Humanized antibody against nectin-4 and its drug conjugate

Abstract

The present invention provides humanized antibodies against Nectin-4, and antibody drug conjugates (ADCs) of the humanized antibodies, and their use in the treatment of diseases, particularly cancer.

Description

[Technical Field]

[0001] The present invention is in the field of immunotherapy and relates to humanized anti-Nectin-4 antibodies and antibody conjugates, as well as therapeutic and diagnostic compositions containing them, for treating diseases, particularly cancer. [Background technology]

[0002] Cancer immunotherapy is used to generate and enhance anti-tumor immune responses, for example, by treatment with antibodies specific for antigens on tumor cells or by the specific activation of anti-tumor T cells. The ability to recruit immune cells (e.g., T cells) against a patient's tumor cells provides a therapeutic modality to combat cancer types and metastases that are otherwise considered incurable.

[0003] Nectin cell adhesion molecule 4 (nectin-4), also known as poliovirus receptor-related 4 (PVRL4), is a type I transmembrane protein and a member of the nectin family of related immunoglobulin-like adhesion molecules. Nectin-4 is a tumor-associated marker for many tumors, including bladder cancer, breast cancer, lung cancer, and other malignancies.

[0004] Chailta-Eid et al., 2016 (Cancer Res. 2016;76:3003-13) disclosed an anti-nectin-4 (enfortumab) antibody-drug conjugate as a highly potent therapeutic agent in multiple preclinical cancer models. The antibody, conjugated with the microtubule inhibitor vedotin, binds to human, rat, and monkey nectin-4 and inhibits the growth of several nectin-4-expressing cell lines and xenografts.

[0005] International Patent Application Publication No. WO 2019 / 215728 discloses a monoclonal antibody that recognizes human nectin-4 with high affinity and specificity and inhibits its binding to the T cell immunoreceptor with Ig and ITIM domains (TIGIT).

[0006] Antibody-drug conjugates (ADCs) are promising tools for both the direct killing of tumor cells and the consequent activation of bystander immune cells. These therapeutic entities consist of a mAb linked to a cytotoxic drug (payload) and are designed, in principle, to extend the therapeutic window of these drugs by restricting their specific delivery to cells expressing the target antigen, thus reducing their systemic exposure and toxicity.

[0007] Auristatins are microtubule-disrupting drugs. They are called dolastatins and were derived from the shellless marine mollusk Dolabella auricularia. Various auristatin derivatives, such as monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF), have been synthesized. MMAE and MMAF were developed by Seattle Genetics and used as payloads for ADCs. MMAF and MMAE have their own advantages and disadvantages. MMAE has higher membrane permeability and a lower IC50 than MMAF. However, MMAF is more hydrophilic and has a lower tendency to aggregate, which results in lower systemic toxicity than MMAE (Park et al., Molecules, 2019, 24, 2754).

[0008] International Patent Application Publication No. WO 2018 / 158398 discloses antibodies with specificity for Nectin-4, such as 14A5.2, and their use in the treatment of cancer.

[0009] International Patent Application Publication No. WO 2017 / 042210 discloses an anti-Nectin-4 antibody conjugated to MMAE that is more effective than enfortumab-vedotin (PADCEV). The antibody used in this application is of murine origin and is less suitable for clinical use.

[0010] There is an unmet need to provide humanized antibodies that recognize human Nectin-4 and ADCs containing the same that are improved, safe, and potent and may be useful in the treatment of cancers that express Nectin-4. Summary of the Invention

[0011] The present invention provides humanized antibodies that specifically bind to Nectin-4 or its antigen-binding portion. The humanized antibodies of the present invention are selected from a larger collection of antibody clones and have improved properties compared to other anti-Nectin-4 antibodies. The present invention also provides, according to some embodiments, conjugates comprising the antibodies and therapeutic or diagnostic agents. In some embodiments, the conjugates comprise a cytotoxic moiety and are useful for treating cancers in which tumor cells display the Nectin-4 receptor on their surface.

[0012] In particular, the present invention demonstrates that the NTX1105 ADC is more effective than the most advanced ADCs with anti-Nectin-4 antibodies published to date (PADCEV or enfortumab-based ADCs). NTX1105-based ADCs have now been shown to have significant antitumor activity in treatment regimens in which PADCEV or other enfortumab-based ADCs have no activity.

[0013] A large collection of humanized antibodies has been created by combining specific sets of complementarity-determining region (CDR) sequences with human framework sequences and introducing specific mutations into these sequences to generate antibodies with altered variable regions and improved properties. The antibodies disclosed herein were designed based on factors including homology, T-cell epitopes, critical residues, and predicted structure. Advantageously, the newly designed humanized variable regions described herein preserve residues important for maintaining antibody conformation and binding affinity while significantly reducing the incidence of potential T-cell epitopes, thus minimizing the risk of adverse immune responses against the antibody.

[0014] The humanized antibodies disclosed herein possessed superior manufacturability and improved developability characteristics (e.g., higher resistance to aggregation as measured by improved tagging). The humanized antibodies disclosed herein were found to be highly suitable for use as targeted therapy with therapeutic toxins. It is now disclosed that the anti-Nectin-4 monoclonal humanized antibodies described herein conjugated to cytotoxic moieties exhibit potent killing of various tumor cell lines. Direct targeting of toxins using the antibodies described herein has the potential to increase the anti-tumor activity of these toxins and improve survival of cancer patients. Some of the ADCs of the present invention contain sequence modifications in their Fc region that significantly reduce their binding by FcγR-bearing normal cells, thereby increasing their safety.

[0015] According to one aspect, the present invention provides a humanized antibody that specifically binds to human Nectin-4, or a fragment thereof comprising at least the antigen-binding site, wherein the humanized antibody specifically binds to human Nectin-4, or a fragment thereof comprising at least the antigen-binding site, comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0016] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:2. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:3. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:4. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:5. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:6.

[0017] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 8. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 9. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 10. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 11. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 12.

[0018] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 13, and the light chain comprises a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO: 14.

[0019] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain comprising a variable region having the amino acid sequence set forth in SEQ ID NO: 13. According to some embodiments, the humanized antibody or fragment thereof comprises a light chain comprising a variable region having the amino acid sequence set forth in SEQ ID NO: 14. According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 13 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0020] Although several methods are known in the art for determining the CDR sequences of a given antibody molecule, there is no standard, defined method. Determination of CDR sequences from the heavy and light chain variable regions of an antibody can be performed according to any method known in the art, including, but not limited to, the methods known as KABAT, Chothia, and IMGT. The selected set of CDRs can include sequences identified by more than one method; for example, some CDR sequences can be determined using KABAT and some can be determined using IMGT. According to some embodiments, the CDR sequences of mAb variable regions are determined using the IMGT method.

[0021] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence SYY (SEQ ID NO: 26), a heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), a light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0022] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences: heavy chain CDR1 comprising the sequence SYYIH (SEQ ID NO: 15), heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprising the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 19), and light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0023] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27). According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFQG ​​(SEQ ID NO: 28).

[0024] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 29). According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence RASQSVNNDVA (SEQ ID NO: 30).

[0025] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence GYTFTSYY (SEQ ID NO: 21), a heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence ARSNPYVMDY (SEQ ID NO: 23), a light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), a light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0026] According to some embodiments, the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a set of four heavy chain (HC) framework (FR) sequences: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 31, 32, and 33, (B) FR-H2 selected from the group consisting of SEQ ID NOs: 34, 35, and 36, (C) FR-H3 selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, and (D) FR-H4, which is SEQ ID NO: 41, and the light chain variable region comprises a set of four light chain (LC) framework (FR) sequences: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 42, 43, and 44, (B) FR-L2 selected from the group consisting of SEQ ID NOs: 45 and 46, (C) FR-L3 selected from the group consisting of SEQ ID NOs: 47, 48, and 49, and (D) FR-L4, which is SEQ ID NO: 50. Each framework possibility or combination represents a separate embodiment.

[0027] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, and the light chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12. Each heavy and light chain possibility or combination represents a separate embodiment of the present invention.

[0028] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, and the light chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12. Each combination of heavy and light chain variable regions represents a separate embodiment of the invention. According to certain exemplary embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 5, and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 12 (designated herein as NTX1105(H4 / k5)). According to further exemplary embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 6, and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 12.

[0029] According to some embodiments, the humanized antibody or fragment thereof is a monoclonal antibody, a Fab, a F(ab)2, a single domain antibody, or a single chain variable fragment (scFv).

[0030] According to some embodiments, the humanized antibody or fragment thereof is an IgG monoclonal antibody. According to some embodiments, the humanized antibody has a heavy chain constant region selected from IgG1, IgG4, and IgG2. In certain embodiments, the humanized antibody or fragment thereof is of the IgG4 subclass. In certain embodiments, the humanized antibody or antigen-binding fragment thereof is of the IgG1 subclass. According to some embodiments, the antibody has a kappa light chain constant region.

[0031] According to some embodiments, the humanized antibody has a variant Fc domain that prevents FcγR-mediated internalization.

[0032] According to some embodiments, the humanized antibody comprises an Fc null domain. According to certain embodiments, the Fc domain is null for binding to Fcγ receptors found on immune cells. According to certain exemplary embodiments, the Fc domain is null for binding to CD64, CD32a, CD32b, CD16a, and / or CD16b.

[0033] According to some embodiments, the humanized antibody comprises an Fc null domain with a LALAPG mutation.

[0034] According to some embodiments, the humanized antibody comprises the heavy chain sequence set forth in SEQ ID NO: 51 and the light chain sequence set forth in SEQ ID NO: 52 (LALAPG variant (FcgR null ) with NTX1105(H4 / k5)).

[0035] According to some embodiments, there is provided a conjugate comprising the above-described humanized antibody or fragment thereof.

[0036] The antibodies or fragments thereof according to the invention may be conjugated to a cytotoxic moiety, a radioactive moiety, or a labeling tag.

[0037] According to some embodiments, the humanized antibody or fragment thereof is conjugated to a toxin (payload).

[0038] According to some embodiments, the toxin is selected from the group consisting of a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, and an RNA polymerase inhibitor.

[0039] According to certain embodiments, the toxin is a microtubule-disrupting agent. According to certain exemplary embodiments, the toxin is an auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0040] According to some embodiments, the toxin is saporin.

[0041] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0042] According to some embodiments, the toxin is a quinoline alkaloid. According to particular embodiments, the quinoline alkaloid is SN-38.

[0043] In some embodiments, the toxin is directly linked to the antibody. In other embodiments, the antibody and toxin are linked via a linker. In some embodiments, the toxin is covalently attached to the humanized antibody directly or via a linker.

[0044] According to some embodiments, the linker is cleavable. According to further embodiments, the linker is not cleavable. According to some embodiments, the linker is an enzymatically cleavable linker. According to particular embodiments, the linker is a pH-sensitive linker. According to some embodiments, the linker is a reducible linker (sulfo-SPDB).

[0045] According to some embodiments, the linker is selected from the group consisting of maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-amino-benzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), and Lys-PAB-CO (lysine-ρ-aminobenzyl-C═O).

[0046] According to some embodiments, the drug-to-antibody ratio (DAR) is between 4 and 8. According to certain embodiments, the DAR is 4 (DAR-4). According to certain embodiments, the DAR is 8 (DAR-8).

[0047] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the heavy chain variable region and light chain variable region described above are provided.

[0048] In a further aspect, the present invention provides a nucleic acid construct comprising a nucleic acid molecule encoding at least one humanized antibody chain or fragment thereof as described herein. According to some embodiments, the nucleic acid construct is a plasmid.

[0049] In some embodiments, there is provided a plasmid for expressing a humanized antibody or fragment thereof described herein, the plasmid comprising a nucleic acid molecule encoding the antibody.

[0050] According to another aspect, the present invention provides a pharmaceutical composition comprising a humanized antibody or antigen-binding fragment described herein, or a conjugate comprising the antibody, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0051] According to some embodiments, the pharmaceutical composition is for use in the treatment of cancer.

[0052] Any mode of administration can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0053] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. According to some embodiments, the pharmaceutical composition is formulated for intravenous administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral administration.

[0054] According to yet another aspect, the present invention provides a method of treating cancer, comprising administering to a subject in need thereof a therapeutically effective amount of at least one humanized antibody, fragment thereof, or conjugate thereof, as described herein.

[0055] According to some embodiments, the cancer comprises a solid tumor.

[0056] According to certain embodiments, the cancer is selected from the group consisting of prostate cancer, colorectal cancer, bladder cancer, liver cancer, ovarian cancer, endometrial cancer, gastric cancer, thyroid cancer, carcinoid tumor, head and neck cancer, breast cancer, pancreatic cancer, testicular cancer, urothelial cancer, cervical cancer, melanoma, lymphoma, and lung cancer, with each possibility representing a separate embodiment of the present invention.

[0057] According to certain embodiments, the cancer is selected from the group consisting of pancreatic ductal adenocarcinoma, clear cell renal cell carcinoma, and cutaneous melanoma.

[0058] According to other embodiments, the cancer is a hematological cancer. According to some embodiments, the hematological cancer is selected from leukemias, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), lymphomas, including Hodgkin's disease and non-Hodgkin's lymphoma, and multiple myeloma.

[0059] According to some embodiments, the subject is a human.

[0060] According to some embodiments, the method of treating cancer comprises administering or administering at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

[0061] According to some embodiments, a method of treating cancer comprises administering a humanized antibody or conjugate described herein and an additional anti-cancer agent, according to some embodiments, the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

[0062] According to some embodiments, the anticancer agent is selected from the group consisting of erbitux, cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, vincristine, vinblastine, vinorelbine, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, plicamycin, etoposide, teniposide, and any combination thereof, wherein each possibility represents a separate embodiment of the present invention.

[0063] According to some embodiments, the method of treating cancer comprises preventing or reducing the formation, growth or spread of metastases in a subject.

[0064] According to another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-Nectin-4 antibody or antigen-binding portion thereof conjugated to a toxin (payload), wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences: heavy chain CDR1 comprising the sequence SYY (SEQ ID NO: 26), heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0065] According to some embodiments, the antibody-drug conjugate (ADR) comprises a humanized anti-Nectin-4 antibody or an antigen-binding portion thereof, comprising a set of six CDR sequences: heavy chain CDR1 comprising the sequence SYYIH (SEQ ID NO: 15), heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprising the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 19), and light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0066] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27). According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFQG ​​(SEQ ID NO: 28).

[0067] According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 29). According to some embodiments, the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence RASQSVNNDVA (SEQ ID NO: 30).

[0068] According to some embodiments, the antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0069] According to some embodiments, the heavy chain variable region of the humanized antibody comprises the amino acid sequence set forth in SEQ ID NO:13, and the light chain variable region of the humanized antibody comprises the amino acid sequence set forth in SEQ ID NO:14.

[0070] According to some embodiments, the heavy chain variable region of the humanized antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, and 6, and the light chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11, and 12.

[0071] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises the sequence set forth in SEQ ID NO: 5, and the light chain variable region of the humanized monoclonal antibody comprises the sequence set forth in SEQ ID NO: 12. According to other embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises the sequence set forth in SEQ ID NO: 6, and the light chain variable region of the humanized monoclonal antibody comprises the sequence set forth in SEQ ID NO: 12.

[0072] According to some embodiments, the antibody-drug conjugate comprises a toxin as described hereinabove.

[0073] Any chemical or biological entity capable of killing or inhibiting the growth of tumor cells in vivo can be used as a toxin in conjunction with the ADCs of the invention. According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors.

[0074] According to certain embodiments, the toxin is a microtubule-disrupting agent. According to certain exemplary embodiments, the toxin is an auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF).

[0075] According to some embodiments, the toxin is saporin.

[0076] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0077] According to some embodiments, the toxin is a quinoline alkaloid. According to particular embodiments, the quinoline alkaloid is SN-38.

[0078] According to some embodiments, the toxin is selected from the group consisting of DM4, MMAE, and SN-38. According to particular embodiments, the toxin is DM4 or MMAE.

[0079] According to some embodiments, the toxin is a topoisomerase I inhibitor. According to some embodiments, the toxin is a derivative of camptothecin. According to certain embodiments, the toxin is exatecan.

[0080] In some embodiments, the toxin is directly linked to the antibody. In other embodiments, the antibody and toxin are linked via a linker. In some embodiments, the toxin is covalently attached to the humanized antibody directly or via a linker.

[0081] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is not cleavable. According to some embodiments, the cleavable linker is selected from the group consisting of an enzymatically cleavable linker, a pH-sensitive linker, and a reducible linker. According to some embodiments, the linker is an enzymatically cleavable linker. According to certain embodiments, the linker is a pH-sensitive linker. According to some embodiments, the linker is a reducible linker.

[0082] According to some embodiments, the linker is selected from the group consisting of maleimidocaproyl (MC), maleimidocaproyl-valine-citrulline-p-amino-benzyloxycarbonyl (MC-VC-PAB), maleimidomethylcyclohexane-1-carboxylate (SMCC), N-succinimidyl-4-(2-pyridyldithio)butanoate (sulfo-SPDB), valine-alanine linker, and Lys-PAB-CO (lysine-ρ-aminobenzyl-C═O).

[0083] In some embodiments, the conjugate comprises the toxin MMAE and the linker MC-VC-PAB (referred to herein as NTX1105-MMAE). In some embodiments, the conjugate comprises the toxin MMAF and the linker MC (referred to herein as NTX1105-MMAF). In some embodiments, the conjugate comprises the toxin DM1 and the linker SMCC (referred to herein as NTX1105-DM1). In some embodiments, the conjugate comprises the toxin DM4 and the linker SPDB (referred to herein as NTX1105-DM4). In some embodiments, the conjugate comprises the toxin SN38 and the linker Lys-PAB-CO (referred to herein as NTX1105-SN38).

[0084] According to some embodiments, the ADC comprises an anti-Nectin-4 antibody that specifically binds to a Nectin-4 molecule in competition with an antibody described herein. According to certain embodiments, the ADC comprises an anti-Nectin-4 antibody that specifically binds to a Nectin-4 molecule in competition with an antibody comprising heavy and light chain variable regions, wherein the heavy chain CDR1 comprises the sequence SYYIH (SEQ ID NO: 15), the heavy chain CDR2 comprises the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), the heavy chain CDR3 comprises the sequence SNPYVMDY (SEQ ID NO: 17), the light chain CDR1 comprises the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), the light chain CDR2 comprises the sequence YASNRFT (SEQ ID NO: 19), and the light chain CDR3 comprises the sequence QQAYRSPYT (SEQ ID NO: 20).

[0085] According to another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0086] Any mode of administration can be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0087] According to some embodiments, the pharmaceutical composition is formulated for injection or infusion. In some embodiments, the pharmaceutical composition is formulated for intravenous (IV) administration. In certain embodiments, the pharmaceutical composition is formulated for intratumoral (IT) administration.

[0088] According to some embodiments, the conjugate or pharmaceutical composition is for use in treating cancer in an individual.

[0089] The cancer is as described above. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of liver cancer, lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, testicular cancer, head and neck cancer, pancreatic cancer, and breast cancer. Each possibility represents a separate embodiment of the present invention.

[0090] According to some embodiments of the invention, the use further comprises use in combination with an additional ADC.

[0091] According to another aspect, the present invention provides a method of treating cancer in an individual in need of such treatment, comprising administering to the individual a therapeutically effective amount of a conjugate or pharmaceutical composition described herein. In certain embodiments, the cancer is a solid tumor. According to further embodiments, the cancer is a non-solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, colon cancer, cervical cancer, prostate cancer, and lung cancer. In certain embodiments, the method of treating cancer comprises preventing or reducing the formation, growth, or spread of metastases in a subject.

[0092] According to other embodiments, the cancer is a hematological cancer. According to some embodiments, the hematological cancer is selected from leukemias, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), lymphomas, including Hodgkin's disease and non-Hodgkin's lymphoma, and multiple myeloma.

[0093] According to some embodiments, the individual is a human.

[0094] According to some embodiments, the method of treating cancer comprises administering or administering at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

[0095] According to some embodiments, a method of treating cancer comprises administering a conjugate described herein and an additional anti-cancer agent, wherein the additional anti-cancer agent is selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

[0096] Also described herein are methods of making a composition for treating cancer in an individual suffering from cancer, the methods comprising admixing an ADC described herein with a pharmaceutically acceptable excipient, carrier, or diluent.

[0097] According to yet another aspect, the present invention provides a method for delivering at least one humanized antibody, fragment thereof or conjugate thereof described herein to a cell, the method comprising contacting the cell with the humanized antibody, fragment thereof or conjugate thereof.

[0098] According to some embodiments, the method comprises administering the humanized antibody, fragment or conjugate thereof to cells of a subject. According to particular embodiments, the subject is a human subject.

[0099] According to another aspect, the present invention provides a method of delivering at least one humanized antibody, fragment thereof, or conjugate thereof described herein to a cell of a subject, the method comprising administering at least one humanized antibody, fragment thereof, or conjugate thereof to a subject. According to some embodiments, the cell is a tumor cell.

[0100] The present invention further provides, according to one aspect, a method for diagnosing or prognosing cancer in a subject, the method comprising determining the expression level of Nectin-4 in a biological sample of said subject using at least one antibody conjugate described herein.

[0101] According to some embodiments, the method includes detecting the bound antibody or antibody fragment and determining the expression level of Nectin-4 in the sample. According to certain embodiments, the method includes comparing the expression level to a control. According to some embodiments, the control is a predefined value. According to certain embodiments, the control is a corresponding non-cancerous tissue. According to some embodiments, the comparison indicates or suggests whether the subject has cancer.

[0102] According to some embodiments, the method is used to diagnose cancer subtypes. According to some embodiments, the method is used to determine a patient's eligibility to receive anti-cancer therapy. According to particular embodiments, the anti-cancer therapy is an antibody-drug conjugate. According to some embodiments, the method is used to determine a patient's eligibility to receive therapy with an ADC described herein.

[0103] The present invention further provides, according to another aspect, a method for diagnosing, determining or quantifying Nectin-4 expression, the method comprising contacting a biological sample with an antibody conjugate described herein and measuring the level of complex formation.

[0104] According to some embodiments, the method for detecting or quantifying Nectin-4 expression comprises: i. incubating the sample with an antibody conjugate described herein ii. detecting bound Nectin-4 using the conjugate.

[0105] According to some embodiments, the method comprises: iii. comparing the amount of step (ii) with a standard curve obtained from reference samples containing known amounts of Nectin-4; and iv. Calculating the amount of Nectin-4 in the sample from the standard curve.

[0106] According to some particular embodiments, the sample is a body fluid or a solid tissue. In some embodiments, the method is performed in vitro or ex vivo.

[0107] Also provided is a kit for measuring Nectin-4 expression in a biological sample, comprising at least one conjugate described herein and a means for measuring Nectin-4 expression. In some embodiments, the kit further comprises instructions for use of the kit.

[0108] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0109] [Figures 1A-1C] Correlation between Nectin-4 mRNA expression (high or low, as indicated) and survival probability is shown for patients with pancreatic ductal adenocarcinoma (Figure 1A), clear cell renal cell carcinoma (Figure 1B), and cutaneous melanoma (Figure 1C). Data sets were obtained from the TCGA website and analyzed using the oncolnc.org website (https: / / doi.org / 10.7717 / peerj-cs.67). N indicates the number of patients included in the analysis. [Figure 2] Figure 1 shows a graph showing the percentage of tumors positive for Nectin-4 expression. Data was obtained from proteinatlas.com using HPA010775 mAb (anti-Nectin-4 Sigma-Aldrich®). Moderate to high membranous expression of Nectin-4 is seen in 13 / 20 indications. [Figure 3] FIG. 1 shows dose-dependent killing of MDA-MB-468 cell line (breast adenocarcinoma cells) in vitro using various linker-payload combinations of NTX1105 (VH0 / Vκ0). [Figure 4] Figure 1 shows the in vivo efficacy of NTX1105 (VH0 / Vκ0) ADC against the triple-negative breast cancer (TNBC) tumor cell line MDA-MB-468 subcutaneously (sc) implanted in NOD-SCID female mice. The effect of treatment with all NTX1105 linker-payload (LP) combinations is shown. [Figure 5] Figure 1 shows improved characteristics of some humanized variants. The productivity of the parent / chimeric (VH0 / Vκ0) antibody and six purified lead humanized variants was evaluated. [Figures 6A-6B] The humanized variants demonstrate potent in vivo activity. The in vivo efficacy of the lead humanized NTX1105 (VH4 / Vκ5 and VH5 / Vκ5) ADCs, both conjugated to MC-VC-PAB-MMAE at a DAR of approximately 4, was evaluated against the TNBC tumor cell line MDA-MB-468. Results are shown as tumor volume (FIG. 6A) and fold change in tumor volume over time (FIG. 6B). [Figures 7A-7B] Figure 7 shows the difference in in vitro activity of NTX1105 (H4κ5) ADC compared to enfortumab-vedotin (PADCEV). Both ADCs were tested for on-cell binding (Figure 7A) and in vitro killing activity (Figure 7B). [Figure 8A-8B] The in vivo antitumor activity of NTX1105 (H4κ5) conjugated to MC-VC-PAB-MMAE at a DAR of approximately 4 is superior to that of enfortumab-vedotin (PADCEV). To compare the ADC with PADCEV, the activity of both ADCs was evaluated in vivo using H322 (NSCLC model). The ADCs were given either as six doses of 1 mg / kg (Figure 8A) or three doses of 3 mg / kg (Figure 8B). [Figure 9]1 shows the in vivo efficacy of humanized NTX1105 (H4κ5-FcgRnull) ADCs containing either the DAR-4 MMAE or DAR-8 exatecan payload against the H322M (lung cancer model) sc tumor model in nude female mice. [Figure 10] Shows the in vivo superiority of the humanized NTX1105 (H4κ5-FcgRnull) anti-nectin-4 ADC with DAR-4 exatecan compared to enfortumab-DAR-4 exatecan against a large (>450 mm3) H322M tumor model in nude female mice. DETAILED DESCRIPTION OF THE INVENTION

[0110] The present invention provides humanized anti-Nectin-4 antibodies and antibody-drug conjugates, or ADCs comprising them, that are useful for the treatment of cancer. Advantageously, the ADCs described herein comprise nearly fully humanized antibodies, thus avoiding the risk of adverse immune responses to the antibodies and therefore potentially safe for use in humans. The ADCs described herein have been found to be highly potent and superior to other known anti-Nectin-4 ADCs.

[0111] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context requires otherwise, throughout the specification and the following claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," should be construed in an open and inclusive sense, i.e., "including but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense, including "and / or," unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments. As used herein, the term "about" refers to an amount within 10% of the stated amount.

[0112] As used herein, the term "nectin-4" or "nectin cell adhesion molecule 4" refers to a single-pass type I membrane protein with 510 amino acids and a molecular weight of 55,454 Da, also known as PVRL4, LNIR, PRR4, and EDSS1. The nectin-4 protein contains two immunoglobulin-like (Ig-like) C2-type domains and one Ig-like V-type domain. It is involved in cell adhesion through trans-homophilic and trans-heterophilic interactions. A soluble form is generated by proteolytic cleavage at the cell surface by the metalloproteinase ADAM17 / TACE, while a secreted form is found in both breast tumor cell lines and breast tumor patients. Exemplary Nectin-4 according to the present invention are designated by SwissPort, UniPort and GenBank symbols or accession numbers: Q96NY8-NECT4_HUMAN, Q96NY8, B4DQW3, Q96K15, Q96NY8-1, Q96NY8-2, ENSP00000356991, NP_112178.2, XP_005245565.1, XP_011508323.1, XP_011508324.1 or XP_011508325.1.

[0113] According to one aspect, the present invention provides a humanized antibody that specifically binds to human Nectin-4, or a fragment thereof comprising at least the antigen-binding site, wherein the humanized antibody specifically binds to human Nectin-4, or a fragment thereof comprising at least the antigen-binding site, comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0114] According to another aspect, the present invention provides an antibody-drug conjugate (ADC) comprising a humanized anti-Nectin-4 antibody or antigen-binding portion thereof conjugated to a toxin (payload), wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences: heavy chain CDR1 comprising the sequence SYY (SEQ ID NO: 26), heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

[0115] According to another aspect, the present invention comprises an antibody-drug conjugate (ADC) comprising a humanized anti-Nectin-4 antibody, or an antigen-binding portion thereof, conjugated to a toxin selected from the group consisting of MMAE, SN-38, DM1, DM4, and MMAFA, wherein the humanized antibody or antigen-binding portion thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

[0116] According to some embodiments, the humanized antibody or antigen-binding fragment thereof has the amino acid sequence

number

number

[0117] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12. SEQ ID NOs:2-6, 8-12 are humanized variants of NTX1105 selected based on improved developability and reduced immunogenicity.

[0118] In general, "NTX1105" includes chimeric antibodies and / or optionally humanized variants thereof. The parent chimeric antibody is disclosed in WO 2019 / 215728 (clone 11). The parent chimeric antibody is designated herein as NTX1105 (VH0 / Vk0).

[0119] The humanized variant of NTX1105 comprises five heavy chains (VH1 to VH5) and five light chains (Vκ1 to Vκ5). According to some embodiments, the humanized variant comprises the sequence formulas shown in SEQ ID NO: 13 (heavy chain) and SEQ ID NO: 14 (light chain).

[0120] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having the amino acid sequence SEQ ID NO: 5 and the light chain comprises a variable region having the amino acid sequence SEQ ID NO: 12 (referred to herein as NTX1105(VH5 / Vk5)).

[0121] According to some embodiments, the humanized antibody comprises a combination of heavy chain variable regions and light chain variable regions selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:8, SEQ ID NO:2 and SEQ ID NO:9, SEQ ID NO:2 and SEQ ID NO:10, SEQ ID NO:2 and SEQ ID NO:11, SEQ ID NO:2 and SEQ ID NO:12, SEQ ID NO:3 and SEQ ID NO:8, SEQ ID NO:3 and SEQ ID NO:9, SEQ ID NO:3 and SEQ ID NO:10, SEQ ID NO:3 and SEQ ID NO:11, SEQ ID NO:3 and SEQ ID NO:12, SEQ ID NO:4 and SEQ ID NO:8, SEQ ID NO:4 and SEQ ID NO:9, SEQ ID NO:4 and SEQ ID NO:10, SEQ ID NO:4 and SEQ ID NO:11, SEQ ID NO:4 and SEQ ID NO:12, SEQ ID NO:5 and SEQ ID NO:8, SEQ ID NO:5 and SEQ ID NO:9, SEQ ID NO:5 and SEQ ID NO:10, SEQ ID NO:5 and SEQ ID NO:11, SEQ ID NO:5 and SEQ ID NO:12, SEQ ID NO:6 and SEQ ID NO:8, SEQ ID NO:6 and SEQ ID NO:9, SEQ ID NO:6 and SEQ ID NO:10, SEQ ID NO:6 and SEQ ID NO:11, and SEQ ID NO:6 and SEQ ID NO: 12. Each heavy and light chain possibility or combination represents a separate embodiment of the present invention.

[0122] According to some embodiments, the humanized antibody comprises a combination of a heavy chain variable region and a light chain variable region, the combination comprising: i. a heavy chain variable region sequence set forth in SEQ ID NO: 5 and a light chain variable region sequence set forth in SEQ ID NO: 12, and ii. The heavy chain variable region sequence shown in SEQ ID NO:6 and the light chain variable region sequence shown in SEQ ID NO:12 are selected from the group consisting of:

[0123] Conjugates of the present invention include humanized antibodies as described herein. Antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Thus, antibodies include, but are not limited to, full-length antibodies and any specific binding portion thereof, including fragments and portions thereof that retain their binding specificity, e.g., those of any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM), as well as biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, Fab, F(ab')2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are generally within a substantially homogeneous antibody composition. Thus, all individual antibodies within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region. The antibody may comprise a human kappa light chain.

[0124] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments, including single-chain variable fragments (sFv or scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term also encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody can comprise a human IgG1 constant region. The antibody can comprise a human IgG4 constant region. The antibody can comprise a human kappa light chain.

[0125] Although several methods are known in the art for determining the CDR sequences of a given antibody molecule, there is no standard, defined method. Determination of CDR sequences from the heavy and light chain variable regions of an antibody can be performed according to any method known in the art, including, but not limited to, the methods known as KABAT, Chothia, and IMGT. The selected set of CDRs can include sequences identified by more than one method; for example, some CDR sequences can be determined using KABAT and some can be determined using IMGT. In some embodiments, the CDR sequences of mAb variable regions are determined using the IMGT method. For example, CDR determination is performed according to Kabat (Wu TT and Kabat EA, J Exp Med, 1970;132:211-50) and IMGT (Lefranc MP, et al., Dev Comp Immunol, 2003, 27:55-77).

[0126] When the term "CDR having a sequence" or similar term is used, it includes options in which the CDR comprises the specified sequence, and also options in which the CDR consists of the specified sequence.

[0127] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv or sFv), and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, comprising a variable heavy chain region and / or a variable light chain region.

[0128] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all framework region (FR) amino acid residues are derived from human FRs. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of a non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parent non-human antibody. According to some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0129] Amino acid residues in the Fc domain can be substituted to be null, meaning that the Fc domain does not bind to an Fc receptor or can bind with such low affinity and / or avidity that it does not trigger Fc receptor signaling as a result of binding. The Fc domain can be null for binding to an Fcγ receptor. Some examples of Fcγ receptors to which the Fc domain can be null for binding include, but are not limited to, FcγRI (CD64), FcγRIIA (CD32a), FcγRIIB (CD32b), FcγRIIIA (CD16a), FcγRIIIA (CD16a) F158 variant, FcγRIIIA (CD16a) V158 variant, or FcγRIIIB (CD16b). The Fc domain can have one or more, two or more, three or more, or four or more amino acid substitutions that reduce binding of the Fc domain to an Fc receptor.

[0130] According to some embodiments, the humanized antibody has a variant Fc domain that prevents FcγR-mediated internalization.

[0131] According to some embodiments, the humanized antibody comprises an Fc null domain. According to particular embodiments, the Fc domain is null for binding to Fcγ receptors.

[0132] As used herein, "Fc null" refers to a domain that exhibits weak to no binding to one or more Fcγ receptors.

[0133] According to some embodiments, the humanized antibody comprises an Fc null domain. According to certain embodiments, the Fc domain is null for binding to Fcγ receptors found on immune cells. According to certain exemplary embodiments, the Fc domain is null for binding to CD64, CD32a, CD32b, CD16a, and / or CD16b.

[0134] According to some embodiments, the humanized antibody comprises an Fc null domain with a LALAPG mutation.

[0135] According to some embodiments, the humanized antibody comprises the heavy chain sequence set forth in SEQ ID NO: 51 and the light chain sequence set forth in SEQ ID NO: 52 (LALAPG variant (FcgR null ) with NTX1105(H4 / k5)).

[0136] The present invention provides a conjugate comprising a humanized antibody disclosed herein and a toxin.

[0137] According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors, with each possibility representing a separate embodiment of the present invention.

[0138] According to certain embodiments, the toxin is a microtubule-disrupting agent. According to certain exemplary embodiments, the toxin is an auristatin or a derivative thereof. According to certain embodiments, the auristatin derivative is monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). According to some embodiments, the toxin is exatecan.

[0139] According to some embodiments, the toxin is a saponin.

[0140] According to some embodiments, the toxin is a maytansine derivative. According to certain embodiments, the maytansine derivative is DM4 or DM1.

[0141] According to some embodiments, the toxin is a quinoline alkaloid. According to particular embodiments, the quinoline alkaloid is SN-38.

[0142] According to further embodiments, the toxin is selected from the group consisting of MMAE, MMAF, saporin, DM4, DM1, SN-38, calicheamicin, DXd, PBD, duocarmycin, sandramycin, alpha-amanitin, chaetocin, CYT997, daunorubicin, 17-AAG, agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, epothilone B, hygrolysin, herboxidien, ferulenol, curvulin, paclitaxel, englerin A, taltobulin, triptolide, cryptophycin, and nemorubicin. Each possibility represents a separate embodiment of the present invention.

[0143] According to some embodiments, the toxin is SN-38. According to some embodiments, the toxin is DM1. According to some embodiments, the toxin is DM4. According to some embodiments, the toxin is MMAE. According to some embodiments, the toxin is MMAF. According to some embodiments, the toxin is exatecan.

[0144] In some embodiments, the antibody is directly linked to the toxin. In other embodiments, the antibody and toxin are linked via a linker. In some embodiments, the humanized antibody described herein is covalently linked to the toxin.

[0145] According to some embodiments, the linker is cleavable. According to further embodiments, the linker is not cleavable.

[0146] In some embodiments, the linker is cleaved in response to a change in pH or redox potential. In some embodiments, the linker is cleaved upon contact with a lysosomal enzyme.

[0147] According to some embodiments, the linker is 6-maleimidocaproyl (MC), maleimidopropionyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)valerate (SPP), N-succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC), N-succinimidyl(4-iodo-acetyl)aminobenzoate (SLAB), 6-maleimidocaproyl-valine-cit and perfluorophenyl 3-(pyridin-2-yldisulfanyl)propanoate. Each possibility represents a separate embodiment of the present invention.

[0148] According to some embodiments, the antibody is conjugated to two or more toxin molecules.

[0149] According to another aspect, the present invention provides a pharmaceutical composition comprising a conjugate described herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0150] According to another embodiment, the pharmaceutical composition according to the invention is for use in the treatment of cancer.

[0151] Cancers amenable to treatment in accordance with the present invention include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various head and neck cancers, as well as B-cell lymphomas (low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL)NHL, including intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-cleaving cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with phakomatosis, edema (e.g., associated with brain tumors), and Meigs syndrome. Preferably, the cancer is selected from the group consisting of breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, non-Hodgkin's lymphoma (NHL), renal cell carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. Cancerous conditions modifiable by treatments of the present invention include metastatic cancer.

[0152] According to another embodiment, the pharmaceutical composition according to the present invention is for use in treating cancers characterized by overexpression of Nectin-4. Cancer types associated with overexpression of Nectin-4 can be identified using known databases, such as The Cancer Genome Atlas (TCGA). According to a particular embodiment, cancers treatable with the composition according to the present invention include adrenocortical carcinoma (ACC), chromophobe renal cell carcinoma (KICH), liver hepatocellular carcinoma (LIHC), colorectal adenocarcinoma (COAD and READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma & paraganglioma (PCPG), papillary renal cell carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine endometrial carcinoma (UCEC), and cervical adenocarcinoma. The cancer is selected from the group consisting of cutaneous melanoma (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell renal cell carcinoma (KIRC), lung squamous cell carcinoma (LUSC), uterine carcinosarcoma (UCS), sarcoma (SARC), ovarian serous cystadenocarcinoma (OV), papillary thyroid carcinoma (THCA), glioblastoma multiforme (GBM), breast cancer (BRCA), low-grade glioma (LGG), and diffuse large B-cell lymphoma (DLBC). Each possibility represents a separate embodiment of the present invention.

[0153] The molecules of the present invention as active ingredients are dissolved, dispersed, or mixed in an excipient that is pharmaceutically acceptable and, as is well known, compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, etc., and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0154] The pharmaceutical composition according to the present invention may be administered together with an anti-neoplastic composition.

[0155] As used herein, the term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. The term "treatment" also relates to alleviation or prevention of symptoms associated with the disease and / or lessening the severity of the disease.

[0156] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include melanoma, lung cancer, thyroid cancer, breast cancer, colon cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, cervical cancer, pancreatic cancer, leukemia, lymphoma, bone marrow cancer, ovarian cancer, uterine cancer, sarcoma, bile duct cancer, or endometrial cancer.

[0157] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes administration of at least one additional anti-cancer agent.

[0158] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease for which the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0159] As used herein, the term "effective amount" refers to the amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, a reduction in tumor growth, a reduction in tumor metastasis, or an increase in the survival time of tumor-bearing animals. A "therapeutic amount" is a concentration of a drug calculated to exert a therapeutic effect. A therapeutic amount encompasses a range of dosages that can induce a therapeutic response in a population of individuals. The mammal may be a human individual. The human individual may have a tumor, be suspected of having a tumor, or be developing a tumor.

[0160] The molecules of the present invention as active ingredients are dissolved, dispersed, or mixed in an excipient that is pharmaceutically acceptable and, as is well known, compatible with the active ingredient. Suitable excipients are, for example, water, saline, phosphate-buffered saline (PBS), dextrose, glycerol, ethanol, etc., and combinations thereof. Other suitable carriers are well known to those skilled in the art. In addition, if desired, the composition can contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like.

[0161] According to some embodiments, the method of treating cancer comprises administering the pharmaceutical composition as part of a treatment regimen that includes administration of at least one additional anti-cancer agent.

[0162] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotic agents, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginase, alkylating agents, antitumor antibiotics, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0163] In some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. In some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. In some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan. In some embodiments, the alkylating agent is selected from the group consisting of busulfan, carmustine, lomustine, chlorambucil, cyclophosphamide, cisplatin, carboplatin, ifosfamide, mechlorethamine, melphalan, thiotepa, dacarbazine, and procarbazine. In some embodiments, the antitumor antibiotic is selected from the group consisting of bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin, mitoxantrone, and plicamycin. According to some embodiments, the topoisomerase II is selected from the group consisting of etoposide and teniposide. Each possibility represents a separate embodiment of the present invention.

[0164] Also described herein are methods of preparing a composition for treating cancer in an individual suffering from cancer, the method comprising admixing a humanized antibody or ADC described herein with a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of glioblastoma, colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, prostate cancer, and lung cancer. [Example]

[0165] Reference is now made to the following examples, which together with the above descriptions illustrate, but do not limit, the invention.

[0166] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Such techniques are well known in the art. Other general references citing well-known procedures are provided throughout this document for the convenience of the reader.

[0167] Example 1. High expression of Nectin-4 mRNA correlates with poor survival in patients with various cancers.

[0168] The correlation between Nectin-4 mRNA expression and survival probability was examined using data from the TCGA website and analyzed using the oncolnc.org website (https: / / doi.org / 10.7717 / peerj-cs.67). The results are presented in Figures 1A-1C. Nectin-4 mRNA expression levels were used to divide patients into two subgroups, low and high, as indicated by the boxes. The survival advantage of low-expressing patients confirms the detrimental role of Nectin-4 and the possibility of improving survival by targeting this protein.

[0169] Example 2. Nectin-4 is expressed at the protein level in the majority of solid tumors The database Proteinatlas.com was searched for all synonyms of nectin-4 (nectin-4, PVRL4). Data using a single mAb was found under the pathology heading (HPA010775). Expression data across various tumors are shown in Figure 2. The graph shows the percentage of tumors positive for nectin-4 expression. In all indications, at least one patient had surface expression of nectin-4, and in 13 / 20 indications, membranous expression of nectin-4 was observed at moderate to high levels.

[0170] Example 3. Various cytotoxic agents can be efficiently conjugated to Nectin-4-targeting mAbs to generate Nectin-4 ADCs Table 1 lists various linker-payload combinations and drug-to-antibody ratios (DARs) used to conjugate the anti-nectin-4 antibody clone-11 (VH0 / Vκ0)-hIgG1 (also described in our WO2019 / 215728) to generate the NTX1105 (VH0 / Vκ0) nectin-4 ADC. The cytotoxic agents listed in Table 1 include tubulin- and TOPO1-targeting agents, and the DARs were selected according to ADCs approved using the aforementioned payloads.

[0171] [Table 1]

[0172] Example 4. Various cytotoxic agents can induce tumor cell killing in vitro when conjugated to Nectin-4-targeting mAb Next, we investigated the dose-dependent killing activity of various linker-payload combinations of NTX1105 (VH0 / Vκ0). We used an in vitro assay of the MDA-MB-468 cell line, a TNBC model. 2 x 10 target cells were cultured per well. 3 Cells were seeded with IgG and allowed to attach for 4-6 hours. The ADC was added at concentrations ranging from 30 nM to 1.9 nM using two-fold dilutions, and the cells were then incubated with the ADC for an additional 72 hours. After 72 hours, the assays were harvested and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega G9242) according to standard protocols. Killing data demonstrated strong potency, with most payloads not reaching EC-50 (except for DM1 / 4, which reached EC-50 at approximately 3.8 nM), suggesting high potency of the ADCs regardless of the linker-payload combination selected (Figure 3).

[0173] Example 5. Various cytotoxic agents can induce tumor cell killing in vivo when conjugated to Nectin-4-targeting mAb The in vivo efficacy of NTX1105 (VH0 / Vκ0) anti-Nectin-4 ADC was evaluated against the TNBC tumor cell line MDA-MB-468 (5M cells / mouse) sc implanted into 6-week-old NOD-SCID female mice. 3 Treatment began when the tumor reached a mean tumor volume of 1000 mg / mL, and animals received three doses (Q4D) of 5 mg / kg NTX1105 containing the indicated linker-payload. Figure 4 compares the effects of treatment with all NTX1105 combinations of linker-payloads (LPs). Three LP combinations had significant effects. SN38 and DM4 produced significant tumor growth inhibition (TGI, p<0.05), while MMAE produced complete tumor regression. The fact that three of the five LPs tested produced significant antitumor activity suggests the unique utility of NTX1105 as an Ab for ADCs. Based on the results of these experiments, further development of a humanized anti-Nectin-4 ADC based on the NTX1105 mAb was undertaken.

[0174] Example 6. Human Nectin-4 Binding and Cross-Reactivity to CynoNectin-4 is Retained in Top Selected Humanized Nectin-4 mAbs To evaluate the binding of all variants to human or cynomolgus nectin-4 antigen and select the lead humanized IgG with the tightest affinity to the chimeric (VH0 / Vκ0) antibody, single-cycle kinetics experiments were performed on supernatants from transfected cell cultures. Kinetic experiments were performed at 25°C on a Biacore T200 (Cytiva, Marlborough, USA) running Biacore T200 Control Software V 2.0.1 and Evaluation Software V3.0. To minimize potential mass transfer effects, single-cycle kinetic data were obtained using recombinant human (Acro Biosystems, Newark, USA) or cynomolgus nectin-4 (Acro Biosystems, Newark, USA) as the analyte, injected at a flow rate of 40 μl / min. A four-point, two-fold dilution range of antigen from 1.25 nM to 10 nM in running buffer was used without regeneration between each concentration. The association phase was monitored for 150 s for each of four injections of increasing concentrations of antigen, and a single dissociation phase was measured for 250 s after the final injection of antigen (dissociation was trimmed to 175 s for human nectin-4 to improve fitting). Regeneration of the sensor chip surface was performed using a single injection of 3 M MgCl2. Based on the relative KDs from Biacore single-cycle kinetic analysis of the supernatant, six humanized variants (VH4 / Vκ1, VH4 / Vκ3, VH4 / Vκ4, VH4 / Vκ5, VH5 / Vκ4, and VH5 / Vκ5) were selected as lead IgGs for scale-up of production and purification. All selected variants were within a 2.5-fold range of the parent (VH0 / Vκ0) clone, suggesting similar activity profiles for the humanized variants (Table 2).

[0175] [Table 2]

[0176] Example 7. Improved characteristics (reduced immunogenicity) of humanized anti-nectin-4 mAb The designed VH and Vκ sequences were analyzed for the occurrence of potential T cell epitopes, as determined by application of Abzena's proprietary in silico technology, iTope™ (Perry et al. 2008). The iTope™ software predicts favorable interactions between peptide amino acid side chains and specific binding pockets (particularly pocket positions p1, p4, p6, p7, and p9) within the open binding groove of 34 human MHC class II alleles. These alleles represent the most common HLA-DR alleles found worldwide and are not weighted toward those most commonly found in specific ethnic groups. Twenty of the alleles contain an "open" p1 configuration, and 14 contain a "closed" configuration, in which glycine at position 83 is replaced by valine. Location of key binding residues is achieved by in silico generation of 9-mer peptides with eight overlapping amino acids spanning the test protein sequence. However, the results should be evaluated in light of the fact that all prediction methods for MHC class II binding inherently overpredict the number of T cell epitopes because they do not account for other important processes during antigen presentation, such as protein / peptide processing, recognition by T cell receptors, or T cell tolerance to peptides. iTope™ analysis was performed using overlapping 9-mer peptides (each overlapping the last peptide by 8 residues). Each 9-mer was scored based on potential "fit" and interaction with each of the 34 MHC class II allotypes, with resulting peptide scores ranging from 0 to 1. MHC class II epitopes are then defined by iTope™ as follows:

[0177] (1) Promiscuous, high-affinity MHC class II binding peptides bind to >50% of alleles in the majority (17 of 34 alleles) and have a binding score of >0.6.

[0178] (2) Promiscuous, intermediate-affinity MHC class II binding peptides bind to >50% of alleles and have a binding score of >0.55 (with no majority >0.6).

[0179] These criteria are modified if a large aromatic amino acid (i.e., F, W, or Y) occurs at the p1 anchor position, allowing the binding of large aromatic residues, as 20 of the 34 alleles have open p1 pockets. When this occurs, a promiscuous peptide is defined as binding to 10 or more of the 20 allele subsets. Germline promiscuous high-affinity and medium-affinity MHC class II epitopes that bind the ligand were identified in the parent antibody and the engineered variants; however, due to T cell tolerance, these epitopes were unlikely to have immunogenic potential and were therefore excluded from further analysis. Overall, the humanization process resulted in significantly improved mAbs with lower immunogenicity scores and therefore lower associated risk. The least immunogenic variants after this step included heavy chain variants 3-5 and light chain 5 (Table 3).

[0180] [Table 3]

[0181] Example 8. Improved characteristics (improved developability) of humanized anti-nectin-4 mAb Thermal ramp stability experiments (Tm and Tagg) are well-established methods for ranking proteins and formulations for stability. The denaturation profile of a protein provides information about its thermal stability and represents a structural "fingerprint" for assessing structural and formulation buffer modifications. A widely used measure of a protein's thermal structural stability is the temperature at which the protein unfolds from the native state to the denatured state.

[0182] For many proteins, this unfolding process occurs over a narrow temperature range, and the midpoint of this transition is called the "melting temperature" or "Tm." To determine a protein's melting temperature, UNcle measures the fluorescence of Sypro Orange (which binds to exposed hydrophobic regions of the protein) as the protein undergoes a conformational change. Purified lead antibodies were diluted in duplicate in PBS to a final test concentration of 0.5 mg / ml, to which Sypro Orange (160x stock solution) was added to make a final concentration of 20x solution. 9 μL of each sample mixture was loaded in duplicate into UniCuvettes. Samples were subjected to a thermal ramp from 25 to 95 °C with a ramp rate of 0.3 °C / min and excitation at 473 nm. Full emission spectra were collected from 250 to 720 nm, and the area under the curve from 510 to 680 nm was used to calculate the inflection points (Tonset and Tm) of the relaxation curve. Monitoring static light scattering (SLS) at 473 nm allowed for the detection of protein aggregation, and Tag (onset of aggregation) was calculated from the resulting SLS profile. Data analysis was performed using UNcle™ software version 4.0. No significant changes in T or T were observed, and significant improvements in stability (>5°C higher than VH0 / Vκ0) were found for the two humanized variants (VH4 / Vκ5 and VH5 / Vκ5) (Table 4).

[0183] [Table 4]

[0184] Example 9. Improved characteristics (productivity improvement) of humanized anti-nectin-4 mAb mAb production is a complex process influenced by the amino acid composition and structure of the molecule. Transient production is a good indicator of the "productivity" of a mAb. Combinations of chimeric (VH0 / Vκ0) and humanized heavy and light chain DNA constructs were transiently transfected into HEK293 EBNA adherent cells (LGC Standards, Teddington, UK) in 6-well plates using the PEI transfection method and incubated for 6 days post-transfection. Samples were collected, and antibody concentrations were measured using an Octet QK 384 protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) with an IgG1 antibody as a standard. As shown in Figure 5, the humanization process generally improved the productivity of all mAbs (calculated by dividing the titer (mg / ml) of the relevant humanized variant by that of the parental / chimeric VH0 / Vκ0), with the productivity of the major clones (VH4 / Vκ5 and VH5 / Vκ5) improving by more than 10-fold.

[0185] Example 10. Potent in vivo antitumor activity of lead humanized mAbs We evaluated the in vivo efficacy of the lead humanized NTX1105 (VH4 / Vκ5 and VH5 / Vκ5) anti-Nectin-4 ADCs against the TNBC tumor cell line MDA-MB-468 (5M cells / mouse) sc-implanted into 6-week-old NOD-SCID female mice. Based on previous results, the MMAE payload was selected. Animals were administered 5 mg / kg of the lead humanized ADC four times (Q4D). As shown in Figure 6A, both lead ADCs resulted in tumor regression (greater than 66% reduction from the treatment initiation volume). Unexpectedly, clone H4κ5 was significantly better than clone H5κ5 at maintaining the response over time. Treatment with clone H5κ5 resulted in tumor volumes similar to those at the initiation of treatment 40 days after the last dose (p=0.8) (static). In contrast, clone H4κ5 significantly reduced tumor volume 40 days after the last treatment compared to the day of treatment initiation (p = 0.038) (Figure 6B). This result was unexpected and suggests the superiority of humanized anti-nectin-4 clone H4κ5 over other anti-nectin-4 humanized mAbs.

[0186] Example 11. Differences in in vitro binding and cell killing between NTX1105 and enfortumab-vedotin in vitro Enfortumab vedotin (PADCEV) is the only approved ADC targeting Nectin-4. Although it has demonstrated excellent clinical results, PADCEV has severe adverse events, some of which may be due to on-target / off-tumor binding, given that PADCEV can act even on the extremely low density of Nectin-4 found in some normal tissues. Here, we hypothesize that some of these adverse events could be prevented by an ADC with significantly lower on-target binding / activity.

[0187] Binding was assessed on MDA-MB-468 cells. Briefly, 5*10 per well 4 Cells were stained with either PADCEV or NTX1105 conjugated to MMAE at the same DAR as PADCEV. The ADCs were added at concentrations starting at 3 μg / ml using 3-fold dilutions. Detection was performed with Alexa Fluor® 647 (AffiniPure Fab Fragment Goat Anti-Human IgG (H+L) (cat 109-607-003 Jackson ImmunoResearch). Both ADCs showed identical maximal binding, consistent with the literature, with an EC-50 value of approximately 10 pM for PADCEV (black arrow), approximately 20-fold more potent than NTX1105 (gray dashed arrow) (Figure 7A). To assess the killing potential of the two ADCs in vitro, MDA-MB-468 cells were plated at 2 x 10 per well. 3Cells were seeded and allowed to adhere for 4-6 hours. Using 4-fold dilutions, the ADC was added at concentrations ranging from 12 to 0.01 μg / ml, and the cells were incubated with the ADC for an additional 72 hours. After 72 hours, the assays were harvested, and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay (Promega G9242). Killing data demonstrated approximately 16-fold higher in vitro potency of PADCEV than NTX1105 (Figure 7B). Cumulatively, these results suggest improved safety of NTX1105 compared to PADCEV, the most advanced Nectin-4-targeting ADC.

[0188] Example 12. Superior in vivo antitumor activity of NTX1105 lead humanized clone compared to enfortumab-vedotin (PADCEV).

[0189] Next, the in vivo activity of the NTX1105 lead humanized clone (VH4 / Vκ5) was compared with enfortumab vedotin (PADCEV), a leading anti-nectin-4 ADC already approved for the treatment of patients with urothelial carcinoma. Using an in vivo efficacy assay against the NSCLC tumor cell line H322, the lead ADC (humanized NTX1105 (VH4 / Vκ5), an anti-nectin-4 ADC) was compared with PADCEV. Tumor cells (5M cells / mouse) were implanted sc into 6-week-old nude female mice. Tumors grew to 140 mm. 3 Treatment began once tumor volume reached 100 μg / mL, and animals received either 6 doses (Q4D) of PBS (vehicle) or 1 mg / kg of either PADCEV or NTX1105-MC-VC-PAB-MMAE DAR4 (same conjugation method and DAR as PADCEV), or 3 doses (Q4D) of 3 mg / kg of PADCEV or NTX1105-MC-VC-PAB-MMAE DAR4 (same conjugation method and DAR as PADCEV). As shown in Figure 8A, the low dose of PADCEV did not affect tumor growth at any time point, and at the end of the study, the mean TV of the PADCEV-treated group was slightly higher than that of the PBS-treated group (924 vs. 693 mm).3 ns, p=0.48), NTX1105 was able to halt tumor growth (220 vs. 693 mm 3 , p=0.04). Furthermore, the NTX1105 mean TV did not significantly increase from the start of treatment (p=0.28, compared to the start of treatment). At the 3 mg / kg dose, both NTX1105 and PADCEV caused tumor regression (mean TV decreased by 46% and 42% by day 20 compared to the start of treatment) (Figure 8B). However, from day 34 after the first administration, the NTX1105 ADC was significantly superior to PADCEV in preventing tumor growth throughout the entire study. Notably, at day 48 after the start of the study, the PADCEV-treated group was no longer significantly superior to PBS in terms of tumor volume. The NTX1105 ADC-treated group caused complete tumor growth inhibition and even had a lower tumor volume at day 48 than at the start of the study (138 mm 3 110) at day 48 compared with 110. These results are unexpected, especially considering the significantly enhanced in vitro activity of PADCEV. Furthermore, this is the first time that a Nectin-4-targeting ADC (NTX1105) has demonstrated significant antitumor activity in a model in which no biological activity was observed for PADCEV.

[0190] Example 13. Humanized NTX1105 (H4K5-FcgR null ) ADCs containing either tubulin- or TOPO1-targeting payloads can regress H322 tumors in vivo.

[0191] Nude female mice (n = 5 per group) received 5 × 10 6 H322 cells were injected SC. The tumors were 140 mm 3Once the tumor reached an average volume of 1000 mg / mL, mice were treated with PBS, 3 mg / kg NTX1105-MMAE-DAR4, or 5 mg / kg NTX1105-exatecan-DAR-8 intravenously for two doses every four days in a blinded fashion. As shown in Figure 9, both ADCs were able to shrink established tumors to the same extent, a surprising result given previous attempts using TOPO1-targeted payloads (Figure 4). This demonstrates the potential utility of the NTX1105 ADC, which uses both MMAE and TOPO1 payloads.

[0192] Example 14. Humanized NTX1105 containing a TOPO1-targeting payload (H4K5-FcgR null ) ADC is superior to enfortumab with the same payload and DAR Nude female mice (n = 5 per group) received 5 × 10 6 H322 cells were injected SC. The tumors were 480 mm 3 Once the tumor reached an average volume of 1000 mcg, mice were treated in a blinded fashion with either PBS (vehicle), NTX1105, or enfortumab-based ADCs (both DAR-4 exatecan) by intravenous injection at 10 mg / kg for two doses, 5 days apart. As shown in Figure 10, after two 10 mg / kg doses, only the NTX1105 ADC was able to halt tumor growth, while enfortumab-exatecan showed no effect compared to PBS. This indicates the potential superiority of the NTX1105 ADC over other anti-nectin-4-based ADCs.

[0193] array [Table 5]

[0194] SEQ ID NO: 13 - Heavy chain - generic sequence including all humanized variants

number

[0195] SEQ ID NO: 14 -Light chain - common sequence including all humanized variants

number

[0196] [Table 6]

[0197] [Table 7]

[0198] [Table 8]

[0199] LALAPG mutant (FcgR null ) Full sequence of NTX1105 (H4 / k5) - Heavy chain - SEQ ID NO: 51:

number

[0200] Complete sequence of NTX1105 (H4 / k5) - light chain - SEQ ID NO: 52

number

Claims

1. A humanized antibody that specifically binds to human Nectin-4, or a fragment thereof comprising at least an antigen-binding site, wherein the antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:

12. A humanized antibody that specifically binds to human Nectin-4, or a fragment thereof comprising at least an antigen-binding site, wherein the antibody or the fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:

12.

2. The humanized antibody of claim 1, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising a variable region having an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, and the light chain comprising a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:

12.

3. 3. The humanized antibody of claim 1, wherein the humanized antibody or fragment thereof comprises a heavy chain and a light chain, the heavy chain variable region comprising the sequence shown in SEQ ID NO: 13, and the light chain variable region comprising the sequence shown in SEQ ID NO:

14.

4. The humanized antibody of any one of claims 1 to 3, wherein the humanized antibody or fragment thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence SYY (SEQ ID NO: 26), a heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), a light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

5. The humanized antibody of any one of claims 1 to 4, wherein the humanized antibody or fragment thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence SYYIH (SEQ ID NO: 15), a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), a light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 19), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

6. The humanized antibody of any one of claims 1 to 5, wherein the humanized antibody or fragment thereof comprises a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERFKG (SEQ ID NO: 27) or WIYPGNVNTKYNERFQG ​​(SEQ ID NO: 28).

7. The humanized antibody of any one of claims 1 to 5, wherein the humanized antibody or fragment thereof comprises a light chain CDR1 comprising the sequence KASQSVNNDVA (SEQ ID NO: 29) or RASQSVNNDVA (SEQ ID NO: 30).

8. The humanized antibody of any one of claims 1 to 7, wherein the humanized antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a set of four heavy chain (HC) framework (FR) sequences: (A) FR-H1 selected from the group consisting of SEQ ID NOs: 31, 32, and 33, (B) FR-H2 selected from the group consisting of SEQ ID NOs: 34, 35, and 36, (C) FR-H3 selected from the group consisting of SEQ ID NOs: 37, 38, 39, and 40, and (D) FR-H4 which is SEQ ID NO: 41, and the light chain variable region comprises a set of four light chain (LC) framework (FR) sequences: (A) FR-L1 selected from the group consisting of SEQ ID NOs: 42, 43, and 44, (B) FR-L2 selected from the group consisting of SEQ ID NOs: 45 and 46, (C) FR-L3 selected from the group consisting of SEQ ID NOs: 47, 48, and 49, and (D) FR-L4 which is SEQ ID NO:

50.

9. The humanized antibody of any one of claims 1 to 8, wherein the heavy chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5 and 6, and the light chain variable region of the humanized monoclonal antibody comprises a sequence selected from the group consisting of SEQ ID NOs: 8, 9, 10, 11 and 12.

10. The humanized antibody of any one of claims 1 to 9, wherein the heavy chain variable region of the humanized monoclonal antibody comprises SEQ ID NO: 5 and the light chain variable region of the humanized monoclonal antibody comprises SEQ ID NO:

12.

11. A conjugate comprising the humanized antibody of any one of claims 1 to 10.

12. 12. The conjugate of claim 11, comprising a cytotoxic moiety, a radioactive moiety, or a labeling tag.

13. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment of any one of claims 1 to 10, or a conjugate comprising said antibody, and a pharmaceutically acceptable excipient, carrier, or diluent.

14. 14. The pharmaceutical composition of claim 13 for use in the treatment of cancer.

15. An antibody-drug conjugate (ADC) comprising a humanized anti-Nectin-4 antibody or an antigen-binding portion thereof conjugated to a toxin, wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence SYY (SEQ ID NO: 26), a heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), a light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

16. The antibody-drug conjugate of claim 15, wherein the antibody or antigen-binding portion thereof comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence SYYIH (SEQ ID NO: 15), a heavy chain CDR2 comprising the sequence WIYPGNVNTKYNERF(K / Q)G (SEQ ID NO: 16), a heavy chain CDR3 comprising the sequence SNPYVMDY (SEQ ID NO: 17), a light chain CDR1 comprising the sequence (K / R)ASQSVNNDVA (SEQ ID NO: 18), a light chain CDR2 comprising the sequence YASNRFT (SEQ ID NO: 19), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

17. The antibody-drug conjugate of claim 15 or claim 16, wherein the antibody or fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the light chain comprising a variable region having an amino acid sequence at least about 90% identical to a sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:

12.

18. The antibody-drug conjugate of any one of claims 15 to 17, wherein the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 13, and the light chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO:

14.

19. The antibody-drug conjugate of any one of claims 15 to 17, wherein the heavy chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO: 5, and the light chain variable region of the humanized antibody comprises the amino acid sequence shown in SEQ ID NO:

12.

20. The antibody-drug conjugate of any one of claims 15 to 19, wherein the toxin is selected from the group consisting of a microtubule inhibitor, a DNA synthesis inhibitor, a topoisomerase inhibitor, and an RNA polymerase inhibitor.

21. 21. The antibody-drug conjugate of claim 20, wherein the toxin is selected from the group consisting of monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), saporin, DM4, DM1, exatecan, and SN-38.

22. The antibody-drug conjugate of any one of claims 15 to 21, wherein the antibody and the toxin are linked via a linker.

23. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 15 to 22 and a pharmaceutically acceptable excipient, carrier, or diluent.

24. 24. The pharmaceutical composition of claim 23 for use in treating cancer in an individual.

25. 25. The pharmaceutical composition of claim 24, wherein the cancer is selected from the group consisting of bladder cancer, liver cancer, lung cancer, colon cancer, glioblastoma, adrenal cancer, uterine cancer, testicular cancer, head and neck cancer, pancreatic cancer, and breast cancer.

26. 26. A method of treating cancer in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of the pharmaceutical composition of claim 13 or 23.

27. 27. The method of claim 26, wherein the method of treating cancer comprises administering or administering at least one additional anti-cancer therapy.

28. 28. The method of claim 27, wherein the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy, or immunotherapy.

29. 25. The method of claim 24, comprising administering an additional anti-cancer agent selected from the group consisting of an immunomodulatory agent, an activated lymphocyte cell, a kinase inhibitor, and a chemotherapeutic agent.

30. The humanized antibody or fragment thereof according to any one of claims 1 to 4, or the antibody-drug conjugate according to claim 11, wherein the humanized antibody comprises a set of six CDR sequences: a heavy chain CDR1 comprising the sequence GYTFTSYY (SEQ ID NO: 21), a heavy chain CDR2 comprising the sequence IYPGNVNT (SEQ ID NO: 22), a heavy chain CDR3 comprising the sequence ARSNPYVMDY (SEQ ID NO: 23), a light chain CDR1 comprising the sequence QSVNND (SEQ ID NO: 24), a light chain CDR2 comprising the amino acid sequence YAS (SEQ ID NO: 25), and a light chain CDR3 comprising the sequence QQAYRSPYT (SEQ ID NO: 20).

31. 11. The humanized antibody of any one of claims 1 to 10, comprising a heavy chain sequence shown in SEQ ID NO: 51 and a light chain sequence shown in SEQ ID NO: 52.

Images