of the joint (X), and the antibody is subsequently reacted with a molecule comprising the cytotoxic agent (Z).

[0287] In some embodiments, the linker (X) can be cut by a cleavage agent present in the intracellular environment (e.g., in a lysosome or endosome or caveolae). The linker can include, for example, a peptidyl linker or an amino acid unit cut by an intracellular peptidase or protease (including but not limited to a lysosomal or endosomal protease). In some embodiments, the peptidyl linker portion is at least two amino acids long or at least three amino acids long. The cleavage agent can include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives, thereby releasing active drugs in target cells. The most typical is a peptidyl linker that can be cut by an enzyme present in the cell. In a specific embodiment, the peptidyl linker that can be cut 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 and valine-citrulline linkers). The valine-citrulline (Val-Cit) element can have a structure as shown below:

[0288]

[0289] In another specific embodiment, the peptidyl linker that can be cleaved by intracellular proteases is a valine-alanine (Val-Ala) linker. The val-ala element can have a structure as shown below:

[0290]

[0291] In another specific embodiment, the peptidyl linker capable of being cleaved by an intracellular protease is a glycine-containing oligopeptide linker, such as an oligopeptide linker containing glycine and phenylalanine, 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), wherein "(D-)D" represents D-aspartic acid.

[0292] In some embodiments, the linker can also be used as a spacer or extension to keep the antibody away from Z so as to avoid interfering with the ability of the antibody to bind to Nectin-4 and / or inhibit cell-to-cell interactions mediated by Nectin-4. The linker can include a spacer unit (Y) and / or a spacer or spacer system (Y'). Thus, the spacer Y can be positioned between the Ab and the cleavable portion. The spacer system (Y') can be positioned between the cleavable portion and Z. The molecule X or spacer Y can optionally be designated as a residue that contains a reactive group (R) or a reactive group R that reacts with an amino acid of the antibody or with a complementary reactive group (R') of an amino acid attached to the antibody. The spacer Y may, for example, be a molecule that forms a bond (e.g., through its reactive group R) with an amino acid of the antibody (e.g., a sulfur atom, a primary or secondary amino group, or a carbohydrate group of the antibody), and the spacer or extender (Y) connects the antibody to a cytotoxic agent (Z) or a cleavable amino acid unit (e.g., a peptidyl linker, a cleavable dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide), the cleavable amino acid unit optionally further having a self-eliminating and / or non-self-eliminating spacer (Y′), which in turn is linked to Z. Thus, when one end of the spacer (Y) is linked to an amino acid unit (e.g., a cleavable dipeptide, a tripeptide, a tetrapeptide, or a pentapeptide), the cleavable amino acid unit may in turn be directly linked to Z or may comprise another spacer (Y′), such as a non-self-eliminating or self-eliminating spacer linking the amino acid unit and Z.

[0293] The spacer (Y) may optionally be designated as or include a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-100 atoms, optionally 2-40, 2-30, 2-20, 4-40, 4-30 or 4-20 atoms, optionally wherein one or more atoms may not be carbon, e.g., oxygen, sulfur, nitrogen or other atoms, optionally wherein any carbon of the chain is substituted with an alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0294] The spacer (Y) may optionally be specified to include a stability enhancing portion. For example, the spacer Y may be orthogonal to a polyethylene glycol (PEG) portion or a polysarcosine (poly-N-methylglycine or PSAR) portion in a linker design (see, e.g., WO2019 / 081455, WO2015 / 057699, and WO2016 / 059377, the disclosures of which are incorporated herein by reference).

[0295] In some embodiments, the spacer (Y) may include one or more ethylene oxide monomers, optionally Y includes a polyethylene oxide moiety, optionally Y includes between 1 and 24 polyethylene oxide moieties, optionally between 1 and 12, optionally between 1 and 8, optionally between 6 and 24 polyethylene oxide moieties, optionally Y includes the structure -(CH2CH2O) x -, wherein x is 1 to 24, optionally 1 to 12, optionally 1 to 6, optionally 6 to 24.

[0296] The example of suitable stability enhancing part, spacer chain Y, may include the stability enhancing part disclosed in PCT Publication No. WO2015 / 057699 or WO2019 / 081455. For example, spacer chain Y may include an orthogonal linker part and a stability enhancing part. The stability enhancing part may be a PEG homopolymer, or generally any single molecular weight homopolymer (such as PEG or polysarcosine homopolymer) that is attached to an orthogonal linker part. Homopolymers may have, for example, 1-4, 1-6, 1-7, 1-8, 1-10, 1-12, at least 6, 8 or 10, or 6-12, 6-24, 6-72 or no more than 6, 7 or 8 PEG units or other monomers. The term orthogonal linker refers to a branched linker unit assembly that connects a linker moiety (e.g., a chain of spacer Y) to a homopolymer unit and to a cytotoxic agent (Z) through a linker (e.g., a cleavable oligopeptide (Pep) and a spacer Y') such that the homopolymer unit is in a parallel configuration (as opposed to a tandem configuration) relative to the cytotoxic agent (homopolymer parallel to the Pep-Y'-Z moiety). The orthogonal linker moiety can, for example, be one or more natural or unnatural amino acids, optionally selected from glutamic acid, lysine, and glycine. Optionally, the amino acid orthogonal linker moiety is placed at the end of the spacer chain Y such that the orthogonal linker moiety amino acid residue is connected by an amino acid sequence. α -Carboxyl group with another amino acid α -amino group is connected to an amino acid residue of a peptidyl linker (e.g., (Pep) in Formula V or VI). Y may, for example, comprise the result of a reaction of an orthogonal linker moiety with a moiety of Formula D:

[0297]

[0298] where R1 and R2 are different, and

[0299] One of R1 and R2 is H or an inert group, the other of R1 and R2 is a functionalized reactive group that is reactive for covalent bonding to a bondable group of an orthogonal linker moiety, under such reaction conditions that the inert group is non-reactive, Z1 and Z2, the same or different, are optional spacers, and n is 1 or greater and k is 2 or greater.

[0300] Optionally, the orthogonal linker is derived from glutamic acid.

[0301] In another example, the spacer Y includes a group disclosed in U.S. Patent Publication No. US2017 / 0072068A1 (the disclosure of which is incorporated herein by reference), such as a group according to formula (E) or a salt thereof:

[0302]

[0303] in

[0304] a is 0 or 1; and

[0305] R 1 Selected from the group consisting of: hydrogen, C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C24 (hetero)aryl, C3-C24 alkyl (hetero)aryl and C3-C 24 (Hetero)arylalkyl, these C1-C 24 Alkyl, C3-C 24 Cycloalkyl, C2-C24 (hetero)aryl, C3-C24 alkyl (hetero)aryl and C3-C 24 (Hetero)arylalkyl is optionally selected from O, S and NR 3 and optionally interrupted by one or more heteroatoms, wherein R 3 independently selected from the group consisting of hydrogen and C1-C4 alkyl; and wherein the group according to formula E or a salt thereof is located between the first and second ends of the spacer chain Y.

[0306] According to this aspect, the spacer Y may include -(succinimide-3-yl-N)-CH2CH2-C(=O)-, -(succinimide-3-yl-N)-CH2CH2CH2-C(=O)-, -(succinimide-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(succinimide-3-yl-N)-CH2CH2CH2CH2-C(=O)-, -(succinimide-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-.

[0307] Optionally, such spacer Y may further comprise the following structure: -NH-(CH2CH2-O)n-CH2CH2-C(=O)-, wherein n is an integer from 1 to 6, preferably from 2 to 4. Such structures include, for example, -NH-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-, -NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C H2CH2-C(=O)-,-NH-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-,-N H-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-O-CH2CH2-C(=O)-.

[0308] The spacer or spacer system (Y') placed between the amino acid unit (e.g., a cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide) and Z can be self-eliminating or non-self-eliminating. For example, the spacer Y' can include a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-30 atoms, optionally 2-20, 4-20, 2-10, or 4-20 atoms, optionally wherein one or more atoms may not be carbon, such as oxygen, sulfur, nitrogen, or other atoms, optionally wherein 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' includes p-aminobenzyloxycarbonyl. In one embodiment, Y' is a non-self-eliminating spacer and comprises a structure of -(CH2)nC(=O)-, wherein n is an integer from 0 to 5, and the structure is connected to the cleavable portion by -O- or a single bond. For example, Y′ may be or may include -C(=O)-, -OC(=O)-, -O-CH2-C(=O)-, -O-CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, -O-CH2CH2CH2CH2-C(=O)-, HO-O-CH2-C(=O)-, -CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2CH2CH2CH2-C(=O)-, -CH2-O-CH2-C(=O)- or -CH2CH2-O-CH2-C(=O)- group.

[0309] "Self-eliminating" spacer units allow the release of drug moieties without the need for a separate hydrolysis step. When using self-eliminating spacers, after the cutting or conversion of the amino acid unit, the side of the spacer connected to the amino acid unit becomes unblocked, which results in the final release of one or more parts Z. Self-eliminating spacer systems can be, for example, those described in WO02 / 083180 and WO2004 / 043493 (the disclosure of which is incorporated herein by reference in its entirety), and other self-eliminating spacers known to those skilled in the art. In certain embodiments, the spacer unit of the linker includes a p-aminobenzyl unit. In one such embodiment, p-aminobenzyl alcohol is attached to the amino acid unit by an amide bond, and carbamate, methyl carbamate or carbonate are prepared between benzyl alcohol and a cytotoxic agent. In one embodiment, the spacer unit is, for example, p-aminobenzyloxycarbonyl (PAB) with the following structure:

[0310]

[0311] Examples of self-immolative spacer units further include, but are not limited to, aromatic compounds that are electronically similar to p-aminobenzyl alcohol (see, e.g., US 2005 / 0256030 A1), such as 2-aminoimidazole-5-methanol derivatives (Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237) and o-aminobenzyl acetal or p-aminobenzyl acetal. Spacers can be used to cyclize upon hydrolysis of the amide bond, 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 alpha position of glycine (Kingsbury et al., J. Med. Chem., 1984, 27, 1447) is also an example of a self-immolative spacer. The p-aminobenzyl self-immolative spacer (e.g., PAB) is particularly suitable for use with a Phe-Lys, Val-Ala or Val-Cit cleavable dipeptide unit (PAB is placed between the dipeptide and the camptothecin derivative (Z)).

[0312] A "non-self-immolative" spacer unit is one in which part or all of the spacer unit remains bound to moiety Z upon enzymatic (e.g., proteolytic) cleavage of the antibody-moiety of interest conjugate. Examples of non-self-immolative spacer units suitable for use as a spacer between the Gly-Gly-Phe-Gly amino acid unit and the exotecan molecule include, but are not limited to, -O-CH2-C(=O)-, HO-O-CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2-O-CH2-C(=O)-, and -CH2CH2-O-CH2-C(=O)- (e.g., to form a GGFG-CH2CH2-O-CH2-C(=O)-exotecan unit). Use of such a spacer between the GGFG amino acid unit and exotecan 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 that are sensitive to sequence-specific enzymatic cleavage can be used in a similar manner. For example, enzymatic cleavage of an antibody-part of interest conjugate containing a glycine-glycine spacer unit by a tumor cell-associated protease will result in the release of the glycine-glycine-drug moiety from the remainder of the antibody-part of interest conjugate. In one such embodiment, the glycine-glycine-drug moiety is subsequently subjected to a separate hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug moiety.

[0313] Exemplary linker-cytotoxic drug moieties (XZ) can comprise any of the structures shown below in Formulae III and IV.

[0314]

[0315]

[0316] Spacers (Y) and (Y′) may optionally be designated as independently selected from the group consisting of: linear or branched C1-C 20 Alkylene, C2-C 20 Alkenylene, C2-C 20 Alkynylidene, C3-C 20 Cycloalkylene, C5-C 20 Cycloalkenylene, C8-C 20 Cycloalkynylene, C7-C 20 Alkyl arylene, C7-C 20 Aryl alkylene, C8-C 20 Arylene and C9-C 20Aryl alkynylene, these alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, alkylarylene, arylalkylene, arylalkenylene and arylalkynylene are optionally selected from O, S and NR 1 and optionally interrupted by one or more heteroatoms, wherein R 1 are independently selected from the group consisting of: hydrogen, C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 Alkynyl and C3-C 24 Cycloalkyl, these alkyl, alkenyl, alkynyl and cycloalkyl groups are optionally substituted.

[0317] Spacers (Y) and (Y') may optionally be specified as being or comprising C1-C 10 Alkylene-, -C1-C 10 heteroalkylene-, -C3-C8 carbocyclyl-, -O-(C1-C8 alkyl)-, -arylene-, -C1-C 10 Alkylene-arylene-, -arylene-C1-C 10 Alkylene-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-, -C3-C8 heterocyclic group-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-, -C1-C 10 Alkylene-C(=O)-, -C1-C 10 heteroalkylene-C(=O)-, -C3-C8 carbocyclyl-C(=O)-, -O-(C1-C8 alkyl)-C(=O)-, -arylene-C(=O)-, -C1-C 10 Alkylene-arylene-C(=O)-, -arylene-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-C(=O)-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-C(=O)-, -C3-C8 heterocyclyl-C(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-C(=O)-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-C(=O)-, -C1-C 10 Alkylene-NH-, -C1-C 10Heteroalkylene-NH-, -C3-C8 carbocyclyl-NH-, -O-(C1-C8 alkyl)-NH-, -arylene-NH-, -C1-C 10 Alkylene-arylene-NH-, -arylene-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-NH-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-NH-, -C3-C8 heterocyclyl-NH-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-NH-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-NH-, -C1-C 10 Alkylene-S-, -C1-C 10 heteroalkylene-S-, -C3-C8 carbocyclyl-S-, -O-(C1-C8 alkyl)-)-S-, -arylene-S-, -C1-C 10 Alkylene-arylene-S-, -arylene-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-S-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-S-, -C3-C8 heterocyclyl-S-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-S-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-S-, -C1-C 10 Alkylene-OC(=O)-, -C3-C8 carbocyclyl-OC(=O)-, -O-(C1-C8 alkyl)-OC(=O)-, -arylene-OC(=O)-, -C1-C 10 Alkylene-arylene-OC(=O)-, -arylene-C1-C 10 Alkylene-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 carbocyclic group)-OC(=O)-, -(C3-C8 carbocyclic group)-C1-C 10 Alkylene-OC(=O)-, -C3-C8 heterocyclyl-OC(=O)-, -C1-C 10 Alkylene-(C3-C8 heterocyclic group)-OC(=O)-, -(C3-C8 heterocyclic group)-C1-C 10 Alkylene-OC(═O)-, these groups are each substituted by one or more substituents selected from the following substituents: -X, -R', -O, -OR', ═O, -SR', -S-, -NR'2, -NR'3 +, =NR′, -CX3, -CN, -OCN, -SCN, -N=C=O, -NCS, -NO, -NO2, =N2, -N3, -NR′C(=O)R′, -C(=O)R′, -C(=O)NR′2, -SO3 - , -SO3H, -S(=O)2R′, -OS(=O)2OR′, -S(=O)2NR′, -S(=O)R′, -OP(=O)(OR′)2, -P(=O)(OR′)2, -PO3, -PO3H2, -C(=O)X, -C(=S)R′, -CO2R′, -CO2, -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: -F, -Cl, -Br or -I; and each R′ is independently -H, -C1-C 20 Alkyl, -C6-C 20 Aryl or -C3-C 14 Heterocyclic ring.

[0318] In one embodiment, the spacer (Y) may optionally be designated as comprising a reactive group (R) that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group or a carbohydrate on the antibody, for example at one end of the chain. In some aspects, the spacer Y comprises an R group -(succinimidyl-3-yl-N)--(CH2) n 3 -C(=O), where n 3 is an integer of 2 to 8, and -(succinimide-3-yl-N)- has a structure represented by the following Formula F:

[0319]

[0320] Position 3 of the above structure may be a linking position to an antibody. The bond to the antibody at position 3 is characterized by bonding through thioether formation.

[0321] In one embodiment, the spacer (Y) may optionally be designated as comprising, for example, at one end of the chain: a reactive group (R) that is reactive with a complementary reactive group (R′) of an amino acid attached to the antibody (e.g., via a free amino, hydroxyl, sulfhydryl or carboxyl group or a carbohydrate); or, when conjugated to an anti-nectin-4 antibody, a reactive group (R) that reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or with a complementary reactive group (R′) of an amino acid attached to the antibody. Examples of reactive groups R and R' include a range of groups that are capable of biorthogonal reactions, preferably cycloadditions, such as Diels-Alder reactions or 1,3-dipolar cycloadditions, such as between azides and cyclooctynes ​​(copper-free click chemistry), between nitrone and cyclooctyne, oxime / hydrazone formation from aldehydes and ketones, and tetrazine linkages (see also WO2013 / 092983 or US2017 / 0072068A1, the disclosures of which are incorporated herein by reference). For example, R can be an alkyne and R' can be an azide, or R can be an azide and R' can be an alkyne. The resulting linker and functionalized antibody or its Y element can therefore include a group (RR') resulting from the reaction of R and R' in any embodiment, for example RR' can be or include a triazole resulting from the reaction of an alkyne and an azide.

[0322] In one embodiment, the reactive groups R and R' are complementary reagents that together are capable of a "click" reaction (i.e., click chemistry reagents or reactive groups). For example, a 1,3-dipole functional compound can react with an alkyne in a cyclization reaction to form a heterocyclic compound, preferably in the substantial absence of an added catalyst (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipole group attached thereto (having a three-atom π-electron system containing 4 electrons delocalized over three atoms) can be used to react with alkynes disclosed herein. Exemplary 1,3-dipole groups include, but are not limited to, azides, nitrile oxides, nitrones, azooxy groups, and acyldiazo groups.

[0323] Examples include o-phosphonoarylates, azides, fulminates, alkynes (including any strained ring alkynes), cyanides, anthracenes, 1,2,4,5-tetrazines, or norbornene (or other strained ring olefins).

[0324] In one embodiment, R is a moiety with a terminal alkyne or azide; such moieties are described, for example, in U.S. Pat. No. 7,763,736, the disclosure of which is incorporated herein by reference. Suitable reaction conditions for the click reaction between a terminal alkyne and an azide using copper (and other metal salts) as catalysts are disclosed in U.S. Pat. No. 7,763,736.

[0325] In one embodiment, R is a substituted or unsubstituted cycloalkyne. Cycloalkynes, including specific compounds, are described, for example, in U.S. Pat. No. 7,807,619, the disclosure of which is incorporated herein by reference.

[0326] In some embodiments, the cycloalkyne can be a compound of formula A:

[0327]

[0328] in:

[0329] R 1 is selected from the group consisting of 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;

[0330] R 1 It can be anywhere on the cyclooctyne group, not just on the two carbons connected by a triple bond.

[0331] In some embodiments, the modified cycloalkyne has formula A, wherein one or more carbon atoms in the cyclooctyne ring, except for the two carbon atoms connected by the triple bond, are substituted with one or more electron withdrawing groups, e.g., halogen (bromine, chloride, fluorine, iodine), nitro, cyano, sulfone, or sulfonic acid. Thus, for example, in some embodiments, the subject modified cycloalkyne has formula B:

[0332]

[0333] in:

[0334] R 2 and R 3 Each independently represents: (a) H; (b) a halogen atom (e.g., bromine, chlorine, fluorine, iodine); (c) -W-(CH2) n -Z (wherein: 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) -(CH2) n -W-(CH2) m -R 4 (wherein: n and m are each independently 1 or 2; W is O, N, S or sulfonyl; if W is O, N or S, then R 4 is nitro, cyano or halogen; and if W is sulfonyl, then R 4 is H); or (e)-CH2) n -R 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

[0335] R 1 R is selected from the group consisting of 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 It can be anywhere on the cyclooctyne group, not just on the two carbons connected by a triple bond.

[0336] In one embodiment, R is a substituted or unsubstituted heterocyclic strained alkyne. Cycloalkynes including specific 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 has the formula C:

[0337]

[0338] in:

[0339] Each R 1 independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, nitrate, nitrite, sulfate and C1-C 10 Alkyl or heteroalkyl;

[0340] Each R 2 independently selected from the group consisting of hydrogen, halogen, hydroxyl, alkoxy, nitrate, nitrite, sulfate and C1-C 10 Organic group; X represents NR 3 R 4 NH-R 4 、CH-N-OR 4 、CN-NR 3 R 4 , CHOR4 or CHNHR4; and each R 3 represents hydrogen or an organic group, and R 4 represents the linking portion C of the linker. In one embodiment, R or R′ is the following DBCO (dibenzylcyclooctyl) group:

[0341]

[0342] Alkynes such as those described 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 added catalyst (e.g., Cu(I)). A variety of compounds having at least one 1,3-dipole group attached thereto (having a three-atom π-electron system containing 4 electrons delocalized over 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, azooxy groups, and acyldiazo groups.

[0343] In the chemical formula herein, Y' may optionally be absent or may be a spacer, optionally a self-eliminating spacer, such as comprising a p-aminobenzyl unit, or a non-self-eliminating spacer. Optionally, Y' is or comprises a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein the chain length of Y is 2-40 atoms, optionally 2-30, 2-20, 4-40, 4-30 or 4-20 atoms, optionally wherein one or more atoms may not be carbon, such as oxygen, sulfur, nitrogen or other atoms, optionally wherein any carbon of the chain is substituted by alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide.

[0344] An exemplary linker-cytotoxic agent molecule (eg, the XZ moiety of Formulas I to XI) that can be conjugated to an anti-nectin-4 antibody can optionally be represented by Formula V:

[0345] (R)-(Y)-(Pep)-(Y')-(Z) Formula (V)

[0346] in,

[0347] R is a group reactive with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or with a complementary reactive group (R′) of an amino acid attached to the antibody, or when conjugated to an anti-nectin-4 binding protein, R is a residue whose reactive group (R) reacts with a free amino, hydroxyl, sulfhydryl or carboxyl group on the antibody or with a complementary reactive group (R′) of an amino acid attached to the antibody;

[0348] Y is optionally absent or a spacer;

[0349] Pep is or includes a peptidyl linker that is cleaved by an intracellular peptidase or protease, such as a valine-citrulline, valine-alanine, or phenylalanine-lysine dipeptide;

[0350] Y' is optionally absent or is a spacer, optionally a self-immolative spacer or a non-self-immolative spacer; and

[0351] Z is a cytotoxic agent, optionally a camptothecin analog, optionally an exotecan molecule, Dxd or SN-38 molecule.

[0352] The resulting nectin-4 binding immunoconjugate according to the present invention can be represented, for example, by formula (VI):

[0353] Ab-(Y)-(Pep)-(Y')-(Z) Formula (VI)

[0354] in,

[0355] Ab is anti-nectin-4 antibody;

[0356] Y is optionally absent or is a spacer. Optionally, Formula VI comprises a residue between (Ab) and (Y) that reacts with a side chain of an amino acid or a carbohydrate of the anti-Nectin-4 antigen binding protein (Ab) (e.g., maleimide, primary amine). Optionally, a residue that reacts with a side chain of an amino acid of the anti-Nectin-4 antigen binding protein (Ab) (e.g., maleimide, primary amine) may be designated as included in Y;

[0357] Pep is or includes an amino acid unit (e.g., a peptidyl linker) that is cleaved by an intracellular peptidase or protease (e.g., (Pep) is a protease-cleavable dipeptide, tripeptide, tetrapeptide, or pentapeptide, such as a valine-citrulline, valine-alanine, or phenylalanine-lysine unit);

[0358] Y' is optionally absent or is a spacer, optionally a self-immolative spacer or a non-self-immolative spacer; and

[0359] Z is a cytotoxic agent, optionally a camptothecin derivative, optionally an exotecan molecule, Dxd or SN-38 molecule.

[0360] Optionally, the formula can be specified as a residue (RR′) comprising (e.g., between the termini of (Ab) and Y (or (Pep or X) if Y is absent)) a reactive group (R) that reacts with a free amino, hydroxyl, sulfhydryl, or carboxyl group on the antibody or with a complementary reactive group (R′) of an amino acid attached to the antibody.

[0361] In one example, where (RR′) is a residue of a reactive group (R) that reacts with a complementary reactive group (R′) attached to an antibody (e.g., R′ is attached to a side chain of an amino acid or a glycan of the antibody), the nectin-4 binding immunoconjugate according to the present invention can be, for example, represented by the formula (VI bis )express:

[0362] Ab-(RR')-(Y)-(Pep)-(Y')-(Z) formula (VI bis )

[0363] wherein 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′ has a structure selected from the group consisting of:

[0364]

[0365] Where X 8is O or NH, X 9 is selected from H, methyl and pyridyl, and in structures (RR'c) and (RR'd), A bond represents a single bond or a double bond.

[0366] In any embodiment, the exotecan molecule (or other 6-ring camptothecin) can be specified to be bound to Y' (or (Pep) if Y' is not present) via the amine at position 1 of exotecan.

[0367] In any embodiment, the SN-38 molecule (or other 5-ring camptothecin) can be specified to be bound to Y′ (or (Pep) if Y′ is not present) via the amine at position 9 of SN-38.

[0368] Cytotoxic agents, also referred to as (Z) moieties, include, for example, cytotoxic agents, such as antitumor 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 generally described in terms of substitution reactions of N, O, and S heteroatom nucleophiles with electrophilic alkylating agents, but Michael addition reactions are also important. Examples of alkylating agents include nitrogen mustards and sulfur mustards, ethyleneimine, mesylate, CC-1065, and duocarmycin, nitrosoureas, platinum-containing agents, agents that achieve topoisomerase II-mediated DNA site-dependent alkylation (e.g., psorospermin and related bisfuranoxanthone), ecteinascidins, and other or related DNA minor groove alkylating agents.

[0369] In one embodiment, Z is a chelated metal, such as a chelate of a positive divalent or positive trivalent metal with a coordination number of 2 to 8 (including end values). 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). Generally speaking, the metal is preferably a radionuclide. Specific radionuclides include 99mTc, 186Re, 188Re, 58Co, 60Co, 67Cu, 195Au, 199Au, 110Ag, 203Pb, 206Bi, 207Bi, 111In, 67Ga, 68Ga, 88Y, 90Y, 160Tb, 153Gd and 47Sc. The chelated metal can be, for example, one of the above types of metals chelated with any suitable polydentate chelator (eg, acyclic or cyclic polyamines, polyethers (eg, crown ethers and derivatives thereof)); polyamides; porphyrins; and carbocyclic derivatives.

[0370] Anti-nectin-4 antibodies or antibody fragments can also be used for diagnosis, for example, to detect nectin-4 tumor cells. In such embodiments, the antibody or antibody fragment can be conjugated to an effector molecule, such as a detectable substance that can be used, for example, for diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for positron emission tomography), and non-radioactive paramagnetic metal ions. For metal ions that can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Patent No. 4,741,900. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazinylamine luciferin, dansyl chloride and phycoerythrin; suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; and suitable radionuclides include 125I, 131I, 111In and 99Tc.

[0371] 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.

[0372] In another embodiment, the cytotoxic agent is selected from the group consisting of taxanes, anthracyclines, camptothecins, epothilones, mitomycins, compretins, vinca alkaloids, nitrogen mustards, maytansinoids, calicheamicins, duocarmycins, tubulysins, dolastats and auristatins, enediynes, amatoxins, pyrrolobenzodiazepines, ethyleneimines, radioisotopes, therapeutic proteins and polypeptides, and toxins or fragments thereof.

[0373] In another embodiment, the cytotoxic agent is selected from 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, inirotecan, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, lutetocan, camptothecin, crisnatol, mitomycin C, mitomycin A A), methotrexate, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, hydroxyurea, deferoxamine, 5-fluorouracil, floxuridine, doxifluridine, raltitrexed, cytarabine, cytosine arabinoside, fludarabine, 6-mercaptopurine, thioguanine, raloxifen, megestrol, goserelin, leuprolide acetate acetate), flutamide, bicalutamide, vertoporfin, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, lovastatin, staurosporine, actinomycin D, bleomycin A2, bleomycin B2, peplomycin, daunorubicin, doxorubicin, N-(5,5-diacetoxyphenyl)doxorubicin, morpholinodoxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone, thapsigargin, N, 8 -acetylspermine, tallysomycin, esperamycin, butyric acid, retinoic acid, 1,8-dihydroxybicyclo[7.3.1]tridec-4-ene-2,6-diyn-13-one, anguidine, podophyllotoxin, Compretin A-4, pancratistatin, tubulysin A, tubulysin D, carminomycin, streptonigrin, elliptmium acetate, maytansine, maytansinol, calicheamicin, mertansine (DM1), N-acetyl-γ1 I -Caliphatic mycin, calicheamicin-γ1 I , calicheamicin-α2 I , calicheamicin-α3 I , duocarmycin SA, duocarmycin A, CC-1065, CBI-TMI, duocarmycin C2, duocarmycin B2, centanamycin, tail sea turtle, auristatin E, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), α-amanitin, β-amanitin, γ-amanitin, ε-amanitin, amanin, amaninamide, amanullin and amanullinic acid and their derivatives.

[0374] Exemplary auristatin embodiments include an N-terminally linked monomethyl auristatin drug moiety comprising the structure of any of the following formulae (a1) and (a2):

[0375]

[0376] Wherein the wavy lines of (a1) and (a2) indicate the site of covalent attachment to a linker (e.g., linker X or moiety Y, Pep or Y′), and independently at each position:

[0377] R 2 Selected from H and C1-C8 alkyl;

[0378] R 3 is selected from H, C1-C8 alkyl, C1-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0379] R 4 is selected from H, C1-C8 alkyl, C1-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0380] R 5 is selected from H and methyl;

[0381] or R 4 and R 5 Together they form a carbocyclic ring having the formula -(CR a R b ) n , where R a and R b is independently selected from H, C1-C8 alkyl and C3-C8 carbocycle, and n is selected from 2, 3, 4, 5 and 6;

[0382] R 6 Selected from H and C1-C8 alkyl;

[0383] R 7 is selected from H, C1-C8 alkyl, C3-C8 carbocycle, aryl, C1-C8 alkyl-aryl, C1-C8 alkyl-(C3-C8 carbocycle), C3-C8 heterocycle and C1-C8 alkyl-(C3-C8 heterocycle);

[0384] Each R 8 Independently selected from H, OH, C1-C8 alkyl, C3-C8 carbocycle and O-(C1-C8 alkyl);

[0385] R 9 Selected from H and C1-C8 alkyl;

[0386] R 10 Selected from aryl or C3-C8 heterocycle;

[0387] Z is O, S, NH or NR 12 , where R 12 is a C1-C8 alkyl group;

[0388] R 11 Selected from H, C1-C 20 Alkyl, aryl, C3-C8 heterocycle, -(R 13 O) m-R 14 or -(R 13 O) m -CH(R 15 )2; m is an integer from 1 to 1000;

[0389] R 13 is a C2-C8 alkyl group;

[0390] R 14 is H or C1-C8 alkyl;

[0391] R 15 Each occurrence is independently H, COOH, -(CH2) n -N(R 16 )2, -(CH2) n -SO3-C1-C8 alkyl;

[0392] R 16 Each occurrence is independently H, C1-C8 alkyl or -(CH2) n -COOH;

[0393] R 18 Selected from -C(R 8 )2-C(R 8 )2-aryl, -C(R 8 )2-C(R 8 )2-(C3-C8 heterocycle) and

[0394] -C(R 8 )2-C(R 8 )2-(C3-C8 carbocyclic ring); and

[0395] n is an integer of 0-6.

[0396] In one embodiment, R 3 , R 4 and R 7 is independently isopropyl or sec-butyl and R 5 is -H or methyl. In an exemplary embodiment. 3 and R 4 Each is isopropyl, R 5 is -H, and R 7 It is sec-butyl.

[0397] In yet another embodiment, R 2 and R 6 Each is a methyl group, and R 9 is -H.

[0398] In yet another embodiment, R 8 Each occurrence is -OCH3.

[0399] In an exemplary embodiment, R 3 and R 4 Each is isopropyl, R 2 and R 6 Each is a methyl group, R 5 For -H, R 7 is sec-butyl, R 8 Each occurrence is -OCH3, and R 9 is -H.

[0400] In one embodiment, Z is -O- or -NH-.

[0401] In one embodiment, R 10 It is an aromatic group.

[0402] In an exemplary embodiment, R 10 It is -phenyl.

[0403] In an exemplary embodiment, when Z is -0-, R 11 is -H, methyl or tert-butyl.

[0404] In one embodiment, when Z is -NH, R 11 -CH(R 15 )2, where R 15 -(CH2) n -N(R 16 )2, and R 16 is -C1-C8 alkyl or -(CH2) n -COOH.

[0405] In another embodiment, when Z is -NH, R 11 -CH(R 15 )2, where R 15 -(CH2) n -SO3H.

[0406] An exemplary auristatin embodiment of formula (a1) is MMAE, wherein the wavy line indicates the covalent attachment to the linker:

[0407]

[0408] An exemplary auristatin embodiment of formula (a2) is MMAF, wherein the wavy line indicates the covalent attachment to the linker (L) of the antibody-drug conjugate (see US 2005 / 0238649 and Doronina et al. (2006) Bioconjugate Chem. 17: 114-124):

[0409]

[0410] Other exemplary Z embodiments include monomethyl valine compounds with a phenylalanine carboxyl modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008848) and monomethyl valine compounds with a phenylalanine side chain modification at the C-terminus of the pentapeptide auristatin drug moiety (WO 2007 / 008603).

[0411] Other drug moieties include the following MMAF derivatives, where the wavy line indicates the covalent attachment to the linker:

[0412]

[0413]

[0414]

[0415] An example of a linker comprising a spacer (Y) comprising a primary amine, valine-citrulline as the (Pep) moiety, PAB as the (Y′) moiety, and MMAF as the (Z) moiety is shown below:

[0416]

[0417] In one embodiment, Z part is a DNA minor groove binder, and optionally Z comprises 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 polymer comprising more than three pyrrolobenzodiazepine units. The structure and formula of PBD and methods for producing the same are described in, for example, the following PCT Publications: WO 2013 / 177481, WO 2011 / 130616, WO 2004 / 043880, WO 2005 / 085251, WO 2012 / 112687, and WO 2011 / 023883, the disclosures of each of which are incorporated herein by reference.

[0418] Pyrrolo[2,1-c][1,4]benzodiazepines are a family of sequence selective minor groove binding DNA-interacting agents that are covalently linked to guanine residues. The S-chirality at the C11a position of PBDs reportedly provides them with the appropriate three-dimensional shape to fit perfectly into the DNA minor groove. PBDs can have different effects and modes of action. PBDs can be DNA binders or DNA alkylating agents that do not cause DNA crosslinking, or PBDs can be DNA crosslinking agents.

[0419] The pyrrolobenzodiazepine unit or monomer may have the following general structure:

[0420]

[0421] The PBD may have different numbers, types and positions 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. 10 -C 11 The position of imine (N=C), methanolamine (NH-CH(OH)) or methanolamine methyl ether (NH-CH(OMe)) is responsible for the electrophilic center of alkylation of DNA.

[0422] The biological activity of PBDs can be enhanced by linking two PBD monomers or units together, usually via their C8 / C8′-hydroxyl functionalities via a flexible alkylene linker.

[0423] In one aspect of any embodiment herein, the pyrrolobenzodiazepine monomer or unit is a pyrrolo[2,1-c][1,4]benzodiazepine. In one aspect of any embodiment herein, the pyrrolobenzodiazepine dimer is a C8 / C8′-linked pyrrolo[2,1-c][1,4]benzodiazepine dimer.

[0424] The PBD can be attached to the linker via any suitable position. For example, the PBD can be connected to the linker via any position in the PBD unit indicated below.

[0425]

[0426] In one embodiment, the PBD dimer comprises a structure of the following general formula, wherein exemplary points of attachment to other substituents or functional groups within the compound are indicated by arrows:

[0427]

[0428] in:

[0429] R 12 and R 12’ and / or R 2 and R2’ together to form a double bond =CH2 or =CH-CH3, respectively; or

[0430] R 2’ and R 12’ does not exist, and R 2 and R 12 Independently selected from:

[0431] (iia)C 1-5 Saturated aliphatic alkyl;

[0432] (iib)C 3-6 Saturated cycloalkyl;

[0433] (iic)

[0434]

[0435] Where R 21 , R 22 and R 23 Each independently selected from H, C 1-3 Saturated alkyl, C 2-3 Alkenyl, C 2-3 Alkynyl and cyclopropyl, where R 12 The total number of carbon atoms in the group does not exceed 5;

[0436] (iid)

[0437]

[0438] Where R 25a and R 25b One of them is H, and the other is selected from:

[0439] phenyl, optionally substituted by a group selected from halogen, methyl, methoxy; pyridyl; and thienyl; and

[0440] (iie)

[0441]

[0442] Where R 24 Selected from: H; C 1-3 Saturated alkyl; C 2-3 Alkenyl; C 2-3 alkynyl; cyclopropyl; phenyl, the phenyl being optionally substituted by a group selected from halogen, methyl, methoxy; pyridyl; and thienyl;

[0443] R 6 and R 9independently selected from H, R, OH, OR, SH, SR, NH2, NHR, NRR', nitro, Me3Sn and halo; wherein R and R' are independently selected from optionally substituted C 1-12 Alkyl, C 3-20 Heterocyclic and C 5-20 Aryl;

[0444] R 7 is selected from H, R, OH, OR, SH, SR, NH2, NHR, NHRR', nitro, Me3Sn and halide;

[0445] or:

[0446] (a)R 10 is H, and R 11 OH, OR A , where R A is an alkyl group;

[0447] (b)R 10 and R 11 Form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached; or

[0448] (c)R 10 is H, and R 11 For SO z M, wherein z is 2 or 3, and M is a monovalent pharmaceutically acceptable cation;

[0449] R″ is C 3-12 Alkylene, the chain may be interrupted by: one or more heteroatoms, such as O, S, NR N2 (where R N2 is H or C1-4 alkyl), and / or an aromatic ring, such as benzene or pyridine;

[0450] Y and Y' are selected from O, S or NH; and

[0451] R 6′ , R 7′ , R 9′ Selected from R 6 , R 7 and R 9 The same group, and R 10’ and R 11’ With R 10 and R 11 Same, if R 11 and R 11 For SO z M, then M represents a divalent pharmaceutically acceptable cation.

[0452] In another example, the PBD dimer comprises a structure of the following general formula:

[0453]

[0454] Where R 6 , R 7 , R 9 , R 6’ , R 7’ , R 9’ , R 10 , R 11 , R 10’ and R 11’ As defined above, and wherein the "K" ring is a substituted or unsubstituted aromatic or non-aromatic ring, optionally a 6-membered ring, optionally phenyl.

[0455] In one embodiment, the cytotoxic agent (Z) is a camptothecin, for example, it has or comprises the structure of a camptothecin or a camptothecin analog. Camptothecin is well known, as are a wide range of camptothecin analogs, which share a core ring system with various substitutions, but preferably have modifications or substitutions in ring A and / or B in the following basic camptothecin structure:

[0456]

[0457] Many camptothecin analogs have been reported, including topotecan, irinotecan, exitecan, DXd, 9-aminocamptothecin, 9-nitrocamptothecin, 10-hydroxycamptothecin, rutotecan, camptothecin, gimatine, belotecan and rubitecan. Additional camptothecin analogs are disclosed in Li et al., ACS Med. Chem. Lett. 2019, 10, 10, 1386-1392, Japanese Journal of Cancer Research (Jpn. J. Cancer Res.) 86: 776-782 and Takiguchi et al. 1997 Japanese Journal of Cancer Research (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 delunotecan-trastuzumab) have been approved by the FDA. In one embodiment, the camptothecin analog is a pentacyclic compound (eg, camptothecin lacks the F ring). In one embodiment, the camptothecin analog is a hexacyclic compound, eg, including the F ring.

[0458] Some examples such as the basic camptothecin structure, the SN-38 molecule (7-ethyl-10-hydroxycamptothecin; an active metabolite of irinotecan), and the 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), for example, via a substituent on the B ring.

[0459] SN-38:

[0460]

[0461] Li et al., Compounds in ACS Med. Chem. Lett., 2019, 10, 10, 1386-139:

[0462]

[0463] Optionally, the camptothecin analog is a six-ring compound (additional F ring), wherein the compound is attached to the linker via a substituent on this F ring. Examples of such six-ring compounds include, but are not limited to, DXd (Catalog No.: 1599440-33-1) and Exitecan.

[0464] Camptothecin analogs thus include exotecan, SN-38, and any of a range of molecules comprising such moieties, for example, exotecan may be unsubstituted or may be substituted at the amine at position 1, for example, wherein the substituent is or comprises a -C(=O)-, -OC(=O)-, -O-CH2-C(=O)-, HO-O-CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2-O-CH2-C(=O)-, -CH2CH2-O-CH2-C(=O)- group, or other groups shown in U.S. Pat. No. 6,835,807, the disclosure of which is incorporated herein by reference.

[0465] In one embodiment, the antibodies of the present disclosure release exotecan molecules having the structure of Compound 1 in vivo or in vitro in the presence of nectin-4 expressing tumor cells (eg, upon enzymatic cleavage of the cleavable moiety followed by self-elimination of spacer Y').

[0466] Camptothecin derivatives or analogs Exotecan are described in Mitsui et al. 1995 "Japanese Journal of Cancer Research (Jpn. J. Cancer Res.)" 86: 776-782 and Takiguchi et al. 1997 "Japanese Journal of Cancer Research (Jpn. J. Cancer Res.)" 88: 760-769, the disclosures of which are incorporated herein by reference. The structure of Exotecan is shown in the following compound 1a:

[0467]

[0468] Exotecan can be coupled to a linker via the nitrogen atom of the amino group at position 1, such that when the exotecan moiety is bound to the linker or is present within a linker-exotecan molecule ((XZ) molecule), for example, when conjugated to an antibody, exotecan will have the structure of compound 1b:

[0469]

[0470] Thus, it will be understood that when the exotecan of compound 1a is attached to the linker via the amine at position 1 (and, for example, when the linker is in turn attached to the antibody), exotecan will be understood as a modified group at position 1 (i.e., the NH2 group at position 1 is replaced by NH, or alternatively by an OH or O group). For example, exotecan can be coupled to an antibody via a linker comprising a cleavable oligopeptide. Examples include dipeptides, tripeptides, tetrapeptides, and pentapeptides, such as peptides containing glycine and phenylalanine as shown in U.S. Pat. No. 6,835,807, or dipeptides valine-citrulline or valine-alanine attached to a PAB molecule, or the disclosure of said U.S. Pat. No. is incorporated herein by reference. It is known that various suitable linker-Z structures can release active exotecan or exotecan derivatives at the amino group at position 1. As shown in Formula VII, VIII and IX or in the (PEG(8U)-Val-Ala-PAB-isotecan) linker of Example 7, isotecan can be linked to a cleavable oligopeptide via a p-aminobenzyloxycarbonyl (PAB) group attached to the amine at position 1, which upon cleavage results in the release of isotecan having the structure of compound 1a. In one example, as shown in Formula III and compound 13 (Dxd) and the ggfg-Dxd linker of Example 7, isotecan can be linked to a cleavable oligopeptide via a (CH2-C(=O)) group attached to the amine at position 1, which upon cleavage results in the release of compound 13 (Dxd) containing isotecan. Examples of the substituent at the NH or NH2 of position 1 of isotecan of compound 1 include -C(=O)-, -OC(=O)-, or (CH2-C(=O)) containing groups such as -O-CH2-C(=O)-, HO-O-CH2-C(=O)-, -CH2CH2-C(=O)-, -CH2CH2CH2-C(=O)-, -CH2-O-CH2-C(=O)-, and -CH2CH2-O-CH2-C(=O)-.

[0471] In one embodiment, the substituted exotecan derivative (eg, derivatized at position 1) has the structure of compound 13.

[0472] The structure of Dxd is shown below:

[0473]

[0474] Dxd can be coupled to the linker via the oxygen atom at the terminal of the carbon chain, such that when the Dxd moiety is bound to the linker or present in a linker-Dxd molecule ((XZ) molecule), for example when conjugated to an antibody, the terminal OH of the carbon chain will be replaced by O.

[0475] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in Formula VII below, wherein (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues). An anti-nectin-4 binding protein functionalized with a linker comprising the structure of Formula VII or Compound 3 or 4 will release or produce (e.g., in a cell, in the presence of a nectin-4 expressing tumor cell) a compound having the structure of Compound 1a.

[0476]

[0477] An exemplary linker having maleimide as the R group may have the structure of the following compound 3. Such linkers can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bonds with a reducing agent (e.g., tris(2-carboxyethyl)phosphine hydrochloride).

[0478]

[0479] The resulting antibody-drug conjugate will include an antibody comprising one or more cysteine ​​residues functionalized with a compound having the structure of Compound 3 (wherein Compound 3 is bound via the S atom of the cysteine ​​residue).

[0480] In another embodiment, the linker can have a primary amine as the R group, and when reacted with an antibody in the presence of a transglutaminase, an antibody comprising one or more acceptor glutamine residues functionalized with a linker can be produced. For example, a linker (XZ) or an antibody functionalized with the linker has or comprises a structure as shown in Compound 4 below:

[0481]

[0482] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in Formula VIII below, wherein (Y) is a spacer comprising a reactive group (R) (e.g., at its terminus) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated, or otherwise modified N-glycan bound to Kabat residue N297 of the antibody). An anti-nectin-4 binding protein functionalized with a linker comprising the structure of Formula VIII or Compound 5, 6, or 7 will release or produce (e.g., within a cell, in the presence of a nectin-4 expressing tumor cell) a compound having the structure of Compound Ia.

[0483]

[0484] An exemplary linker having maleimide as the R group may have the structure of the following compound 5 or 6. Such a linker can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bond with a reducing agent.

[0485]

[0486] In another embodiment, the linker can have a primary amine as the R group, and when reacted with an antibody in the presence of a transglutaminase, an antibody comprising one or more acceptor glutamine residues functionalized with a linker can be produced. For example, a linker (XZ) or an antibody functionalized with the linker has or comprises a structure as shown in Compound 7 below:

[0487]

[0488] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in the following formula IX, wherein (Y) is a spacer comprising a reactive group (R) (e.g., at its terminus) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated, or otherwise modified N-glycan bound to Kabat residue N297 of the antibody). An anti-nectin-4 binding protein functionalized with a linker comprising the structure of formula IX will release (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells) a compound having the structure of compound Ia.

[0489]

[0490] An exemplary linker having maleimide as the R group may have the structure of the following compound 8. Such a linker can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bonds with a reducing agent.

[0491]

[0492] In another embodiment, the linker can have a primary amine as the R group, and when reacted with an antibody in the presence of a transglutaminase, an antibody comprising one or more acceptor glutamine residues functionalized with a linker can be produced. For example, a linker (XZ) or an antibody functionalized with the linker has or comprises a structure as shown in Compound 9 below:

[0493]

[0494] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in the following formula X, wherein (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) that reacts with 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 acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated or otherwise modified N-glycan bound to Kabat residue N297 of the antibody).

[0495]

[0496] An exemplary linker having maleimide as the R group may have the structure of the following compound 10. Such linkers can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bonds with a reducing agent (eg, tris(2-carboxyethyl)phosphine hydrochloride).

[0497]

[0498] In one embodiment, the linker moiety (XZ) is or comprises the structure shown in Formula XI below, wherein (Y) is a spacer comprising (e.g., at its terminus) a reactive group (R) that reacts with 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 acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated or otherwise modified N-glycan bound to Kabat residue N297 of the antibody).

[0499]

[0500] An exemplary linker having maleimide as the R group may have the structure of the following compound 11. Such a linker can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bonds with a reducing agent.

[0501]

[0502] In one embodiment, the linker moiety (XZ) is or comprises a structure according to Formula XII below, wherein (Y) is a spacer comprising a reactive group (R) (e.g., at its terminus) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated or otherwise modified N-glycan bound to Kabat residue N297 of an antibody). The linker may comprise, for example, 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, the number of PEG units n can be, for example, 1-72, 5-23, 1-15, 1-16, 1-11, 1-12, 1-8, 1-7, 1-6, 7, 15, 17 or 23.

[0503]

[0504] In such embodiments, the immunoconjugate or antibody-drug conjugate comprising a linker moiety having maleimide as the R group may have the structure of Formula XII bis, wherein the maleimide moiety is bound to the thiol group (S) of an amino acid (i.e., cysteine) of the antibody (Ab) after reduction of the interchain disulfide bonds with a reducing agent.

[0505]

[0506] In the variant of Formula XII where the intracellular cleavable peptide is valine-citrulline, the immunoconjugate or antibody-drug conjugate comprising a linker moiety having maleimide as the R group may have the structure of Formula XIIter:

[0507]

[0508] An exemplary linker having maleimide as the R group may have the structure of the following compound 14a. After reducing the interchain disulfide bonds with a reducing agent, such a linker can be conjugated to the antibody via the cysteine ​​residues in the antibody. In this example, the linker moiety comprises 8 PEG units.

[0509]

[0510] According to this embodiment, an immunoconjugate or an antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 14a bis.

[0511]

[0512] Another exemplary linker having maleimide as the R group may have the structure of the following compound 14b. After reducing the interchain disulfide bonds with a reducing agent, such a linker can be conjugated to the antibody via the cysteine ​​residues in the antibody. In this example, the linker moiety comprises 7 PEG units.

[0513]

[0514] According to this embodiment, an immunoconjugate or an antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 14b bis.

[0515]

[0516] Another exemplary linker having maleimide as the R group may have the structure of the following compound 14c. After reducing the interchain disulfide bonds with a reducing agent, such a linker can be conjugated to the antibody via the cysteine ​​residues in the antibody. In this example, the linker moiety comprises 16 PEG units.

[0517]

[0518] According to this embodiment, an immunoconjugate or antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 14c bis.

[0519]

[0520] In one embodiment, the linker moiety (XZ) is or comprises a structure according to Formula XIII below, wherein (Y) is a spacer comprising a reactive group (R) (e.g., at its terminus) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated or otherwise modified N-glycan bound to Kabat residue N297 of an antibody). The linker may comprise, for example, 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, the number of PEG units n can be, for example, 1-72, 5-23, 1-15, 1-16, 1-11, 1-12, 1-8, 1-7, 1-6, 7, 15, 17 or 23.

[0521]

[0522] In such embodiments, the immunoconjugate or antibody-drug conjugate comprising a linker moiety having maleimide as the R group may have the following formula XIII bis, wherein the maleimide moiety is bound to the thiol group (S) of an amino acid (i.e., cysteine) of the antibody (Ab) after reduction of the interchain disulfide bonds with a reducing agent.

[0523]

[0524] An exemplary linker having maleimide as the R group may have the structure of the following compound 15a. After reducing the interchain disulfide bonds with a reducing agent, such a linker can be conjugated to the antibody via the cysteine ​​residues in the antibody. In this example, the linker portion comprises 8 PEG units.

[0525]

[0526] According to this embodiment, the immunoconjugate or antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 15a bis.

[0527]

[0528] Another exemplary linker having maleimide as the R group may have the structure of the following compound 15b. After reducing the interchain disulfide bonds with a reducing agent, such a linker can be conjugated to the antibody via the cysteine ​​residues in the antibody. In this example, the linker portion comprises 7 PEG units.

[0529]

[0530] According to this embodiment, an immunoconjugate or antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 15b bis.

[0531]

[0532] In one embodiment, the linker moiety (XZ) is or comprises a structure according to the following formula XIV, wherein (Y) is a spacer comprising a reactive group (R) (e.g., at its terminus) that reacts with 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 carboxyl group of one or more glutamic acid or aspartic acid residues, or the S atom of one or more cysteinyl residues), or a glycan structure such as a glycosylated amino acid residue (e.g., a native, truncated or otherwise modified N-glycan bound to Kabat residue N297 of an antibody). In the following formula, the number of PSAR units n can be, for example, 1-72, 1-24, 1-16, 1-10, 1-12, 1-8, 1-6, 8, 16, 18 or 24.

[0533]

[0534] An exemplary linker having maleimide as the R group may have the structure of the following compound 16. Such a linker can be conjugated to the antibody via a cysteine ​​residue in the antibody after reducing the interchain disulfide bonds with a reducing agent.

[0535]

[0536] According to this embodiment, an immunoconjugate or antibody-drug conjugate comprising such a linker moiety may have the structure of the following compound 16bis.

[0537]

[0538] In another embodiment, the linker can have a primary amine as the R group, and when reacted with an antibody in the presence of a transglutaminase, an antibody comprising one or more acceptor glutamine residues functionalized with a linker can be produced. For example, a linker (XZ) or an antibody functionalized with the linker has or comprises a structure as shown in the following compound 12:

[0539]

[0540] Anti-nectin-4 binding proteins functionalized with oligopeptide linkers of Formula XI or compounds 11 and 12 result in the release (e.g., intracellularly, in the presence of nectin-4 expressing tumor cells) of substituted exotecan having an OH-CH2-C(=O) substituent present at the amine at position 1, as shown in the structure below:

[0541]

[0542] When prepared as a structure with a primary amine, the exemplary linker of formula III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII or XIV can react with an antibody in the presence of a transglutaminase (e.g., bacterial transglutaminase, BTG) so that the transglutaminase catalyzes the conjugation of a linker to an acceptor glutamine residue in the primary structure of the antibody (e.g., in an immunoglobulin constant domain or in a TGase recognition tag inserted into or attached to (e.g., fused to) a constant region). Methods and linkers for conjugation with antibodies mediated by BTG are described in PCT Publication No. WO2014 / 202773, the disclosure of which is incorporated by reference. The conjugation catalyzed by BTG allows precise control of the average drug in the composition: antibody ratio. The term "transglutaminase", which can be used interchangeably with "TGase" or "TG", refers to an enzyme that can cross-link proteins to produce ε-(γ-glutamyl) lysine isopeptide bonds by an acyl transfer reaction between the ε-amino group of the γ-carboxamide group of peptide-bound glutamine and the ε-amino group of lysine or a structurally related primary amine (such as aminopentyl, such as peptide-bound lysine). TGase especially includes bacterial transglutaminases (BTG), such as enzymes (protein-glutamine-γ-glutamyl transferase) with EC reference EC 2.3.2.13. The term "acceptor glutamine" residue, when referring to the glutamine residues of an antibody, refers to a glutamine residue that is recognized by TGase and can be cross-linked by TGase by a reaction between glutamine and lysine or a structurally related primary amine (such as aminopentyl). Preferably, the acceptor glutamine residue is a surface-exposed glutamine residue. The term "TGase recognition tag" refers to an amino acid sequence including an acceptor glutamine residue, and when incorporated into (e.g., attached to) a polypeptide sequence under suitable conditions, the amino acid sequence is recognized by TGase and causes TGase cross-linking by the reaction between the amino acid side chains in the amino acid sequence and the reaction partner. The recognition tag can be a peptide sequence that is not naturally present in the polypeptide including the enzyme recognition tag. The example of the TGase recognition tag includes the amino acid sequence disclosed in WO2012 / 059882 and WO2014 / 072482.

[0543] As exemplified in WO2013 / 092983 and WO2020 / 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 in which a moiety comprising a primary amine and a first reactive group (R) is conjugated to the antibody in the presence of BTG; and a subsequent step of reacting the antibody-linker conjugate with a molecule comprising: (i) a second reactive group (R′) reactive with the first reactive group; and (ii) a cytotoxic agent (Z). Examples of reactive group pairs R and R′ include a range of groups capable of biorthogonal reactions, such as 1,3-dipolar cycloadditions between azides and cyclooctynes ​​(copper-free click chemistry), 1,3-dipolar cycloadditions between nitrones and cyclooctynes, oxime / hydrazone formation from aldehydes and ketones, and tetrazine linkages (see also WO2013 / 092983). The resulting linker and functionalized antibody or its Y element may thus include an RR' group resulting from the reaction of R and R', such as a triazole.

[0544] The anti-Nectin-4 immunoconjugate can be incorporated into a pharmaceutical formulation at a concentration of 1 mg / ml to 500 mg / ml, wherein the pH of the formulation is 2.0 to 10.0. The formulation may further include a buffer system, one or more preservatives, one or more tonicity agents, one or more chelating agents, stabilizers, and surfactants. In one embodiment, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation comprising water. Such formulations are typically solutions or suspensions. In another 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.

[0545] In another embodiment, the pharmaceutical formulation is a lyophilized formulation and the physician or patient adds solvents and / or diluents prior to use.

[0546] In another embodiment, the pharmaceutical formulation is a dry formulation (eg, freeze-dried or spray-dried) and is ready for use without any prior dissolution.

[0547] In another aspect, a pharmaceutical formulation comprises an aqueous solution of such an antibody and a buffer, wherein the antibody is present at a concentration of 1 mg / ml or greater, and wherein the pH of the formulation is from about 2.0 to about 10.0.

[0548] In another embodiment, the pH of the formulation is in a 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.

[0549] In another embodiment, the buffer 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, trimethylglycine, malic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, aspartic acid or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the present invention.

[0550] In another embodiment, the formulation further comprises a pharmaceutically acceptable preservative. In another embodiment, the formulation further comprises an isotonic agent. In another embodiment, the formulation further comprises a chelating agent. In another embodiment of the present invention, the formulation further comprises a stabilizer. In another embodiment, the formulation further comprises a surfactant. For convenience, reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0551] Other ingredients may be present in the pharmaceutical formulations of the present invention. Such additional ingredients may include wetting agents, emulsifiers, antioxidants, fillers, tension modifiers, chelating agents, metal ions, oily vehicles, proteins (e.g., human serum albumin, gelatin or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine and histidine). Of course, such additional ingredients should not adversely affect the overall stability of the pharmaceutical formulations of the present invention.

[0552] Administration of the pharmaceutical composition according to the invention can be by several routes of administration, such as intravenous administration. Suitable antibody formulations can also be determined by examining experience with other already developed therapeutic ADCs.

[0553] In any embodiment, the composition can be characterized as comprising a plurality of Nectin-4 binding immunoconjugates of the present disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the sample have at least 4, 6 or 8 amino acid residues per antibody that are functionalized with a linker disclosed herein. In any embodiment, the composition can be characterized as comprising a plurality of Nectin-4 binding immunoconjugates of the present disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the sample have at least 2, 4, 6 or 8 amino acid residues per antibody that are functionalized with a linker-camptothecin moiety, such as a (XZ) unit or a (-(Y)-(Pep)-(Y')-(Z)) unit of the chemical formula herein. In any embodiment, the composition can be characterized as comprising a plurality of nectin-4 binding immunoconjugates of the present disclosure, wherein at least 70%, 80%, 90%, 95%, 98% or 99% of the immunoconjugates in the sample have the same number of functionalized amino acids per antibody, optionally wherein the number is 4, 6 or 8.

[0554] Diagnosis, prognosis and treatment of malignant tumors

[0555] In some aspects, methods and antibodies, antibody fragments and immunoconjugates useful for diagnosing, prognosing, monitoring and treating cancers characterized by tumor cells expressing Nectin-4 at the surface are described. In the method of treatment, the treatment comprises administering the antibodies of the present disclosure to a human subject or individual. In some embodiments, improved methods for improving the delivery of cytotoxic agents (particularly camptothecin analogs) to Nectin-4 positive tumors are provided. The improved delivery of cytotoxic agents may be a result of antibody-mediated inhibition of cluster formation and / or anchorage-independent growth of Nectin-4 expressing tumor cells, resulting in improved tumor penetration of cytotoxic agents and / or ADCs containing such agents. Treatment with the ADCs of the present disclosure is particularly advantageous for treating diseases with low or heterogeneous Nectin-4 expression, for patients with particularly resistant diseases or other ADCs for which they are not suitable, and / or for use in combination with additional therapeutic agents (e.g., chemotherapeutic agents) that mediate toxicity as a single agent.

[0556] In one embodiment, the anti-Nectin-4 antibody or antibody fragment can be used to sensitize tumors to cytotoxic or chemotherapeutic agents. Compared with the anticancer agent itself, by reducing the chemoresistance of cancer cells, the anti-Nectin-4 antibody can be used to enhance or increase the toxicity of anticancer agents to cancer. Cancer sensitizing compositions comprising anti-Nectin-4 antibodies or antibody fragments can be used to sensitize tumors (e.g., Pgp-expressing tumors) to anticancer agents to reduce chemoresistance and improve the therapeutic effect of anticancer agents.

[0557] In one embodiment, the anti-Nectin-4 antibody or antibody fragment of the present disclosure may be designated for use in combination with a chemotherapeutic agent for treating a tumor in a human subject, wherein the anti-Nectin-4 antibody, antibody fragment and chemotherapeutic agent are formulated for separate administration and are administered simultaneously or sequentially. For example, the treatment may include administering to the subject an effective amount of each of the following: (a) an anti-Nectin-4 antibody or antibody fragment of the present disclosure, and (b) an anticancer agent, optionally a chemotherapeutic agent, optionally a chemotherapeutic agent known to be transported by P-glycoprotein (Pgp), optionally an anthracycline, a vinca alkaloid, an etoposide, a taxane, a platinum compound or optionally a camptothecin.

[0558] In one embodiment, the antibody or antibody fragment is conjugated to a cytotoxic agent, such as camptothecin, exotecan or SN-38 molecules. Antibodies or antibody fragments are typically conjugated to multiple molecules of cytotoxic agents (e.g., camptothecin analogs, exotecan). Cytotoxic agents (e.g., camptothecin or exotecan) can be conjugated to antibodies by a linker (e.g., a linker-toxin of any one of formula VII-XIV) comprising an oligopeptide linker cleavable by a protease. Exemplary pharmaceutical compositions may include an average of 1 to 8 cytotoxic agent molecules (e.g., camptothecin derivatives, exotecan, linkers-toxins of any one of formula 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 the antibody or antibody fragment is conjugated to exotecan (i.e., via a linker, such as a linker-toxin of any of Formulae VII-XIV), such immunoconjugates are particularly useful 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, for example, treatment with ADCs comprising an auristatin (MMAE) payload has been shown to induce PgP expression on tumors. In addition, a range of tumors characterized by native PgP / MDR1 expression are known, for example, the following cancers express significant levels of MDR1: renal cancer, adrenocortical carcinoma, bile duct cancer, 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.

[0559] Nectin-4 binding agents (e.g., anti-nectin-4 antibodies or antibody fragments) conjugated to cytotoxic agents can be advantageously used to treat individuals with nectin-4 expressing cancers characterized by tumor cells expressing nectin-4 (e.g., at the tumor cell membrane or cell surface). Examples of such cancers are urothelial carcinoma, 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.

[0560] Nectin-4 binding agents conjugated with cytotoxic agents can be used in tumors with high Nectin-4 expression.

[0561] Nectin-4 binding agents conjugated to cytotoxic agents may also be used in heterogeneous and / or low Nectin-4 expressing tumors. In such tumors, the immunoconjugates of the present disclosure may provide advantageous efficacy, optionally by avoiding MDR1-mediated resistance and / or bystander anti-tumor effects.

[0562] Nectin-4 binding agents conjugated to cytotoxic agents can be advantageously used to treat individuals regardless of the level of nectin-4 expression, regardless of the heterogeneity of nectin-4 expression levels on tumor cells within an individual, and / or regardless of whether the individual has been previously treated with vetin-enfortumab. When the cytotoxic agent is isotecan, the nectin-4 binding agents conjugated to the cytotoxic agent can also be advantageously used to treat individuals regardless of tumor Pgp expression and / or regardless of whether the individual has been previously treated with an ADC comprising a cytotoxic agent that can be transported by PgP (e.g., an ADC comprising an anti-nectin-4 antibody conjugated to a camptothecin analog other than isotecan, an ADC comprising an anti-nectin-4 antibody conjugated to Dxd (e.g., via a GGFG-containing linker), or an ADC comprising an anti-nectin-4 antibody conjugated to an auristatin, an anthracycline, a vinca alkaloid, an etoposide, a taxane, or a platinum compound).

[0563] In one embodiment, a nectin-4 binder conjugated to a camptothecin derivative molecule may be advantageously used to treat an individual who has previously been treated with Vitin-Enfortuzumab. After Vitin-Enfortuzumab treatment, such an individual may optionally have a cancer characterized by a heterogeneous and / or low nectin4 expressing tumor. After Vitin-Enfortuzumab treatment, such an individual may optionally have a cancer characterized by a Pgp expressing tumor. An individual may have a cancer that is resistant, unresponsive, recurrent, and / or progressive despite treatment with an antibody conjugated to an auristatin or MMAE molecule (e.g., Vitin-Enfortuzumab). For example, an individual may have locally advanced or metastatic urothelial carcinoma and has previously been treated with an antibody conjugated to an auristatin or MMAE molecule (e.g., Vitin-Enfortuzumab).

[0564] In one embodiment, an immunoconjugate comprising a nectin-4 binder conjugated to ixotecan via a linker is advantageously used to treat an individual who has previously been treated with an immunoconjugate comprising a nectin-4 binder conjugated to a cytotoxic agent capable of being transported by Pgp via a linker. In one embodiment, an immunoconjugate comprising a nectin-4 binder conjugated to ixotecan via a linker is advantageously used to treat an individual having a cancer characterized by a heterogeneous and / or low nectin4 expressing tumor following immunoconjugate treatment, the immunoconjugate comprising a nectin-4 binder conjugated to a cytotoxic agent capable of being transported by Pgp via a linker. In one embodiment, an immunoconjugate comprising a nectin-4 binder conjugated to ixotecan via a linker is advantageously used to treat an individual with a cancer that is resistant, unresponsive, relapsed, and / or progresses despite treatment with a nectin-4 binder that is conjugated to a cytotoxic agent that can be transported by Pgp via a linker. Optionally, the nectin-4 binder conjugated to a cytotoxic agent that can be transported by Pgp via a linker comprises an anti-nectin-4 antibody conjugated to a camptothecin analog via a linker, so that a camptothecin analog other than ixotecan (e.g., Dxd or delutecan) is released upon cleavage of the linker. Optionally, the nectin-4 binder conjugated to a cytotoxic agent that can be transported by Pgp via a linker comprises an anti-nectin-4 antibody conjugated to Dxd (e.g., via a GGFG-containing linker). Optionally, the Nectin-4 binder conjugated to a cytotoxic agent capable of being transported by Pgp via a linker comprises 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 binder conjugated to a cytotoxic agent capable of being transported by Pgp via a linker comprises an anti-Nectin-4 antibody that binds to the IgV domain of Nectin-4. Optionally, the Nectin-4 binder conjugated to a cytotoxic agent capable of being transported by Pgp via a linker comprises an anti-Nectin-4 antibody that binds to the VC1 bridging domain of Nectin-4. In one embodiment, the immunoconjugate comprising a Nectin-4 binder conjugated to exotecan via a linker comprises a linker having an enzymatically cleavable portion (and optionally a self-immolative spacer) that results in the release of exotecan upon cleavage.

[0565] In advanced recurrent or metastatic urothelial carcinoma, a large proportion of individuals will express high levels of nectin-4 on tumor cells, for example, an H score of at least 290 (see EV-201 clinical trial cohort 1 Nectin-4 expression). However, a portion of patients have an H score of less than 250, and some have an H score of less than 200. A small number of patients have an H score of less than 150, and some have an H score of less than 100. In triple-negative breast cancer (TNBC), 62% of patients were reported to have high nectin-4 expression on tumor cells, and 38% had low nectin-4 expression on tumor cells (Rabat et al., 2017 Annals Onc. 28: 769-776). In other cancer types, the median H score values ​​for nectin-4 expression are generally 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.

[0566] In one embodiment, the individual treated in accordance with the present disclosure has advanced recurrent or metastatic cancer, optionally has advanced recurrent or metastatic urothelial carcinoma.

[0567] In one embodiment, an individual treated in accordance with the present disclosure has a cancer (e.g., breast cancer) that tests positive for estrogen receptor and / or progesterone receptor but tests negative for epidermal growth factor receptor 2 (HER2) or excess HER2 protein, optionally the cancer tests positive for HER2 but expresses HER2 at low levels.

[0568] In one embodiment, an individual treated in accordance with the present disclosure has triple negative breast cancer (TNBC), e.g., breast cancer that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein.

[0569] In one embodiment, the individual treated according to the present disclosure has a cancer that tests positive for HER2 protein (e.g., breast cancer), optionally wherein the cancer expresses an excess of HER2 protein (HER2 overexpression), optionally wherein the cancer expresses a low level 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); optionally, wherein the agent that binds to HER2 is an ADC; optionally, wherein the antibody that binds to HER2 is conjugated to a cytotoxic agent, optionally auristatin, maytansinoid (e.g., DM1), or a camptothecin analog (e.g., Compound 1, 2, or 13); optionally, wherein the antibody that binds to HER2 is trastuzumab emtansine or trastuzumab deruxtecan (DS-8201a; Enhertu TM ).

[0570] In one embodiment, the individual treated in accordance with the present disclosure has non-small cell lung cancer, optionally lung adenocarcinoma.

[0571] In one embodiment, the individual treated in accordance with the present disclosure has pancreatic cancer.

[0572] In one embodiment, the individual treated in accordance with the present disclosure has ovarian cancer.

[0573] In one embodiment, the individual treated in accordance with the present disclosure has head and neck squamous cell carcinoma.

[0574] In one embodiment, the individual treated in accordance with the present disclosure has esophageal cancer.

[0575] In one embodiment, the individual treated in accordance with the present disclosure has colorectal cancer.As used herein, colorectal cancer (CRC) refers to colon cancer, rectal cancer, and colorectal cancer (cancer of both the colon and rectal regions).

[0576] In one embodiment, the individual treated according to the present disclosure has NSCLC or lung adenocarcinoma, gastric cancer, colorectal cancer, pancreatic cancer, urothelial carcinoma or bladder cancer that tests positive for HER2 protein, optionally wherein the cancer expresses an excess of HER2 protein (HER2 overexpression), optionally wherein the cancer expresses a low level of HER2 protein (less than excess HER2 expression). In one embodiment, the individual is treated with an anti-Nectin-4 ADC according to the present disclosure in combination with an agent (e.g., antibody) that binds to a HER2 polypeptide (e.g., an antibody comprising heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab). Optionally, the agent that binds HER2 is an ADC; optionally, the antibody that binds HER2 is conjugated to a cytotoxic agent, optionally auristatin, maytansinoid (e.g., DM1) or a camptothecin analog (e.g., compound 1, 2 or 13); optionally, the antibody that binds Her2 is trastuzumab enmetuzumab or delutican-trastuzumab (DS-8201a).

[0577] As shown herein, ADCs that release exotecan upon cleavage (e.g., upon cleavage of an intracellular cleavable linker comprising a cytotoxic agent Z) are particularly advantageous in treating tumors resistant to ADCs with linkers that release a cytotoxic agent (other than exotecan) transported by Pgp, such as auristatin or Dxd, upon cleavage. Thus, in one embodiment, when treating HER2-positive cancers with a combination of an anti-Nectin-4 binder (e.g., an anti-Nectin-4 ADC) and an anti-HER2 ADC, the anti-HER2 ADC can be designed to release exotecan upon cleavage (e.g., upon cleavage of an intracellular cleavable linker comprising a cytotoxic agent Z). An anti-nectin-4 binder (e.g., an anti-nectin-4 ADC) can be designed to release exotecan upon cleavage (e.g., upon cleavage of an intracellularly cleavable linker comprising a cytotoxic agent Z), or can release any other cytotoxic agent (Z), for example, wherein Z is a taxane, anthracycline, camptothecin, epothilone, mitomycin, compretin, vinca alkaloid, nitrogen mustard, maytansinoid, duocarmycin, tubulysin, dolastatin, auristatin, enediyne, pyrrolobenzodiazepine, amatoxin, or ethyleneimine.

[0578] Therefore, in one aspect, the present disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide, which is used to treat cancer (e.g., HER2-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human Nectin-4 polypeptide is represented by the following formula:

[0579] Ab N4 -(X N4 -(Z N4 ))

[0580] in,

[0581] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0582] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4 For example, comprising a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide under physiological conditions, optionally under intracellular conditions; and

[0583] Z N4 contains a cytotoxic agent;

[0584] The immunoconjugate binding to human Nectin-4 polypeptide is used in combination with the immunoconjugate binding to human HER2 polypeptide represented by the following formula:

[0585] Ab HER2 -(X HER2 -(Z HER2 ))

[0586] in,

[0587] Ab HER2 is a polypeptide, peptide or antibody that specifically binds to a human HER2 polypeptide, wherein Ab HER2 comprising the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab;

[0588] X HER2 Is connected to Ab HER2 and Z HER2 The numerator, where X HER2 For example, comprising a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide under physiological conditions, optionally under intracellular conditions; and

[0589] Z HER2 The immunoconjugate bound to the human HER2 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0590] In addition, in one aspect, the present disclosure provides an immunoconjugate that binds to a human HER2 polypeptide, which is used to treat cancer (e.g., HER2-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human HER2 polypeptide is represented by the following formula:

[0591] AbHER2 -(X HER2 -(Z HER2 ))

[0592] in,

[0593] Ab HER2 is a polypeptide, peptide or antibody that specifically binds to a human HER2 polypeptide, wherein Ab HER2 comprising the heavy and light chain CDRs or variable regions of trastuzumab or pertuzumab;

[0594] X HER2 Is connected to Ab HER2 and Z HER2 The numerator, where X HER2 For example, comprising a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide under physiological conditions, optionally under intracellular conditions; and

[0595] Z HER2 It is ixitecan;

[0596] The immunoconjugate binding to human HER2 polypeptide is used in combination with the immunoconjugate binding to human Nectin-4 polypeptide represented by the following formula:

[0597] Ab N4 -(X N4 -(Z N4 ))

[0598] in,

[0599] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0600] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4 For example, comprising a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide under physiological conditions, optionally under intracellular conditions; and

[0601] Z N4 The immunoconjugate that binds to the human HER2 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0602] Therefore, in one aspect, the present disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide, which is used to treat cancer (e.g., TROP-2-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human Nectin-4 polypeptide is represented by the following formula:

[0603] Ab N4 -(X N4 -(Z N4 ))

[0604] in,

[0605] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0606] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0607] Z N4 contains a cytotoxic agent;

[0608] The immunoconjugate binding to human nectin-4 polypeptide is used in combination with the immunoconjugate binding to human TROP-2 polypeptide represented by the following formula:

[0609] Ab TROP-2 -(X TROP-2 -(Z TROP-2 ))

[0610] in,

[0611] Ab TROP-2 is a polypeptide, peptide or antibody that specifically binds to a human TROP-2 polypeptide, wherein Ab TROP-2 comprising the heavy and light chain CDRs or variable regions of datopotamab or sacituzumab;

[0612] X TROP-2 Is connected to Ab TROP-2 and Z TROP-2 The numerator, where X TROP-2 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0613] Z TROP-2The immunoconjugate bound to the human TROP-2 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0614] In addition, in one aspect, the present disclosure provides an immunoconjugate that binds to a human TROP-2 polypeptide, which is used to treat cancer (e.g., TROP-2-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human TROP-2 polypeptide is represented by the following formula:

[0615] Ab TROP-2 -(X TROP-2 -(Z TROP-2 ))

[0616] in,

[0617] Ab TROP-2 is a polypeptide, peptide or antibody that specifically binds to a human TROP-2 polypeptide, wherein Ab TROP-2 comprising the heavy and light chain CDRs or variable regions of datopotamab or sacituzumab;

[0618] X TROP-2 Is connected to Ab TROP-2 and Z TROP-2 The numerator, where X TROP-2 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0619] Z TROP-2 It is ixitecan;

[0620] The immunoconjugate binding to human TROP-2 polypeptide is used in combination with the immunoconjugate binding to human Nectin-4 polypeptide represented by the following formula:

[0621] Ab N4 -(X N4 -(Z N4 ))

[0622] in,

[0623] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0624] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0625] Z N4 The immunoconjugate that binds to the human TROP-2 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0626] Therefore, in one aspect, the present disclosure provides an immunoconjugate that binds to a human Nectin-4 polypeptide, which is used to treat cancer (e.g., B7H3-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human Nectin-4 polypeptide is represented by the following formula:

[0627] Ab N4 -(X N4 -(Z N4 ))

[0628] in,

[0629] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0630] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0631] Z N4 contains a cytotoxic agent;

[0632] The immunoconjugate binding to human nectin-4 polypeptide is used in combination with the immunoconjugate binding to human B7H3 polypeptide represented by the following formula:

[0633] Ab B7H3 -(X B7H3 -(Z B7H3 ))

[0634] in,

[0635] Ab B7H3 is a polypeptide, peptide or antibody that specifically binds to a human B7H3 polypeptide, optionally wherein Ab B7H3comprising the heavy and light chain CDRs or variable regions of enoblituzumab, ifinatamab, mirzotamab, obrindatamab, omburtamab, or vobramitamab;

[0636] X B7H3 Is connected to Ab B7H3 and Z B7H3 The numerator, where X B7H3 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0637] Z B7H3 The immunoconjugate bound to the human B7H3 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0638] In addition, in one aspect, the present disclosure provides an immunoconjugate that binds to a human B7H3 polypeptide, which is used to treat cancer (e.g., B7H3-positive Nectin-4-positive cancer), wherein the immunoconjugate that binds to a human B7H3 polypeptide is represented by the following formula:

[0639] Ab B7H3 -(X B7H3 -(Z B7H3 ))

[0640] in,

[0641] Ab B7H3 is a polypeptide, peptide or antibody that specifically binds to a human B7H3 polypeptide, optionally wherein Ab B7H3 comprising the heavy and light chain CDRs or variable regions of enoblituzumab, ifinatamab, mirzotamab, obrindatamab, omburtamab, or vobramitamab;

[0642] X B7H3 Is connected to Ab B7H3 and Z B7H3 The numerator, where X B7H3 comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g. under physiological conditions, optionally under intracellular conditions; and

[0643] Z B7H3 It is ixitecan;

[0644] The immunoconjugate binding to human B7H3 polypeptide is used in combination with the immunoconjugate binding to human nectin-4 polypeptide represented by the following formula:

[0645] Ab N4 -(X N4 -(Z N4 ))

[0646] in,

[0647] Ab N4 It is a polypeptide, peptide or antibody that specifically binds to human nectin-4 polypeptide;

[0648] X N4 Is connected to Ab N4 and Z N4 The numerator, where X N4 For example, comprising a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide under physiological conditions, optionally under intracellular conditions; and

[0649] Z N4 The immunoconjugate that binds to the human B7H3 polypeptide releases exotecan upon cleavage of the cleavable moiety. Optionally, Z N4 The immunoconjugate is exotecan, and optionally, the immunoconjugate bound to the human nectin-4 polypeptide releases exotecan upon cleavage of the cleavable moiety.

[0650] In one aspect, the methods of treatment disclosed herein are independent of the assessment or detection of Nectin-4 expression in tumor tissue and / or are independent of the level of Nectin-4 expression on tumor cells and / or the frequency or number of Nectin-4 expressing tumor cells in a tissue sample from the individual. In one aspect, the methods of treatment disclosed herein are independent of the assessment or detection of tumor PgP (MDR1) expression.

[0651] In one aspect, the present invention provides a method for treating cancer and / or eliciting 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), wherein the method does not require a prior determination of whether the individual has tumor tissue containing cells expressing nectin-4 (e.g., tumor cells).

[0652] In one aspect, the 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), wherein the method does not require a prior determination of whether the individual has tumor tissue containing cells (e.g., tumor cells) expressing high levels of nectin-4, as defined by immunohistochemical evaluation (e.g., H-score or other appropriate IHC scoring method).

[0653] In one aspect, the method of treating cancer and / or killing tumor cells in an individual does not require a priori determination of the level of nectin-4 expression by the tumor cells.

[0654] In one aspect, a method of treating cancer, optionally advanced recurrent or metastatic urothelial carcinoma or breast cancer (e.g., TNBC, HER2-positive cancer) in an individual comprises treating an individual having a cancer characterized by an H-score of no greater than or less than 290, 250, 200, 150, or 100 for Nectin-4 expression.

[0655] In any embodiment for treating or preventing cancer in an individual, the method can be specified to include the steps of: (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.

[0656] In any embodiment for treating or preventing cancer in an individual, the method can be specified to include the steps of: (i) identifying an individual whose tumor cells express (a) nectin-4 (e.g., as determined by immunohistochemistry) and (b) HER2, optionally wherein the tumor cells express low levels of HER2 (e.g., as determined by immunohistochemistry; as determined by Herceptest TM Determined); and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure, optionally in combination with an agent (e.g., antibody) that binds to a Her2 polypeptide (e.g., trastuzumab, pertuzumab); optionally, wherein the antibody that binds to Her2 is an ADC; optionally, wherein the antibody that binds to Her2 is conjugated to a cytotoxic agent, optionally an auristatin, a maytansinoid (e.g., DM1), or a camptothecin derivative (e.g., Compound 1, 2, or 13). Optionally, wherein the antibody that binds to Her2 is trastuzumab enmetuzumab or delutec-trastuzumab (DS-8201a).

[0657] In any embodiment for treating or preventing cancer in an individual, the method can be specified to include the steps of: (i) identifying an individual whose tumor cells have low or intermediate 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).

[0658] In any embodiment for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method can be specified as comprising the steps of: (i) identifying an individual whose cancer is characterized by low level 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 Nectin-4 expression of no greater than or less than 150 or 100.

[0659] In any embodiment for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual, the method may be specified as comprising the steps of: (i) identifying an individual whose cancer is characterized by an intermediate 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 of Nectin-4 expression of no greater than or less than 290, 250, 200, 150, optionally further wherein the cancer is characterized by an H score of Nectin-4 expression of at least 100.

[0660] In yet another embodiment, a method for treating or preventing cancer (e.g., Nectin-4 positive cancer) in an individual is provided, the method comprising: (i) identifying an individual whose cancer is characterized by an H score of tumor Nectin-4 expression of no greater than or less than 290, 250, 200, 150, 120, or 100; and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure. Optionally, step (i) can be specified as comprising a step of assessing Nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0661] In yet another embodiment, a method for treating or preventing cancer (e.g., Nectin-4 positive cancer; breast cancer) in an individual is provided, the method comprising: (i) identifying an individual whose cancer is characterized by a QS score of tumor Nectin-4 expression of no greater than or less than 200, 150, 120, or 100; and (ii) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure. Optionally, step (i) can be specified as comprising a step of assessing Nectin-4 expression on tumor cells by histochemistry (e.g., IHC).

[0662] Biological samples from individuals, such as biological samples from biopsies, can be obtained and evaluated. Optionally, the samples are stored as formaldehyde (e.g., formalin) fixed paraffin embedded (FFPE) samples. After dewaxing, the slides are suitable for methods of detecting Nectin-4 (and / or HER2, TROP-2, B7H3) expression.

[0663] 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, the assay comprises an immunohistochemistry (IHC) assay, a fluorescence activated cell sorting (FACS) assay, such as quantitative FACS, ELISA, immunoblotting (e.g., Western blot, dot blot or intracellular Western blot) and other immunoassays.

[0664] IHC staining of tissue sections has been shown to be a reliable method for evaluating or detecting the presence of proteins in samples. Immunohistochemical techniques utilize antibodies to detect and visualize cellular antigens in situ, usually by color development or fluorescent methods. Therefore, antibodies or antisera (in some embodiments, polyclonal antisera, and in some embodiments, monoclonal antibodies specific to each marker) are used to detect expression. Antibodies can be detected by directly labeling the antibody itself, for example, by labeling 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 in conjunction with labeled secondary antibodies, and the secondary antibodies include antisera, polyclonal antisera or monoclonal antibodies specific to the primary antibodies. Immunohistochemical protocols and kits are well known in the art and are commercially available.

[0665] In some embodiments, the IHC assay is a direct assay, in which the binding of an antibody to a target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be observed without the need for additional antibody interactions. In some embodiments, the IHC assay is an indirect assay. In a typical indirect assay, an unconjugated primary antibody binds to the antigen, and then a labeled secondary antibody binds to the primary antibody. When the secondary antibody is combined with an enzyme label, a color developing or fluorescent substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. Primary antibodies and / or secondary antibodies used for immunohistochemistry are typically labeled with detectable molecules. There are a variety of labels to choose from, including radioisotopes, colloidal gold particles, fluorescent labels, and enzyme substrate labels.

[0666] Strong staining, medium staining and weak staining are descriptions well known to those skilled in the art. In some respects, strong staining, medium staining and weak staining are the staining levels of calibration, wherein a scope is established and the staining intensity is graded within the scope. In some embodiments, strong staining is the staining above the 75th percentile of the intensity range, medium staining is the staining of the 25th to the 75th percentile of the intensity range, and low staining is the staining below the 25th percentile of the intensity range. In some respects, those skilled in the art who are familiar with specific staining techniques adjust the case size and define the staining category.

[0667] Control cell lines (e.g., centrifuged into pellets, formalin-fixed and paraffin-embedded, e.g., prepared into tissue microarrays, and, e.g., stained with anti-Nectin-4 antibodies) with different staining intensities (e.g., when stained with anti-Nectin-4 antibodies) can be used as controls for IHC analysis. One of ordinary skill will appreciate that other control cell pellets with negative, weak, moderate, and high c-met staining intensities can be readily identified using the teachings and methods of the present application known in the art and disclosed herein.

[0668] In some embodiments, when a cancer or tumor is (eg, determined to be) Nectin-4 positive, it is considered a Nectin-4 expressing cancer tumor.

[0669] In some embodiments, when 5% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 10% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 20% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 30% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is Nectin-4 positive. In some embodiments, a cancer or tumor in an individual 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, a cancer or tumor in an individual 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, a cancer or tumor in an individual 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, a cancer or tumor in an individual 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 some embodiments, when 80% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is positive for Nectin-4. In some embodiments, when 90% or more of the tumor cells in the sample express Nectin-4 protein (e.g., express Nectin-4 protein at any intensity), the individual's cancer or tumor is positive for Nectin-4.

[0670] In some embodiments, when 5% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 10% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 20% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 30% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 40% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 50% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 60% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 70% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 80% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive. In some embodiments, when 90% or more of the tumor cells in the sample express Nectin-4 protein with moderate and / or strong staining intensity, the individual's cancer or tumor is Nectin-4 positive.

[0671] Evaluation of immunohistochemical assays to determine whether an individual's cancer or tumor is characterized by high nectin-4 expression (eg, low or moderate nectin-4 expression) will generally involve the application of known scoring methods.

[0672] Low, moderate, and high tumor nectin-4 expression can be determined based on the "H score" described in U.S. Patent Publication No. 2013 / 0005678. The H score is obtained by the following formula: (3×percentage of strongly stained cells)+(2×percentage of moderately stained cells)+(percentage of weakly stained cells), resulting in a range of 0 to 300. The H score is particularly useful in UC.

[0673] In some 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 nectin-4 expression level) 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.

[0674] In some embodiments of any of the methods herein, low nectin-4 expression (e.g., a tumor or tumor cell having a low nectin-4 expression level) 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.

[0675] In some embodiments of any of the methods herein, high Nectin-4 expression (eg, a tumor or tumor cell having a high Nectin-4 expression level) corresponds to an H-score of about 290 or higher.

[0676] In another example, Nectin-4 staining can be scored according to 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 the Nectin-4 low expression group as QS = or <100. In some embodiments of any of the methods herein, low or moderate Nectin-4 expression (e.g., tumors or tumor cells with low or moderate Nectin-4 expression levels) 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.

[0677] Assays for evaluating tumor cell expression of HER2 are well known in the art. For example, assays such as the FDA-approved SPoT-Light HER2 CISH can be used to detect HER2 overexpression. Chromogenic in situ hybridization (CISH) detects HER2 gene amplification. This technique is also known as subtraction probe technique chromogenic in situ hybridization, which is a test used to observe whether breast cancer cells overexpress the HER2 receptor protein on the cell surface.

[0678] Another widely used HER2 assay is the HercepTest TM(Dako North America, Inc.), which is a semiquantitative immunohistochemical assay used to determine overexpression of HER2 protein in formalin-fixed, paraffin-embedded cancer tissue. For example, tumors expressing low levels of HER2 can be detected by the HercepTest TM The evaluation was based on a score of +1 to +2.

[0679] In one aspect, the treatment is for individuals with existing neuropathy, diabetes or hyperglycemia, cardiac insufficiency, eye disease. Such conditions may make individuals unsuitable for treatment with anti-nectin-4 ADCs (such as Vitin-Enfortumab) that have higher toxicity or narrower therapeutic windows than the anti-nectin-4 antibody drug conjugates of the present disclosure.

[0680] In any embodiment, the method of treatment may optionally include the steps of: (a) assessing the cancer stage and / or disease progression in the individual; and (b) administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure if the individual suffers from recurrent, metastatic and / or progressive cancer.

[0681] In some embodiments, the invention includes a method of treating a tumor in an individual having urothelial carcinoma, the method comprising: (a) assessing the individual's cancer stage and / or disease progression; and (b) if the individual has recurrent, metastatic and / or progressive cancer, administering to the individual an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure.

[0682] Optionally, the individual treated with the anti-nectin-4 antibody drug conjugates of the present disclosure may have a cancer (e.g., urothelial carcinoma, 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) that is resistant, unresponsive, relapsed, and / or progressed (e.g., during or after) despite surgery and / or treatment with a therapeutic agent (e.g., a chemotherapeutic agent, an antibody, an ADC, or radiation therapy).

[0683] In any of the embodiments herein, the therapeutic response can be defined and / or assessed according to well-known criteria, such as Response Evaluation Criteria in Solid Tumors (RECIST), such as version 1.1, see 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.

[0684] Optionally, the individual treated with the anti-nectin-4 antibody drug conjugate of the present disclosure has a tumor or cancer that has become resistant, unresponsive to treatment, or has progressed after treatment with a chemotherapeutic agent (e.g., a chemotherapeutic agent known to be transported by P-glycoprotein (PgP), such as anthracyclines (doxorubicin, daunorubicin, taxanes (paclitaxel, docetaxel), vinca alkaloids (vincristine, vinblastine, vindesine), and etoposide). Compounds recognized by PgP are generally characterized as being moderately hydrophobic (octanol to water partition coefficient, logP>1), generally contain titratable protons with a net cationic charge under physiological conditions, and are primarily "natural products" with aromatic moieties.

[0685] In some embodiments, an ADC comprising an anti-Nectin-4 antibody, antibody fragment is used or administered without co-administration of a chemotherapeutic agent.

[0686] In other embodiments, the anti-nectin-4 antibody, antibody fragment, or ADC comprising them is optionally 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, hydroxyurea, idarubicin, ifosfamide, irinotecan, leucovorin, liposomal doxorubicin, liposomal daunorubicin, lomustine, melphalan, mercaptopurine In one embodiment, the anti-nectin-4 antibody, antibody fragment (or ADC comprising the same) and the chemotherapeutic agent are formulated for separate administration and are administered simultaneously or sequentially.

[0687] Optionally, the individual may be characterized as having a cancer that had progressed, relapsed, or was unresponsive to a prior treatment with a prior therapy, optionally further wherein the prior therapy included administration of vestin-enfortumomab and / or administration of a PD-1 neutralizing agent (e.g., pembrolizumab, atezolizumab, nivolumab), optionally wherein the prior therapy was a chemotherapeutic agent.

[0688] Optionally, in any embodiment, the individual may be characterized as not suitable for treatment with Vitin-Enfortuzumab, and / or as having a cancer that is not suitable or amenable to treatment with Vitin-Enfortuzumab.

[0689] Exemplary treatment regimens for treating humans with anti-Nectin-4 antibodies conjugated to camptothecin derivative molecules include, for example, administering to a patient an effective amount of an anti-Nectin-4 antibody drug conjugate of the present disclosure, wherein the method includes at least one administration cycle, wherein 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, such as at least 2, 3, 4, 5, 6, 8, 10 doses. In one embodiment, the doses are administered at intervals of at least 2 weeks, 3 weeks, or 4 weeks. In one embodiment, administration is weekly, every two weeks, every three weeks, or every four weeks.

[0690] In one embodiment, the anti-nectin-4 antibody drug conjugates of the present disclosure are administered by intravenous injection.

[0691] Example

[0692] Example 1: Human tumor cells co-expressing Her2, TROP-2, B7H3 and Nectin-4

[0693] Based on a multidimensional map of key genomic changes in different types of cancer, HER2 and Nectin-4 gene expression was studied using the Cancer Genome Atlas (a collaboration between the National Cancer Institute and the National Human Genome Research Institute). In particular, significant correlations between HER2 and Nectin-4 expression were observed in samples from pancreatic cancer, lung adenocarcinoma patients, breast cancer, and bladder cancer patients. The highest correlation observed was in pancreatic cancer, with correlation values: Spearman 0.71 and Pearson 0.78.

[0694] HER2 and Nectin-4 expression on SUM185 and SUM190 human breast cancer tumor cell lines (Biovitinc.) was determined by flow cytometry. SUM185 was derived from the pleural effusion of a patient with ER-negative, PR-negative, and HER2-positive breast anaplastic carcinoma. The cell line overexpresses Her2. SUM190 was derived from a primary tumor of a patient with ER-negative, PR-negative, and HER2-positive (amplified) breast cancer. Tumor cells were stained with anti-Nectin-4 antibody (ASG-22ME, which was modified to contain the N297Q mutation of human IgG1 isotype, which has reduced Fcγ receptor binding), anti-TROP-2 antibody, anti-B7H3 antibody or anti-Her2 antibody (trastuzumab, which was modified to contain the N297Q mutation of human IgG1 isotype, which has reduced Fcγ receptor binding) and isotype control at a concentration of 10 μg / ml (at 4°C), followed by 1:200 dilution of PE-conjugated polyclonal goat anti-human antibody. Samples were analyzed by cytofluorimetry using Canto II (HTS).

[0695] For HER2 and Nectin-4, Figure 1 Representative results for SUM190 human breast cancer tumor cells are shown in Figure 2 Representative results for SUM185 human breast cancer tumor cells are shown. MFI: Median Fluorescence Intensity. SUM190 tumor cells express HER2 at low to moderate levels (median fluorescence units of 1777) and Nectin-4 at low levels (median fluorescence units of 991). SUM185 cells express HER2 at moderate to high levels (median fluorescence units of 2880) and Nectin-4 at high levels (median fluorescence units of 4326). In addition, SUM185 cells express TROP-2 and B7H3 at high levels (median fluorescence units of 17327 and 11481, respectively). The expression data are presented in Table 1 below.

[0696] Table 1 :

[0697]

[0698] Example 2: Efficacy of anti-nectin-4 camptothecin derivative ADC in combination with anti-HER2 ADC

[0699] Anti-Nectin-4 antibody-drug conjugates were prepared and compared with trastuzumab antibody-drug conjugates for efficacy against HER2+Nectin4+ human tumor cells.An anti-Nectin-4 antibody-drug conjugate having VH and VL of SEQ ID NOs: 6 and 7 as human IgG1 isotype was prepared.

[0700] N41 VH:

[0701] QVQLKQSGPGLVQPSQSLSITCTVSGFSLT NYGVH WVRQSPGKGLEWLG VIWSGGSTDYN

[0702] AAFIS RLSISKDTSKSQVFFKMNSLQADDTAIYYCAR ELIHAMDN WGQGTSVTVSS

[0703] (SEQ ID NO: 6).

[0704] N41 VL:

[0705] DIQMTQSPASLSVSVGETVTITC RASENIYSNLA WYQQKQGNSPQLLVF AATNLAD GVPS

[0706] RFSGSGSGTQYSLKINSLQSEDFGTYYC QHFWGTPT FGGGTKLEIK

[0707] (SEQ ID NO: 7).

[0708] An anti-HER2 antibody-drug conjugate with the heavy and light chains of trastuzumab (human IgG1 isotype) was prepared. After partial reduction of the interchain disulfide bonds, the anti-Nectin-4 antibody and the anti-HER2 antibody were each randomly conjugated to the linker-camptothecin derivative through the cysteine ​​residues in the antibody. The reducing agent tris(2-carboxyethyl)phosphine hydrochloride in the range of 2-10 molar equivalents was incubated with the antibody (3 mg / mL) under stirring (350-400 rpm, +37°C) for 2 hours to reduce the disulfide. The linker-toxin conjugation was performed by adding a molar excess (9.2 or 12 molar equivalents) of the linker-toxin, which was incubated overnight on a stirring wheel at +37°C. The average drug loading (drug: antibody ratio) of the resulting ADC was approximately 8. In another example, the anti-Nectin-4 antibody was also conjugated to a second camptothecin (SN-38) containing a linker using the same method. The ADC used in this example is as follows.

[0709] N4 ADC1: Anti-Nectin-4 conjugated to a linker with the following structure:

[0710]

[0711] Her2 ADC1: anti-HER2 conjugated to a linker with the following structure:

[0712]

[0713] N4 ADC2: Anti-Nectin-4 conjugated to a linker with the following structure:

[0714]

[0715] The resulting ADCs were tested for their ability to induce cell death in the Nectin-4 / HER2-expressing SUM190 (when used in combination) and MCF-7 tumor cells of 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 culture medium (concentration 5x) were tested in a 1:2 serial dilution starting from (530 nM to 30 nM), and N4 ADC1 and isotype control were tested in a 1:5 serial dilution (7 nM to 7x10-2 nM). N4 ADC2 and isotype control were tested in a 1:10 serial dilution starting from (530 nM to 5.3 10-2). The ability of the ADC to cause cell death was determined by assessing confluence using an Incucyte S3-2 device; the results were analyzed using Cell Titer Glo TM (CTG) assays were performed using the Enspire 2 device to determine viability on day 6 after treatment. IC50 values ​​for each ADC were determined using GraphPad Prism 8 using luminescent cell viability data on day 6. The experiment was repeated twice.

[0716] The results showed that the combination of anti-Nectin-4 ADC and anti-Her2 ADC had improved potency (lower IC50) in causing death of Nectin-4+Her2+ tumor cells compared to either ADC alone.

[0717] Example 3: Modeling and generation of the first set of anti-huNectin-4 antibodies with human framework sequences

[0718] To introduce the CDRs of antibody 5E7, human VH and VK templates were identified. Each VH, VJ, VK and JK framework was analyzed separately. The parent antibody with the VH and VL amino acid sequences of SEQ ID NOs: 19 and 20 was then modified by introducing the heavy chain framework (FR1, FR2, FR3) from human subgroup IGHV1-46*01 together with IGHJ4*01 (FR4) into VH and the light chain framework (FR1, FR2, FR3) from human subgroup IGKV2-28*01 together with IGKJ4*01 (FR4) into VL.

[0719] The parental chimeric antibody Fab (HPLP) was modeled using the following heavy and light chain sequences:

[0720] HP heavy chain (variable domain underlined):

[0721] QVQLQQPGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKA TLTLDKSSSTTYMQLSSSLTSEDSAVYYCVRGYGNYGDYWGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK(SEQ ID NO: 67)

[0722] LP heavy chain (variable domain underlined):

[0723] DVVMTQAAFSNPVTLGTSASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFS SSGSGTDFTLRISRVEAEDVGVYYCAQNLELPWTTFGGGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 68)

[0724] The following heavy and light chain sequences were used to model the humanized antibody Fab H0L0.

[0725] H0 Heavy Chain:

[0726] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGEIDPSDSYTNYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGYGNYGDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK(SEQ ID NO: 69)

[0727] L0 Heavy Chain:

[0728] DIVMTQSPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPWTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:70)

[0729] For the design of light chain and heavy chain humanized variants, 5E7 HPLP and H0L0 three-dimensional models were superimposed and all amino acid differences were checked one by one. Intra-chain and extra-chain connections between residues were also evaluated to identify and avoid destroying any important low-energy bonds by introducing back mutations in a given chain.

[0730] Starting from the original CDR grafting, potential back mutations were identified by observing the framework difference residues between human and mouse.

[0731] Heavy chain design :

[0732] In order to study the role of the residue at position 72 (residue 71 according to Kabat numbering), a back mutation was performed on this residue, thereby retaining the leucine present in the parent antibody. Compared with the TRD of the H0L0 antibody, the parent antibody has the sequence TLD. This back mutation should be coupled with the back mutation on residue 79 in order to avoid steric conflicts.

[0733] In order to study the effect of the residue at position 79, back mutation was carried out to this residue, thereby retaining the Threonine present in the parental antibody.Compared with the TVY of the H0L0 antibody, the parental antibody has the sequence TTY.The Threonine in position 79 is directed in the VH domain and does not contact any other residue.Although the Valine in position 79 overlaps well with Threonine, this residue forms many keys with R72, M34, C22 and I51.The rotamer position of I51 is explained by contacting with V79 (V78 according to Kabat numbering).Residue 72 (R72L; According to the residue 71 of Kabat numbering), introducing back mutation can introduce space conflict between I51 and L72, if V79 exists.

[0734] To investigate the role of the residue at position 74, a back mutation was performed on this residue, thereby retaining the lysine present in the parent antibody. Compared to the DTS of the H0L0 antibody, the parent antibody has the sequence DKS. The lysine residue in position 74 is exposed on the surface of the molecule and occupies a critical position, although it is not close to the potential binding site.

[0735] To investigate the role of the residue at position 28, a back mutation was performed on this residue, thereby retaining the isoleucine present in the parent antibody. The parent antibody has the sequence YIF compared to YTF of the H0L0 antibody. The isoleucine and threonine at position 28 overlap well. They are exposed to the molecular surface and are located at the top of the antibody, close to the paratope. It cannot be ruled out that this residue may be involved in binding. In addition, according to the IMGT definition, this residue is included in CDR-H1.

[0736] The first heavy chain variant (H1) having the amino acid sequence shown in SEQ ID NO: 39 has R71L and V78TK substitutions. The second heavy chain variant (H2) having the amino acid sequence shown in SEQ ID NO: 41 has R71L, T73K and V78T substitutions. The third heavy chain variant (H3) having the amino acid sequence shown in SEQ ID NO: 43 has T28I, R71L, T73K and V78T substitutions. Substitutions are numbered according to Kabat.

[0737] Light chain design :

[0738] To investigate the role of the residue at position 2, a back mutation was performed on this residue, thereby retaining the valine residue in the parent antibody. The parent antibody has the sequence DVV compared to DIV of the H0L0 antibody. Residue V2 interacts with residue K27 located in CDR-L1, which further interacts with residue E98 located in CDR-L3.

[0739] In order to study the effect of the residue at position 69, this residue was back mutated, thereby retaining the serine residue in the parental antibody.Compared with the SGS of H0L0 antibody, the parental antibody has sequence SSS.Residue S69 is directed inside the VL domain and forms many keys with adjacent residue W40 and M56 (being positioned in CDR-L2), and G69 (according to the G64 of Kabat numbering) forms h-combination with M56.All these residues overlap well.Residue W40 is the center of the whole internal network.

[0740] To investigate the role of the residue at position 11, a back mutation was performed on this residue, thereby retaining the asparagine residue in the parent antibody. The parent antibody has a sequence SNP compared to the SLP of the H0L0 antibody. Residue L11 interacts with residue P8 and may stiffen the P8 β-strand.

[0741] To investigate the role of the residue at position 8, this residue was backmutated to retain the alanine residue in the parent antibody. The parent antibody had the sequence SAL compared to SPL of the H0L0 antibody. As described above, residue P8 interacts with residue L11, and such interaction may stiffen the P8 β-strand.

[0742] The first light chain variant (L1) having the amino acid sequence of SEQ ID NO: 61 has I2V and G64S substitutions. The second light chain variant (L2) having the amino acid sequence of SEQ ID NO: 63 has I2V, L11N and G64S substitutions. The third light chain variant (L3) having the amino acid sequence of SEQ ID NO: 65 has I2V, P8A, L11N and G64S substitutions. Substitutions are numbered according to Kabat.

[0743] The amino acid sequences of the corresponding heavy chain ("H" chain in Table 2) and light chain ("L" chain in Table 2) variable regions are shown in Table 2 below.

[0744] Table 2

[0745]

[0746] Antibodies having the heavy and light chain combinations shown in Table 3 below were prepared.

[0747] Table 3

[0748]

[0749] Example 4: Characterization of binding to nectin-4 by SPR

[0750] The antibodies in Table 3 of Example 3 were cloned as human IgG1 isotype antibodies, produced and purified, and then tested for binding to human Nectin-4. The affinities of 16 humanized variants and their association constants 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)).

[0751] The H0L0 antibody with the entire human IGKHV-46*01 and IGHJ4*01 heavy chain framework and the entire human IGKV-28*01 and IGKJ4*01 light chain framework would result in a kD of 80.2 nM. As shown in Table 4, other variants exhibited lower KD than H0L0.

[0752] Heavy chain H3 restored a KD value close to that of the parent antibody. Therefore, the back mutation introduced in H3 is important for the stability of the humanized antibody. Note the gradation of dissociation constant values ​​from H0 to H3.

[0753] Table 4

[0754] Antibody KD(nM) ka(1 / Ms) kd(1 / s) 5E7 5.9±1.2 4.8E+5±1.0E-5 2.7E-3±0.3E-3 H0L0 80.2 1.7E+5 1.4E-2 H1L0 67.3 1.1E+5 7.2E-3 H2L0 46.4 1.3E+5 6.2E-3 H3L0 35.8 1.1E+5 4.1E-3 H3L1 22.0±0.4 4.0E+5±0.3E+5 8.7E-3±0.4E-3 H3L2 25.9 3.1E+5 8.1E-3 H3L3 27.0 2.6E+5 7.0E-3

[0755] 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 antibodies generated in Example 3, new antibodies were designed.

[0756] Heavy chain design

[0757] To investigate the role of the residue at position 38, a back mutation was performed on this residue, thereby retaining the lysine residue in the parent antibody. The parent antibody has the sequence VKQ compared to VRQ of the H0L0 antibody. K38 will be part of a salt bridge with residue E46.

[0758] To investigate the role of the residue at position 40, a back mutation was performed on this residue, thereby retaining the arginine residue in the parent antibody. Compared to QAP of the H0L0 antibody, the parent antibody has the sequence QRP. Residue R40 contacts Q43, which contacts Q39, which forms two hydrogen bonds with residue Q43 of the light chain. With this type of back mutation, residue Q43 of the heavy chain occupies a divergent position, but remains in contact with Q39, and two hydrogen bonds also remain in contact with L0-Q43.

[0759] To investigate the role of the residue at position 48, a back mutation was performed on this residue, thereby retaining the isoleucine residue in the parent antibody. The parent antibody has the sequence WIG compared to WMG of the H0L0 antibody. Residue I48 interacts with A68, M81 and F64 (located in CDR-H2).

[0760] In order to study the effect of the residue at position 70, back mutation was carried out to this residue, thereby retaining the leucine residue in the parental antibody.Compared with the TMT of H0L0 antibody, the parental antibody has sequence TLT.Residue L70 interacts with residue M81, I51 (it is positioned in CDR-H2), Y60 (being positioned in CDR-H2) and W36.Residue M70 (according to the M69 of Kabat numbering) interacts with residue I51 (being positioned in CDR-H2), Y60 (being positioned in CDR-H2) and W36.Except that residue I51 occupies different rotamer positions, all other residues overlap completely, and two networks are almost equal.

[0761] The fourth heavy chain variant (H4) having the amino acid sequence shown in SEQ ID NO: 45 has an R38K substitution. The fifth heavy chain variant (H5) having the amino acid sequence of SEQ ID NO: 47 has R38K and A40R substitutions. The sixth heavy chain variant (H6) having the amino acid sequence of SEQ ID NO: 49 has R38K, A40R and M48I substitutions. The seventh heavy chain variant (H7) having the amino acid sequence of SEQ ID NO: 51 has R38K, A40R, M48I and M69L substitutions. Further eighth (H8; SEQ ID NO: 53), ninth (H9; SEQ ID NO: 55) and tenth (H10; SEQ ID NO: 57) chain designs have different substitution combinations. The numbering of amino acid residues is according to Kabat.

[0762] The amino acid sequences of the corresponding heavy chain ("H" chain in Table 5) and light chain ("L" chain in Table 5) variable regions are shown in Table 5 below.

[0763] Table 5

[0764]

[0765] Antibodies having the heavy chain and light chain combinations shown in Table 6 below were prepared.

[0766] Table 6

[0767]

[0768] Example 6: Characterization of binding to nectin-4 by SPR (Group 2)

[0769] The antibodies in Table 6 of Example 5 were cloned into human IgG1 isotype antibodies, produced and purified, and then tested for binding to human nectin-4. The affinity of 16 humanized variants and their association constants 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)).

[0770] The H0L0 antibody with the entire human IGKHV-46*01 and IGHJ4*01 heavy chain framework and the entire human IGKV-28*01 and IGKJ4*01 light chain framework would result in a kD of 80.2 nM. As shown in Table 7, the other variants exhibited a lower KD than H0L0. In particular, variants H4L1, H5L3, and H8L1 had a KD closer to the chimeric parent antibody (which had a KD of 5.9 nM ± 1.2).

[0771] Table 7

[0772]

[0773]

[0774] Example 7: Characterization of Binding to Nectin-4 by Flow Cytometry Assay

[0775] The humanized antibodies in Table 3 of Example 3 and Table 6 of Example 5 were cloned, produced and purified, and then tested for binding to cells expressing Nectin-4 by flow cytometry. The binding of the humanized variants in human IgG1 format was determined on the SUM190 cell line expressing high levels of Nectin-4. The EC50 and maximum MedFI representing the MedFi at the saturation stage are shown in Table 8 below.

[0776] All humanized variants with L0 or L1 chains had lower binding capacity (lower plateau) than the other variants. In contrast, all humanized variants carrying L2 or L3 chains had similar binding capacity compared to the parent chimeric 5E7 antibody (ch5E7). Variant H5L3 showed the highest MedFi value.

[0777] Table 8

[0778] Antibody <![CDATA[EC 50 ]]> Max MedFi ch5E7 1.10 25213 H0L0 0.542 15507 H1L0 0.64 17251 H2L0 0.45 16704 H3L0 0.88 17596 H3L1 1.79 17208 H3L2 1.19 22887 H3L3 1.45 23814 H4L1 1.49 18399 H4L2 1.44 24352 H4L3 1.60 24840 H5L1 1.43 18263 H5L2 1.14 25276 H5L3 1.29 27095 H6L1 1.15 18263 H6L2 1.30 24779 H6L3 1.01 24840 H7L1 1.27 17816 H7L2 1.30 23404 H7L3 1.23 25026 H8L1 1.63 17251

[0779] Example 8: Intracellular internalization assay

[0780] The humanized variants in Table 3 of Example 3 and Table 6 of Example 5 were cloned, produced and 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, using Cell Titer Glo TM (CTG) was measured as readout. The experiment was performed on two cell lines expressing different levels of Nectin-4. MDA-MB-468 has lower Nectin-4 expression than SUM190.

[0781] Internalization assay on SUM190 cell line

[0782] The results of the internalization assay on the SUM190 cell line are shown in Table 9 below, which presents the internalization efficiency (normalized to the chimeric parent antibody internalization efficiency on the SUM190 cell line). This experiment was performed twice (two independent experiments).

[0783] Table 9

[0784]

[0785] The internalization efficiency determined in experiments performed on the SUM190 cell line showed that the humanized variants H3L0, H4L2, H5L2, H5L3, H6L3 and H7L2 induced 75% or more internalization compared to the parent antibody (5E7), thus showing an interesting internalization potential. It should be noted that in the first experiment, the H8L1 and H7L1 variants were more effective than the chimeric 5E7, but this result was not confirmed in the second experiment.

[0786] Internalization assay on MDA-M-468 cell line

[0787] The results of the internalization assay on the SUM190 cell line are shown in Table 10 below, which presents the internalization efficiency (normalized to the chimeric parent antibody internalization efficiency on the MDA-M-468 cell line). This experiment was performed twice (two independent experiments).

[0788] Table 10

[0789]

[0790] The results obtained showed that only the H5L3 humanized variant consistently achieved more than 75% of the efficacy of the parental chimeric 5E7 antibody, while all other variants had lower efficacy and many of them had heterogeneous results between experiments.

[0791] Example 9: In vitro cytotoxicity of antibodies as ADCs against tumor cell lines

[0792] Nectin-4 Low / SUM 190 Breast Cancer Model

[0793] We evaluated the ability of 5E7 antibody conjugated to a camptothecin analog (Dxd or exotecan) to kill SUM190 cells. In this experiment, the 5E7 antibody was tested together with anti-Ig-like V domain antibodies Enfortuzumab and N41 conjugated to the same toxin at an equal drug / antibody ratio and a control antibody. The first ADC was prepared in which the antibody was conjugated (to a cysteine ​​residue) to a camptothecin analog (Dxd) via a linker with 8 toxins / antibody (DAR=8), which contained an intracellularly cleavable tetrapeptide linker (GGFG) having the structure shown below, referred to as ggfg-Dxd:

[0794]

[0795] A second ADC was prepared in which the antibody was conjugated to another camptothecin analog (exotecan) at 8 toxins / antibody (DAR=8) via a cleavable linker having the following structure, referred to as PEG(8U)-Val-Ala-PAB-exotecan:

[0796]

[0797] Briefly, each test antibody at different dose ranges (starting point 150 nM, 3 points of dilution factor 2, then 5 points of dilution factor 5) was incubated with cells for 5 days and then cell viability was measured by adding CTG substrate. Luminescence versus antibody concentration was plotted on a graph.

[0798] For each ADC, a range of ADC concentrations were incubated with nectin-4 expressing cells. After incubation, CTG substrate was added at a 1 / 1 ratio and the luminescent signal was read with a plate reader (Enspire). This allowed quantification of the ATP present (an indicator of metabolically active cells), which is directly proportional to cell viability.

[0799] The results are shown in Figure 3A middle. Figure 3A Killing of human breast cancer cells by 5E7 antibody, conjugated to camptothecin analog Dxd (via GGFG-Dxd linker) or exotecan (via (PEG(8U)-Val-Ala-PAB-exotecan) linker), all at equal drug / antibody ratio (DAR=8), and to V domain binding Enhertu, as well as isotype control antibody (IC), is shown. TM (Trastuzumab delutec (anti-HER2)) Comparison.

[0800] All nectin-4-targeted ADCs reduced cell viability more efficiently than the non-targeted ADC (IC-GGFG-camptothecin).

[0801] SUM185, MDA-MB-468, MC38 and B16F10 cell lines

[0802] Antibody 5E7 conjugated to exotecan (via a PEG(8U)-Val-Ala-PAB-exotecan linker) was further tested for cell killing against other cancer cell lines. Figure 3B The efficacy of "5E7-Ixitecan" (5E7 conjugated to an Ixitecan linker (PEG (8U) -Val-Ala-PAB-Ixitecan)) in causing death of HER-2 and Nectin-4 expressing SUM185 and SUM190 and MDA-MB-468 (TNBC) human tumor cells and human Nectin-4 expressing MC38 (colon cancer) and B16F10 (melanoma) mouse tumor cells is shown. The EC50 values ​​of cell viability are shown in Table 11 below.

[0803] Table 11

[0804] Cell lines MDA-MB-468 SUM185 SUM190 MC38-huN4 B16F10-huN4 <![CDATA[EC 50 (nM)]]> 1.5E+05 3E+05 7E+05 2.3E+5 9.8E+5

[0805] Example 10: In vivo efficacy of ADC in a human breast cancer mouse model (Nectin-4 low / SUM190 model)

[0806] We compared the efficacy of the 5E7 antibody as a camptothecin ADC with Enfortuzumab and N41 in a mouse model of human breast cancer. In this experiment, the 5E7 antibody was tested together with Enfortuzumab and N41 conjugated to a ggfg-Dxd linker and a control antibody at an equal drug / antibody ratio of 8 toxins / antibody (DAR=8).

[0807] SUM190 cells were transplanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl Matrigel and growth factors were added in PBS at 4% CO. 1 The mice were randomly divided into groups of 9 mice and treated intravenously with a single injection of 3 mg / kg camptothecin ADC. Tumor growth was followed twice a week. GraphPad Prism V7 software was used to establish a Kaplan Meier survival curve according to the following criteria: when the tumor volume reached 1500 mm, mice were euthanized and considered dead (D) on the same day of execution. When signs of necrosis appeared in the tumor, mice were euthanized and considered dead (D) on the same day (indicated by red asterisks on the graph of individual tumor growth).

[0808] The results showed that at a dose of 10 mg / kg, all ADCs were similarly efficient in preventing tumor volume increase. However, at a lower dose of ADC (3 mg / kg), antibody 5E7 showed a strong ability to prevent tumor growth, while both N41 and Enfortuzumab no longer showed the ability to control tumor growth. Figure 4 Results for the 3 mg / kg dose are shown.

[0809] Example 11: ADC in a drug-resistant breast cancer model (human breast cancer, HER2 / Nectin-4 high / SUM185 model) Comparative in vitro efficacy in

[0810] We next evaluated the TM (Vitin-Enfortumab) and ENHERTU TM The ability of 5E7 antibody conjugated to a camptothecin analog to kill SUM185 cells was compared. This setting was used as a model of anti-HER2 resistance. SUM185 cells express nectin-4 at relatively high levels, with the nectin-4 expression level in these cells being approximately twice that of HER2 expression (see Example 1). In this experiment, the 5E7 antibody was used as an ADC with a DAR=8 in combination with PADCEV. TM (DAR = 4, FDA-approved PADCEV TM 5E7 was conjugated to the camptothecin analog Dxd via a ggfg-Dxd linker.

[0811] Briefly, each test antibody at different dose ranges (starting point 150 nM, 3 points of dilution factor 2, then 5 points of dilution factor 5) was incubated on cells for 5 days and then cell viability was measured by adding CTG substrate. Luminescence versus antibody concentration was plotted on a graph.

[0812] For each ADC, a range of ADC concentrations were incubated with nectin-4 expressing cells. Following incubation, CTG substrate was added at a 1:1 ratio and the luminescent signal was read with a plate reader (Enspire), allowing quantification of the ATP present (an indicator of metabolically active cells), which is directly proportional to cell viability.

[0813] The results are shown in Figure 5 5E7-GGFG-Dxd and PADCEV TM Can be better than ENHERTU TM More efficient killing of SUM185. In each case, the Nectin 4-targeted ADCs reduced cell viability more efficiently than their non-targeted ADC (IC) counterparts.

[0814] Example 12: Characterization of species cross-reactivity of anti-Nectin-4 antibodies

[0815] The binding of anti-Nectin-4 antibodies to different CHO cell lines prepared to express mouse, cynomolgus monkey and rat Nectin-4 proteins, respectively (including an N-terminal V5 tag not shown in the following sequences) was tested by flow cytometry.

[0816] The mature amino acid sequence of the protein expressed by the cells is as follows:

[0817] Mouse Nectin-4:

[0818] ELETSDVVTVVLGQDAKLPCFYRGDPDEQVGQVAWARVDPNEGIRELALLHSKYGLHVNPAYEDRVEQPPPPRDPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARMRLRVLVPPLPS LNPGPPLEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTQSSRSFTHPRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDRRITHTLQVAFLAEASVRGLEDQNLWQVGREGATLKCLS EGQPPPKYNWTRLDGPLPSGVRVKGDTLGFPPLTTEHSGVYVCHVSNELSSRDSQVTVEVLDPEDPGKQVDLVSASVIIVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLT RENSIRRLHSHHSDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEDDDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV(SEQ ID NO:12)

[0819] Rat Nectin-4:

[0820] MPLSLGAEMWGPEAWLLLLFLASFTGRYSAGELETSDLVTVVLGQDAKLPCFYRGDPDEQVGQVAWARVDPNEGTRELALLHSKYGLHVSPAYEDRVEQPPPPRDPLDGSILLRNAVQADEGEYECRVSTFPAGSFQARMRLRVLVPPLPSLNPGPPLEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTQSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHTLQVAFLAEASVRGLEDQNLWHVGREGATLKCLSEGQPPPKYNWTRLDGPLPSGVRVKGDTLGFPPLTTEHSGVYVCHVSNELSSRASQVTVEVLDPEDPGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLTRENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEEDDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV (SEQ ID NO: 13)

[0821] Cynomolgus monkey Nectin-4:

[0822] GELETSDVVTVVLGQDAKLPCFYRGDSGEQVGQVAWARADAGEGAQELALLHSKYGLHVSPAYEGRVEQPPPPRNPLDGSVLLRNAVQADEGEYECRVSTFPAGSFQARLRLRVLVPPLP SLNPGPALEEGQGLTLAASCTAEGSPAPSVTWDTEVKGTTSSRSFKHSRSAAVTSEFHLVPSRSMNGQPLTCVVSHPGLLQDQRITHILHVSFLAEASVRGLEDQNLWHVGREGAMLKCLS EGQPPPSYNWTRLDGPLPSGVRVDGDTLGFPPLTTEHSGIYVCHVSNEFSSRDSQVTVDVLDPQEDSGKQVDLVSASVVVVGVIAALLFCLLVVVVVLMSRYHRRKAQQMTQKYEEELTLT RENSIRRLHSHHTDPRSQPEESVGLRAEGHPDSLKDNSSCSVMSEEPEGRSYSTLTTVREIETQTELLSPGSGRTEEEEDQDEGIKQAMNHFVQENGTLRAKPTGNGIYINGRGHLV(SEQ ID NO:14)

[0823] Antibody 5E7 binds to human, cynomolgus monkey, and rat nectin 4 proteins, but lacks binding to mouse nectin 4 protein. Fig. 6A and Figure 6B Binding of anti-Nectin-4 antibodies on rat and cynomolgus nectin-4 expressing CHO cell lines is shown.

[0824] Example 13: Characterization of Nectin Family Cross-Reactivity

[0825] The binding of anti-Nectin-4 antibodies to different CHO cell lines prepared to express human Nectin 1, Nectin 2, Nectin 3 and PVR proteins was tested by flow cytometry. The 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 sequence of the protein expressed by the cells is as follows:

[0826] Nectin-1:

[0827] MGLAGAAGRWWGLALGLTAFFLPGVHSQVVQVNDSMYGFIGTDVVLHCSFANPLPSVKITQVTWQKSTNGSKQNVAIYNPSMGVSVLAPYRERVEFLRPSFTDGTIRLSLELEDEGVYICEFATFPTGNRESQLNLTVMAKPTNWIEGTQAVLRAKKGQDDKVLVATCTSANGKPPSVVSWETRLKGEAEYQEIRNPNGTVTVISRYRLVPSREAHQQSLACIVNYHMDRFKESLTNVQYEPEVTIEGFDGNWYLQR MDVKLTCKADANPPATEYHWTTLNGSLPKGVEAQNRTLFFKGPINYSLAGTYICEATNPIGTRSGQVEVNITEFPYTPSPPEHGRRAGPVPTAIIGGVAGSILLVLIVVGGIVVALRRRRHTFKGDYSTKKHVYGNGYSKAGIPQHHPPMAQNLQYPDDSDDEKKAGPLGGSSYEEEEEEEEEGGGGGGERKVGGPHPKYDEDAKRPYFTVDEAEARQDGYGDRTLGYQYDPEQLDLAENMVSQNDGSFISKKEWYV(SEQ ID NO: 15)

[0828] Nectin-2:

[0829] MARAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPPVPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEAELQDATLALHGLTVEDEGNYTCEFATFPKGS

[0830]

[0831] Nectin-3:

[0832]

[0833] PVR(Nectin-5):

[0834]

[0835] The results showed that the anti-Nectin-4 antibody had no cross-reactivity to human nectin family members. The figure shows the flow cytometry results of the anti-Nectin-4 antibody on CHO cells expressing human nectin 1, human nectin 2, human nectin 3 and human PVR. No antibody showed binding to each cell line.

[0836] Example 14: Epitope mapping of nectin-4 by competition binding via SPR

[0837] The chimeric antibody 5E7 and previously reported antibody N4.1 (N41), antibody 14A5 and Enfamoduzumab were tested for their ability to compete with each other for binding to wild-type human Nectin-4 protein by SPR (surface plasmon resonance) method and 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 diluted to 10 μg / mL in Kinetic buffer 10X was 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 diluted to 10 μg / mL in Kinetic buffer 10X was injected.

[0838] The results are shown in Table 12 below. Black squares indicate substantial or direct competition between the first and second antibodies (the first antibody prevents binding of the second antibody / causes loss of binding of the second antibody), squares with Xs indicate potential partial competition (the first antibody causes a potential reduction in binding of the second antibody but not loss), and white squares indicate no competition. Antibodies 5E7 compete with each other for binding to Nectin-4.

[0839] Table 12

[0840]

[0841] Example 15: Epitope mapping of antibodies using nectin-4 point mutants

[0842] Human nectin-4 point mutants expressed on the cell surface

[0843] The binding profiles of anti-nectin-4 antibodies on full-length and Ig-like V domain-deficient proteins, as well as the species binding profiles of the antibodies (binding to human, cynomolgus monkey and rat nectin-4, but not mouse nectin-4), and interspecies differences between non-human nectin-4 proteins, allowed the identification of residues at the junction of the Ig-like V domain and the Ig-like C21-type domain (also known as "C1"). In combination with the structure of the published nectin-4 domains, nectin-4 mutations at surface-exposed amino acid residues were designed. The nectin-4 domain structure was modeled based on the Protein Data Bank reference: 4FRW (domains V and C1). The human nectin-4 used was the NCBI reference sequence: NP_112178.2, the mouse nectin-4 used was the NCBI reference sequence: AAL79833.1, the cynomolgus monkey nectin-4 used was the NCBI reference sequence: XP_005541277.1, and the rat nectin-4 used was the NCBI reference sequence: NP_001102546.1. Cells expressing the nectin-4 mutants were then used to test the loss of binding of anti-nectin-4 antibodies to the different nectin-4 mutants to identify antibodies that bind to the same site on nectin-4. In particular, mutants with substitutions at the C1-V junction at residues K197T and / or S199A, or mutants 7, 7bis and 9 with additional and / or adjacent substitutions at the junction of domains C1 and V, can identify antibodies with advantageous applications as immunoconjugates. Mutant 7 has the substitutions S195A / K197T / S199A. Mutant 7bis has the substitutions A72P / G73N / K197T / S199A. Mutant 9 includes a substitution of the key residue Q234 and has the substitutions L150S / S152A / Q234R / 1236S. Fig. 7A and Figure 7B Molecular model of human nectin-4 protein is shown indicating the positions of substituted residues in mutant 7 (7A) and mutant 7bis (7B); these mutants identify two sites in the C1 domain at the junction of domains C1 and V domains on opposite faces of the nectin-4 protein. Fig. 8A and Figure 8B Different views of the molecular model of human nectin-4 protein are shown, indicating the positions of the substituted residues in mutants 1, 2, 3, 4, 5, 6, 7, 8 and 9.

[0844] Nectin-4 mutants were generated by PCR. The amplified sequences were run on agarose gels and purified using the Macherey Nagel PCR Clean-Up Gel Extraction Kit. The purified PCR products generated for each mutant were then ligated into expression vectors using the ClonTech InFusion system. The vectors containing the mutant sequences were prepared as Minipreps and sequenced. After sequencing, the vectors were purified using the Promega PureYield TM Plasmid Midiprep system, the vector containing the mutant sequence is prepared into Midiprep. Before testing transgenic expression, HEK293T cells were grown in DMEM medium (Invitrogen), transfected with the vector using Invitrogen's Lipofectamine 2000 and incubated at 37°C in a CO2 incubator for 48 hours. Mutants were transfected in Hek-293T cells as shown in the table below. Targeted amino acid mutations are shown in Table 13 below, which lists the residues present in wild-type Nectin-4 / residues present in mutant Nectin-4 / residue positions, and the position reference is the Nectin-4 protein with the leader peptide shown in SEQ ID NO: 1.

[0845] Table 13

[0846] Mutants Reference to amino acid substitutions of Nectin-4 having SEQ ID NO: 1 1 S58P / G59D / Q61A / N106D / P107A 2 V90S / P92A / A93S / E95A / G96D 3 A76S / Q77R / E78A / S9 1N 4 A72P / G73N / G75S 5 A158P / E160G / S243A / F244S 6 H238A / I239T / H241Q 7 S195A / K197T / S199A 7bis A72P / G73N / K197T / S199A 8 T190A / T191Q / S193A 9 L150S / S152A / Q234R / I236S

[0847] The results of the association between the antibodies and the Nectin-4 mutants are shown in Table 14 below. (+) means that antibody-Nectin-4 association occurs. (-) means that antibody-Nectin-4 association does not occur. The results related to mutant 5 are irrelevant because there is no expression of the protein.

[0848] Table 14

[0849]

[0850] Thus, antibody 5E7 has a binding site on nectin-4 that encompasses the C1 domain residues mutated in mutants 7 and 7bis (S195A / K197T / S199A and A72P / G73N / K197T / S199A). Thus, antibody 5E7 binds to an epitope on nectin-4 that is different from Enfortuzumab, N41, and 14A5, which bind to an epitope on the V domain of nectin-4.

[0851] Example 16: In vivo efficacy of exitecan ADC

[0852] The human breast cancer cell line SUM190PT was cultured in the following cell culture medium containing Ham's F12, FBS 1g / L, HEPES 10mM, ethanolamine 5mM, insulin 5μg / mL, hydrocortisone 1μg / mL, apotransferrin 5μg / mL, triiodothyronine (T3) 6.7ng / mL, and sodium selenite 8.7ng / mL.

[0853] Immunodeficient CB17-SCID mice aged 7 to 8 weeks were used. Anti-nectin-4 antibodies (enfortumab, 5E7 and 6A7, each with a human Fc region) were generated and conjugated to the payload Dxd (delutecan) or isotecan via an intracellular cleavable linker ggfg-Dxd or PEG (8U) -Val-Ala-PAB-isotecan. Antibody 6A7 shares most of the CDRs with 5E7, has comparable nectin-4 binding affinity and binds to the same site on nectin-4 (for the 6A7 amino acid sequence, see PCT / EP2021 / 082872 filed on November 24, 2021). The ggfg-Dxd linker will release Dxd (delutecan) upon cleavage, and the PEG (8U) -Val-Ala-PAB-isotecan linker will release isotecan upon cleavage.

[0854] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million in 100 μl Matrigel containing growth factors diluted 1 / 2 in PBS. On day 21, when tumors reached 146.9 mm according to the experiment, 3 ±63.2mm 3 or 213.2mm 3 ±77.5mm 3 When the mean volume of the tumor reached 1500 mm, the mice were randomly divided into groups of 8 or 9 mice according to the experiment and treated intravenously by a single injection of 3 mg / kg body weight or 10 mg / kg body weight of ADC or PBS as a control. Tumor growth was followed twice a week. Kaplan-Meier survival curves were constructed using GraphPad Prism V7 software according to the following criteria: when the tumor volume reached 1500 mm 3 When the tumor was highly necrotic, the mice were euthanized and considered dead on the same day (D).

[0855] Fig.9A and Fig. 9BResults for the 3 mg / kg dose are presented in , where it can be seen that the anti-IgVC1 ADC 5E7-ggfg-Dxd, which was found to lose binding to Nectin-4 with K197T / S199A mutations, showed the highest efficiency in limiting tumor growth in mice. In addition, 5E7 conjugated to PEG(8U)-Val-Ala-PAB-exitecan, which is designed to release exitecan upon linker cleavage, showed higher efficiency in controlling tumor growth in mice compared to 5E7 conjugated to ggfg-Dxd, which is designed to release Dxd upon linker cleavage, as shown in Fig. 9C The 10 mg / kg dose is shown.

[0856] Example 17: In vitro efficacy of ADC in Pg-p expressing cancer models

[0857] MC-38 cells endogenously expressing MDR1 P-glycoprotein (PgP) were engineered to express Nectin-4 and cultured in DMEM + 10% FBS. Cells were treated with vehicle (DSMO) or with cyclosporin A (5 μM, stock solution in DMSO), a known PgP inhibitor, in the presence of ADC. The ADCs tested were as follows:

[0858] (a) PADCEV TM ,

[0859] (b) antibody 5E7 conjugated to delutecan (Dxd) via a ggfg-Dxd linker that releases Dxd upon cleavage (5E7-GGFG-DxD), and

[0860] (c) Antibody 5E7 conjugated to isotecan via a PEG(8U)-Val-Ala-PAB-isotecan linker that releases isotecan upon cleavage (5E7-isotecan).

[0861] ADC and equivalent isotype control ADC were tested at 150 nM to 2.310 -3 After 5 days of incubation with cells, the cells were incubated with Cell Titer Glo TM (CTG) substrate to measure cell viability. Luminescence was plotted against antibody concentration.

[0862] Fig. 10A Shown with Padcev TM Luminescence (indicating cell viability) of cells treated with (vitin-enfortumab), antibody 5E7 conjugated to Dxd, or 5E7 conjugated to isotecan. ADCs with isotecan (5E7-isotecan) as the payload were highly effective in reducing cell viability in this drug resistance setting. Fig. 10BFigure 2 shows the effect of Padcev on ADC at 150 nM in the presence or absence of the Pgp inhibitor cyclosporine and normalized to control antibody. TM Tumor growth (area under the curve) of MC38 cells treated with 5E7 conjugated to Dxd or 5E7 conjugated to exotecan. The results showed that at concentrations where 5E7 conjugated to exotecan was highly effective, Padcev TM The antitumor activity of antibody 5E7 conjugated to Dxd was negatively affected by Pgp.

[0863] Example 18: In vivo efficacy of branched PEG-dipeptide-exitecan ADC

[0864] The anti-VC domain anti-Nectin-4 antibody was conjugated to Exotecan via different branched PEG linkers, and the resulting ADCs were evaluated in the in vivo SUM190 tumor model (Nectin-4 low / SUM190 breast cancer model).

[0865] ADCs were prepared in which the anti-VC domain anti-Nectin-4 (N4) human IgG1 isotype antibody 6A7 (at cysteine ​​residues) was conjugated to exotecan at 8 toxins per antibody (DAR=8) via different linkers. Linker-toxins were tested as follows:

[0866] VA-PAB-Exitecan-PEG (8U):

[0867]

[0868] FK-PAB-Exitecan-PEG (8U):

[0869]

[0870] VA-PAB-exitecan-PEG (16U):

[0871]

[0872] PSAR10-Exitecan:

[0873]

[0874] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl Matrigel with growth factors diluted 1 / 2 in PBS. 3 and 250mm 3When the tumor volume reached 1500 mm, the mice were randomly divided into groups of 10 mice and treated intravenously with a single injection of 3 mg / kg ADC. Tumor growth was followed twice a week. Kaplan Meier survival curves were constructed using GraphPad Prism V7 software according to the following criteria: 3 When the tumors showed signs of necrosis, the mice were euthanized and considered dead on the same day (D). When the tumors showed signs of necrosis, the mice were euthanized and considered dead on the same day (D) (indicated by red asterisks on the graphs of individual tumor growth).

[0875] The results showed that at a dose of 3 mg / kg, all ADCs were highly effective in preventing tumor volume increase. Different linker-toxins administered as free toxins (not conjugated to anti-Nectin-4 antibody, denoted as "IC") were less effective in preventing tumor growth. Fig.11 The results of IC (free toxin) are shown in . Fig.12 The results of ADC are shown in .

[0876] Example 19: In vivo pharmacokinetics and efficacy of branched PEG-dipeptide-exitecan ADC

[0877] The anti-VC domain anti-Nectin-4 antibody 6A7 was conjugated to Exotecan at (DAR=8) via a VA-PAB-Exotecan-PEG (16U) linker (structure shown below) and evaluated at different lower doses in the in vivo SUM190 tumor model (Nectin-4 low / SUM190 breast cancer model). The experiment tested different dosing regimens up to 1 mg / kg body weight and evaluated the correlation of antitumor efficacy with ADC concentration in circulation.

[0878]

[0879] SUM190 cells were implanted subcutaneously into CB17-SCID immunodeficient mice at a dose of 0.5 million cells in 100 μl of Matrigel with growth factors diluted 1 / 2 in PBS. 3 and 250mm 3 The mice were randomly divided into groups of 20 mice each and treated intravenously with PBS as a control or a single injection of ADC at 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 obtained on the same day, and plasma samples were processed at 5 minutes, 5 hours, and 24 hours later, and then plasma samples and tumor growth were measured at 72 hours, on the 7th day, and then weekly.

[0880] The results are shown in Fig.13 middle. Fig.13 , upper left panel, shows that PBS did not prevent the increase in tumor volume. Fig.13 , upper right panel, shows the results of 1 mg / kg dose of ADC, which indicates strong anti-tumor efficacy. Fig.13 , lower panel, shows the change in the concentration of ADC in plasma over time, indicating that the ADC remained detectable during the period during which the ADC exhibited anti-tumor activity.

[0881] Example 20: In vivo safety of branched PEG-dipeptide-exitecan ADC

[0882] A dose ranging study of the anti-VC domain anti-nectin-4 antibody conjugated to the VA-PAB-exitecan-PEG(16U) linker at DAR-8 was conducted at a dose range of 3 mg / kg body weight to 30 mg / kg body weight.

[0883] In the first experiment, Sprague Dawley rats were injected with ADC at 3 mg / kg, 10 mg / kg or 30 mg / kg body weight by intravenous bolus on days 1 and 22. Plasma samples (n=3 males / time point / dose level) were collected during the study, and the concentrations of total anti-nectin-4 antibodies (total antibodies; TA) (DAR≥0) and ADC (DAR≥1) were measured by ELISA, and free exitecan (Exa) was measured by LC-MS.

[0884] Fig.14A Results for the 3 mg / kg dose (upper graph) and the 10 mg / kg dose (lower graph) are shown, and Fig. 14B Results for a 30 mg / kg dose are shown. For each analyte, concentration values ​​below the lower limit of quantitation (LLOQ) are plotted at LLOQ / 2. The Y axis represents the analyte plasma concentration in ng / mL, and the X axis represents the time in days. Symbols and bars show the mean and standard deviation of each group.

[0885] In the second experiment, ADC was injected into Mauritian cynomolgus monkeys by intravenous bolus at 3 mg / kg, 10 mg / kg or 30 mg / kg body weight on days 1 and 22. Plasma samples (n=1 male and 1 female / dose level) were collected during the study, and the concentrations of total anti-nectin-4 antibodies (total antibodies; TA) (DAR≥0) and ADC (DAR≥1) were measured by ELISA, and free exitecan (Exa) was measured by LC-MS.

[0886] Fig.15A Results for the 3 mg / kg dose (upper graph) and the 10 mg / kg dose (lower graph) are shown, and Fig. 15BResults for a 30 mg / kg dose are shown. For each analyte, concentration values ​​below the lower limit of quantitation (LLOQ) are plotted at LLOQ / 2. The Y axis represents the analyte plasma concentration in ng / mL, and the X axis represents the time in days. Symbols and bars show the mean and standard deviation of each group.

[0887] 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).

[0888] All references (including publications, patent applications, and patents) cited herein are hereby incorporated by reference in their entirety to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was fully set forth herein (to the maximum extent permitted by law), regardless of any individually provided incorporation for a specific document elsewhere herein.

[0889] Unless otherwise indicated, all exact values ​​provided herein represent corresponding approximate values ​​(e.g., all exact exemplary values ​​provided with respect to a particular factor or measurement may be considered to also provide corresponding approximate measurements, modified by "about" where appropriate). Where "about" is used in conjunction with a number, this may be specified as including values ​​corresponding to + / -10% of the specified number.

[0890] The description of any aspect or embodiment of the invention herein using terms such as “comprising,” “having,” “including,” or “containing,” is intended to provide support for similar aspects or embodiments of the invention “consisting of,” “consisting essentially of,” or “consisting essentially of,” unless otherwise specified or clearly contradicted by context (e.g., compositions described herein comprising a particular element should be understood to also describe compositions consisting of that element, unless otherwise specified or clearly contradicted by context).

[0891] Unless otherwise protected by the claims, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the invention and does not limit the scope of the invention. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0892] Table 15

[0893]

[0894]

[0895]

[0896]

[0897]

[0898]

[0899]

[0900]

[0901]

[0902]

[0903]

Claims

1. An antibody or antibody fragment thereof that specifically binds to a human nectin-4 polypeptide, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), the heavy chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47, 49, 51, 37, 39, 41, 43, 45; and a light chain variable region (VL), the light chain variable region comprising an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 65, 63, 61, 59.

2. The antibody or antibody fragment according to claim 1, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), wherein the heavy chain variable region comprises CDR1, CDR2, CDR3 having the corresponding amino acid sequences shown in SEQ ID NOs: 21, 22 and 23; and a light chain variable region CDR1, CDR2, CDR3, wherein the light chain variable region CDR1, CDR2, CDR3 has the corresponding amino acid sequences shown in SEQ ID NOs: 24, 25, 26.

3. An antibody or antibody fragment according to claim 2, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), wherein the heavy chain variable region comprises the framework FR1, FR2 and FR3 amino acid sequences from the human gene IGKHV-46*01; and a light chain variable region (VL), wherein the light chain variable region comprises the framework FR1, FR2 and FR3 amino acid sequences from the human gene IGKV-28*01.

4. The antibody or antibody fragment according to claim 3, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), which further comprises a framework FR4 having the amino acid sequence of the human gene IGHJ4*01; and a light chain variable region (VL), which further comprises a framework FR4 having the amino acid sequence of the human gene IGKJ4*01.

5. The antibody or antibody fragment of any one of claims 2 to 4, wherein the amino acid at Kabat heavy chain position 71 is leucine and the amino acid at Kabat heavy chain position 78 is threonine.

6. The antibody or antibody fragment of any one of claims 2 to 5, wherein the amino acid at Kabat heavy chain position 73 is lysine.

7. The antibody or antibody fragment of any one of claims 2 to 6, wherein the amino acid at Kabat heavy chain position 28 is isoleucine.

8. The antibody or antibody fragment of any one of claims 2 to 7, wherein the amino acid at Kabat heavy chain position 38 is lysine.

9. The antibody or antibody fragment of any one of claims 2 to 8, wherein the amino acid at Kabat heavy chain position 40 is arginine.

10. The antibody or antibody fragment of any one of claims 2 to 9, wherein the amino acid at Kabat light chain position 2 is valine and the amino acid at Kabat light chain position 64 is serine.

11. The antibody or antibody fragment of any one of claims 2 to 10, wherein the amino acid at Kabat light chain position 11 is asparagine.

12. The antibody or antibody fragment of any one of claims 2 to 11, wherein the amino acid at Kabat light chain position 8 is alanine.

13. The antibody or antibody fragment according to any one of claims 1 to 12, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 47; and a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of SEQ ID NO:

65.

14. The antibody or antibody fragment of any one of claims 1 to 13, wherein the antibody or antibody fragment is conjugated to a linker-payload moiety (XZ), wherein the antibody-linker-payload has the following structure: Where n is 1 5.

15. An immunoconjugate binding to human nectin-4 polypeptide, the immunoconjugate being represented by formula (I): Ab-(X-(Z)) Formula (I) in, Ab is an antibody or antibody fragment according to any one of claims 1 to 13, X is a molecule linking Ab and Z, wherein X comprises a cleavable moiety, optionally a protease-cleavable dipeptide, tripeptide, tetrapeptide or pentapeptide, e.g., under physiological conditions, optionally under intracellular conditions; Z comprises a cytotoxic agent, optionally a camptothecin analog, optionally a pentacyclic camptothecin analog, optionally a hexacyclic camptothecin analog, optionally exotecan, SN-38 or Dxd.

16. The immunoconjugate of claim 15, wherein the immunoconjugate is represented by the following formula: Ab-(Y)-(X)-(Y')-(Z) in, Ab is a polypeptide, peptide, or antibody that specifically binds to the VC1 bridging domain of human nectin-4 polypeptide; Y is optionally absent or is a spacer, optionally is a substituted or unsubstituted alkyl or heteroalkyl chain, optionally wherein one or more atoms may not be carbon, such as oxygen, sulfur, nitrogen or other atoms, optionally wherein any carbon of the chain is substituted by alkoxy, hydroxyl, alkylcarbonyloxy, alkyl-S-, thiol, alkyl-C(O)S-, amine, alkylamine, amide or alkylamide, wherein Y has a chain length of 2-100 atoms, optionally further wherein Y comprises a reactive group that reacts with the residue of an amino acid side chain of said antibody; X is or comprises a peptidyl linker cleaved by an intracellular peptidase or protease; Y' is optionally absent or is a spacer, optionally comprising a self-immolative spacer or a non-self-immolative spacer; and Z comprises a cytotoxic agent, optionally a camptothecin analog, optionally a pentacyclic camptothecin analog, optionally a hexacyclic camptothecin analog, optionally exotecan, SN-38 or Dxd.

17. The immunoconjugate of any one of claims 15 to 16, wherein the protease-cleavable peptide or peptidyl linker comprises valine-alanine.

18. The immunoconjugate of any one of claims 15 to 16, wherein the protease-cleavable peptide or peptidyl linker comprises phenylalanine-lysine or valine-citrulline.

19. The immunoconjugate of any one of claims 15 to 18, wherein the spacer (Y) comprises an orthogonal linker portion and a stability enhancing moiety bound to the orthogonal linker portion, optionally wherein the stability enhancing moiety is a PEG or PSAR homopolymer.

20. The immunoconjugate of claim 19, wherein the PEG or PSAR homopolymer comprises no more than 1 6 PEG or PSAR units, optionally between 8 and 1 6 PEG or PSAR units.

21. The immunoconjugate of any one of claims 18 to 20, wherein the orthogonal linker is derived from glutamic acid.

22. The immunoconjugate according to any one of claims 15 to 21, wherein the immunoconjugate is represented by any one of the following formulae: wherein n is 5-23; and Wherein Ab is an antibody or an antibody fragment thereof that specifically binds to human nectin-4 polypeptide.

23. The immunoconjugate of any one of claims 15 to 22, wherein the antibody or antibody fragment comprises: a heavy chain variable region (VH), the heavy chain variable region comprising the amino acid sequence of SEQ ID NOS: 47; and a light chain variable region (VL), the light chain variable region comprising the amino acid sequence of SEQ ID NOS:

65.

24. The immunoconjugate of any one of claims 15 to 23, wherein the antibody or antibody fragment comprises: a heavy chain comprising the amino acid sequence of SEQ ID NOS: 77; and a light chain comprising the amino acid sequence of SEQ ID NOS:

78.

25. The immunoconjugate of any one of claims 15 to 24, wherein n is 1 5, and the immunoconjugate is characterized in that the DAR is between 6 and 8, optionally wherein the DAR is 6, optionally wherein the DAR is 8.

26. A method for preparing an antibody-drug-conjugate (ADC), the method comprising conjugating a cytotoxic agent (Z) to an anti-Nectin-4 antibody according to any one of claims 1 to 13, wherein a linker (X) connects the anti-Nectin-4 antibody (Ab) and the cytotoxic agent (Z).

27. The method of claim 26, wherein the method comprises providing an anti-Nectin-4 antibody according to any one of claims 1 to 13, contacting and / or reacting the anti-Nectin-4 antibody with a cytotoxic agent (Z) under conditions suitable for forming an antibody drug conjugate, and isolating the antibody drug conjugate.

28. The method according to any one of claims 26 to 27, wherein the method comprises providing an anti-Nectin-4 antibody according to any one of claims 1 to 13, contacting and / or reacting the anti-Nectin-4 antibody with a linker payload moiety (XZ) having the formula:

29. A method of treating cancer, killing tumor cells and / or delivering a cytotoxic agent to a tumor in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an immunoconjugate according to any one of claims 14 to 25 or an antibody drug conjugate prepared by the method of any one of claims 26 to 28.

30. The method of claim 29, wherein the individual has a Nectin-4 expressing cancer characterized by low or moderate Nectin-4 expression on tumor cells, optionally as determined by immunochemistry.

31. The method of any one of claims 29 to 30, 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.

32. The method of any one of claims 29 to 31, wherein the individual has received prior treatment with a chemotherapeutic agent, optionally a chemotherapeutic agent transported by P-glycoprotein (Pgp), a platinum agent, or a taxane.

33. The method of any one of claims 29 to 32, wherein the individual has received prior treatment with a nectin-4 binder conjugated to an auristatin, optionally vestin-enfortumab.

34. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 13, the immunoconjugate according to any one of claims 14 to 25, or the antibody drug conjugate prepared by the method according to any one of claims 26 to 38, and a pharmaceutically acceptable carrier.

35. A nucleic acid or a set of nucleic acids encoding the heavy chain and / or light chain of the antibody or antibody fragment according to any one of claims 1 to 13.

36. A hybridoma or recombinant host cell that produces the antibody or antibody fragment according to any one of claims 1 to 13.

37. A method of preparing the antibody or antibody fragment according to any one of claims 1 to 13, the method comprising culturing the cell according to claim 36, and recovering the antibody or antibody fragment.

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