Humanized antibody against nectin-2 and its drug conjugate

Abstract

The present disclosure provides humanized monoclonal antibodies that recognize human Nectin-2 with high affinity and specificity and inhibit its binding to CD 112R. These antibodies carry a cytotoxic payload and do not interact with Fc gamma receptors. The present disclosure further provides pharmaceutical compositions comprising the antibodies and methods of their use in cancer immunotherapy.

Description

[Technical field]

[0001] The present invention is in the field of immunotherapy and relates to anti-Nectin-2 antibodies and antibody-conjugates, as well as therapeutic and diagnostic compositions containing them, for treating diseases, particularly cancer. [Background technology]

[0002] Cancer immunotherapy is utilized to generate and enhance anti-tumor immune responses, for example, by treatment with antibodies specific for antigens on tumor cells or by 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] T cell-mediated immune responses involve multiple sequential steps that are regulated by the balance between costimulatory and co-inhibitory signals that control the magnitude of the immune response. Inhibitory signals, called immune checkpoints, are important for maintaining self-tolerance and limiting immune-mediated collateral tissue damage. These inhibitory signals affect the response of T cells and reshape the immune response.

[0004] Nectin-2, also called poliovirus receptor-related protein 2, poliovirus receptor-like 2, CD112 or PRR-2, is a single-pass transmembrane glycoprotein with two Ig-like C2-type domains and one Ig-like V-type domain. Nectin-2 is involved in mediating cell adhesion to extracellular matrix molecules and functions as one of the plasma membrane components of adherens junctions. It also functions as an entry receptor for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Mutations in this gene are associated with differences in the severity of multiple sclerosis. Importantly, nectin-2 may also function as a regulator of T cell signaling. It can be either a co-stimulator or co-inhibitor of T cell function depending on the receptors it binds to on its target cells: upon binding to CD226 (DNAM-1) it stimulates T cell proliferation and cytokine production, including that of IL-2 and IFNγ, but upon interacting with PVRIG (CD112R) and / or TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains) it inhibits T cell proliferation and activation. These opposing interactions are competitive.

[0005] Nectin-2 has been shown to be overexpressed in various tumors, including breast and ovarian cancer (Oshima et al. Molecular Cancer, 2013, 12:60). The presence of Nectin-2 on tumor cells results in poor prognosis and reduced T cell activity (Stamm et al. Oncogene (2018) 37:5269-5280).

[0006] US Patent Application No. 2017 / 0037133 discloses inhibitors (e.g., antibodies) against CD112 (Nectin-2, PVRL2), CD155 (PVR), Galectin-9, TIM-3 and / or TIGIT for use in the treatment of blood-borne cancers, in particular acute myeloid leukemia (AML).

[0007] International Patent Application No. WO 2020 / 144697 discloses a monoclonal antibody (mAb) that recognizes human nectin-2 and blocks its interaction with TIGIT and CD112R for use in the treatment of cancer.

[0008] Antibody-drug conjugates (ADCs) are promising tools for both direct tumor cell killing and consequent activation of bystander immune cells. These therapeutic entities consist of mAbs linked to cytotoxic drugs (payloads) and are designed, in principle, to broaden the therapeutic window of these drugs by specifically restricting their delivery to cells expressing the target antigen, thus reducing their systemic exposure and toxicity.

[0009] One example of a possible payload is auristatin, a microtubule-disrupting drug. It was derived from the marine shellless mollusc Dolabella auricularia, where it is called dolastatin. Various derivatives of auristatin, 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 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 indicates lower systemic toxicity than MMAE (park et al.Molecules 2019,24,2754).

[0010] There is an unmet need to provide humanized antibodies recognizing human Nectin-2 that are safe, potent and can be used alone or as targeting tools for diagnostic and therapeutic use in diseases in which Nectin-2 expression is involved, e.g., as ADCs in cancer. Summary of the Invention

[0011] The present invention provides a humanized antibody that specifically binds to Nectin-2. The humanized antibody of the present invention, selected from a larger collection of antibody clones, has improved properties compared to other anti-Nectin-2 antibodies. The present invention further provides, according to some embodiments, a conjugate comprising an antibody and a diagnostic or therapeutic agent. In some embodiments, the conjugate comprises a cytotoxic moiety that is targeted by the humanized antibody to tumor cells that display the Nectin-2 receptor on their surface.

[0012] A large collection of humanized antibodies was created by combining specific sets of complementarity determining region (CDR) sequences and human framework (FW) sequences and introducing specific mutations into these sequences to generate antibodies with altered variable regions and improved properties. Advantageously, the newly designed humanized variable regions described herein preserve residues important for maintaining the conformation and binding affinity of the antibody while lowering the incidence of potential T-cell epitopes, thus minimizing the risk of adverse immune responses against the antibody. The antibodies disclosed herein were designed based on factors including homology, T-cell epitopes, critical residues, and predicted structure. Unexpectedly, the humanized antibodies disclosed herein exhibit improved biostability compared to the parent chimeric antibodies.

[0013] The humanized antibody disclosed herein has been found to be highly suitable for use as targeted anti-cancer therapy using therapeutic toxins.It is disclosed herein that the anti-Nectin-2 monoclonal humanized antibody conjugated to a cytotoxic moiety exhibits potent killing of various tumor cell lines.Direct targeting of toxins using the antibodies described herein has the potential to increase the anti-tumor effect of these toxins and reduce their systemic toxicity, thus improving the survival of cancer patients.

[0014] Human Fc receptors (FcγR) activate Fc effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Some of the ADCs of the present invention contain one or more Fc mutations that significantly reduce their binding by FcγR expressed on immune cells, thereby increasing their safety and specificity. It is disclosed herein that ADCs comprising anti-Nectin-2 monoclonal humanized antibodies with specific Fc mutations and linked to toxins are highly effective and safe, and suitable for anti-cancer therapy.

[0015] The inventors of the present invention have shown for the first time that ADCs targeting Nectin-2 are highly useful for the treatment of cancer, particularly solid tumors.

[0016] According to a first aspect, the present invention provides a humanized antibody that specifically binds to human Nectin-2, or a fragment thereof comprising at least an antigen-binding site, comprising a heavy chain (HC) and a light chain (LC), 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:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; 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:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

[0017] 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 90% identical to SEQ ID NO:11, and the light chain comprises a variable region having an amino acid sequence at least about 90% 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 variable region comprises the sequence set forth in SEQ ID NO:11 and the light chain variable region comprises the sequence set forth in SEQ ID NO:12.

[0019] There are several methods known in the art for determining the CDR sequences of a given antibody molecule, but no standard, defined method. The 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 may include sequences identified by more than one method, i.e., for example, some CDR sequences may be determined using KABAT and some using IMGT. According to some embodiments, the CDR sequences of the mAb variable regions are determined using the IMGT method.

[0020] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences, with a heavy chain CDR1 comprising the sequence SYW, a heavy chain CDR2 comprising the sequence VYPGNSDS (SEQ ID NO: 8), a heavy chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO: 3), a light chain CDR1 comprising the sequence QNVGIN (SEQ ID NO: 10), a light chain CDR2 comprising the sequence SAS, and a light chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO: 6).

[0021] According to some embodiments, the humanized antibody or fragment thereof comprises a set of six CDR sequences, with a heavy chain CDR1 comprising the sequence SYWIH (SEQ ID NO:1), a heavy chain CDR2 comprising the sequence AVYPGNSDSNYNQKF(KA / QG) (SEQ ID NO:2), a heavy chain CDR3 comprising the sequence LVGTFDY (SEQ ID NO:3), a light chain CDR1 comprising the sequence (K / R)ASQNVGINV(V / A) (SEQ ID NO:4), a light chain CDR2 comprising the sequence SASYRYS (SEQ ID NO:5), and a light chain CDR3 comprising the sequence QQYNTNPFT (SEQ ID NO:6).

[0022] According to some embodiments, the heavy chain CDR2 comprises the sequence AVYPGNSDSNYNQKFKA (SEQ ID NO: 41) or AVYPGNSDSNYNQKFQG (SEQ ID NO: 42). According to certain embodiments, the light chain CDR1 comprises a sequence selected from the group consisting of KASQNVGINVV (SEQ ID NO: 43), KASQNVGINVA (SEQ ID NO: 44), RASQNVGINVV (SEQ ID NO: 45) and RASQNVGINVA (SEQ ID NO: 46).

[0023] According to some embodiments, the humanized antibody or fragment thereof comprises the set of six CDR sequences set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6. According to additional exemplary embodiments, the humanized antibody or fragment thereof comprises the set of six CDR sequences set forth in SEQ ID NOs: 7, 8, 9, 10, (SAS), and 6. According to additional embodiments, the humanized antibody or fragment thereof comprises the set of six CDR sequences set forth in SEQ ID NOs: 1, 41, 3, 43, 5, and 6.

[0024] According to some embodiments, the humanized antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1 to 3; and ii. A set of four heavy chain framework (FR) sequences, wherein FR-H1 is selected from the group consisting of SEQ ID NO:21, 25, and 27; FR-H2 is SEQ ID NO:22; FR-H3 is selected from the group consisting of SEQ ID NO:23, 26, 28, and 29; and FR-H4 is SEQ ID NO:24. Includes.

[0025] According to some embodiments, the humanized antibody, or antigen-binding fragment thereof, comprises a light chain variable region, the light chain variable region comprising: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4 to 6; and ii. A set of four light chain framework (FR) sequences, wherein FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33. Includes.

[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: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1 to 3; and ii. A set of four heavy chain framework sequences, wherein FR-H1 is selected from the group consisting of SEQ ID NO:21, 25 and 27; FR-H2 is SEQ ID NO:22; FR-H3 is selected from the group consisting of SEQ ID NO:23, 26, 28 and 29; and FR-H4 is SEQ ID NO:24; Including, The light chain variable region is i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4 to 6; and ii. A set of four light chain framework sequences, wherein FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33. Includes.

[0027] 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: i. a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 7 to 9; and ii. A set of four heavy chain framework sequences, wherein FR-H1 is selected from the group consisting of SEQ ID NO:21, 25 and 27; FR-H2 is SEQ ID NO:22; FR-H3 is selected from the group consisting of SEQ ID NO:23, 26, 28 and 29; and FR-H4 is SEQ ID NO:24; Including, The light chain variable region is i. a set of three CDR sequences comprising the sequences shown in SEQ ID NO:10, sequence SAS, and SEQ ID NO:6; and ii. A set of four light chain framework sequences, wherein FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33. Includes.

[0028] According to some embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and the light chain variable region comprises an amino acid sequence at least about 95% identical to a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In certain 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 NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and the light chain variable region comprises an amino acid sequence at least about 97% identical to a sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20. In certain embodiments, the heavy chain variable region of the humanized monoclonal antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[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 monoclonal antibody comprises a heavy chain constant region selected from IgG4, IgG1, 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.

[0031] According to some embodiments, the humanized monoclonal antibody comprises a kappa light chain constant region.

[0032] According to some embodiments, the humanized monoclonal antibody is an IgG1 having a heavy chain comprising an amino acid sequence at least about 90%, 95% or 98% identical to the sequence set forth in SEQ ID NO: 47. According to certain exemplary embodiments, the humanized monoclonal antibody is an IgG1 having a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 47.

[0033] According to some embodiments, the humanized monoclonal antibody has a kappa light chain comprising an amino acid sequence at least about 90%, 95%, or 98% identical to the sequence set forth in SEQ ID NO: 49. According to certain exemplary embodiments, the humanized monoclonal antibody has a kappa light chain comprising an amino acid sequence set forth in SEQ ID NO: 49.

[0034] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0035] 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.

[0036] According to some embodiments, the humanized antibody comprises an IgG1 Fc domain. According to certain embodiments, the IgG1 Fc domain is null for binding to Fcγ receptors. According to additional embodiments, the IgG1 Fc domain is null for binding to one or more Fcγ receptors. According to certain exemplary embodiments, the IgG1 Fc domain is null for binding to CD64, CD32a, CD32b, CD16a and / or CD16b.

[0037] According to some embodiments, the humanized antibody comprises a human IgG selected from the group consisting of: (i) a human IgG1 with the L235S mutation; (ii) a human IgG1 with the L235S and E272K mutations; (iii) a human IgG1 with the G237I mutation; (iv) a human IgG1 with the G237I and E272I mutations; (v) a human IgG1 with the G237I and V264R mutations; (vi) a human IgG1 with the V215A, E269R and K322A mutations; (vii) a human IgG1 with the L234A, L235A and P329G mutations; (viii) a human IgG4 with the S228P, L235P and V264R mutations; (ix) a human IgG2 with the P238H mutation; or (x) a human IgG2 with the P238H and V264R mutations. Each possibility represents a separate embodiment of the invention.

[0038] According to some embodiments, the humanized antibody comprises a heavy chain sequence selected from the group consisting of SEQ ID NOs: 61 to 70. Each possibility represents a separate embodiment of the present invention.

[0039] According to some embodiments, there is provided a conjugate comprising the above-described humanized antibody or fragment thereof.

[0040] The antibodies or fragments thereof according to the invention are, according to some embodiments, conjugated to a cytotoxic moiety, a radioactive moiety, or an affinity or labeling tag.

[0041] An antibody-drug conjugate (ADC) according to the invention comprises a humanized antibody as described herein, optionally a linker, and a toxin.

[0042] According to some embodiments, the humanized antibody or fragment thereof is conjugated to a toxin (payload) directly or via a linker.

[0043] According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors.

[0044] 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 monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

[0045] According to some embodiments, the toxin is saporin.

[0046] According to some embodiments, the toxin is a maytansine derivative. According to particular embodiments, the maytansine derivative is DM4 or DM1.

[0047] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0048] In some embodiments, the toxin is a DNA topoisomerase I (TOP1) inhibitor. In some embodiments, the DNA topoisomerase I (TOP1) inhibitor is exatecan or an exatecan derivative. In certain embodiments, the DNA topoisomerase I (TOP1) inhibitor is DXd.

[0049] According to some embodiments, the toxin is directly attached to the antibody. According to other embodiments, the antibody and the toxin are attached via a linker or spacer. According to some embodiments, the toxin is covalently attached to the humanized antibody directly or via a linker or spacer. Each possibility represents a separate embodiment of the present invention.

[0050] According to some embodiments, the linker is cleavable. According to other embodiments, the linker is not cleavable. 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 (sulfo-SPDB).

[0051] 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). Each possibility represents a separate embodiment of the present invention.

[0052] According to some embodiments, polynucleotide sequences are provided that encode the amino acid sequences of the heavy and light chain variable regions described above.

[0053] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 51-55, or a variant thereof having at least 80% sequence identity, with each possibility representing a separate embodiment of the present invention.

[0054] According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, wherein the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 56-60, or a variant thereof having at least 80% sequence identity, with each possibility representing a separate embodiment of the present invention.

[0055] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 51 to 55. According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, and the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs: 56 to 60.

[0056] According to some embodiments, the polynucleotide sequence encodes a humanized antibody heavy chain variable region, and the polynucleotide comprises the sequence set forth in SEQ ID NO: 51. According to some embodiments, the polynucleotide sequence encodes a humanized antibody light chain variable region, and the polynucleotide comprises the sequence set forth in SEQ ID NO: 56.

[0057] According to some embodiments, polynucleotide sequences encoding the amino acid sequences of the heavy and light chains of the above-mentioned humanized antibodies are provided.

[0058] According to some embodiments, the polynucleotide sequence encoding the humanized antibody heavy chain comprises the sequence set forth in SEQ ID NO: 48, or a variant thereof having at least 80% sequence identity. According to some embodiments, the polynucleotide sequence encoding the humanized antibody light chain comprises the sequence set forth in SEQ ID NO: 50, or a variant thereof having at least 80% sequence identity.

[0059] According to some embodiments, the DNA sequence encoding the amino acid chain of the humanized antibody described herein comprises a leader sequence. According to certain exemplary embodiments, the DNA sequence encodes the leader peptide sequence set forth in SEQ ID NO: 71.

[0060] In a further aspect, the 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.

[0061] Also described are cell lines comprising nucleic acids encoding the antibodies of the invention. The cell lines are for expressing the humanized antibodies or fragments thereof described herein. In certain embodiments, the cell lines are mammalian cell lines, such as Chinese Hamster Ovary (CHO) cell lines.

[0062] According to another aspect, the invention provides a pharmaceutical composition comprising the humanized antibody or antigen-binding fragment described herein, or a conjugate comprising the antibody and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0063] According to some embodiments, the pharmaceutical composition is for use in the treatment of cancer.

[0064] Any mode of administration may be used to deliver the compositions of the present invention to a subject in need thereof, including parenteral and enteral modes of administration.

[0065] 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.

[0066] 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 or conjugate thereof described herein.

[0067] According to some embodiments, the cancer is characterized by overexpression of Nectin-2.

[0068] In certain embodiments, the cancer comprises a solid tumor.

[0069] In certain embodiments, the cancer is selected from the group consisting of prostate cancer, colorectal cancer, liver cancer, ovarian cancer, endometrial cancer, gastric cancer, thyroid cancer, carcinoid tumors, 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.

[0070] In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colorectal adenocarcinoma or lung adenocarcinoma.

[0071] In other embodiments, the cancer is a hematological cancer. In 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.

[0072] According to some embodiments, the subject is a human.

[0073] According to some embodiments, the method of treating cancer comprises administering or performing at least one additional anti-cancer therapy. According to certain embodiments, the additional anti-cancer therapy is surgery, chemotherapy, radiation therapy or immunotherapy.

[0074] According to some embodiments, the method of treating cancer comprises administering a humanized antibody 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.

[0075] According to other embodiments, the additional immunomodulatory agent is an antibody, antibody fragment, or antibody conjugate that binds to an antigen other than human Nectin-2.

[0076] According to some embodiments, the additional immunomodulatory agent is an antibody against an immune checkpoint molecule. According to some embodiments, the additional immunomodulatory agent is an antibody against an immune checkpoint molecule selected from the group consisting of human programmed cell death protein 1 (PD-1), PD-L1 and PD-L2, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), lymphocyte activation gene 3 (LAG3), CD137, OX40 (also called CD134), killer cell immunoglobulin-like receptor (KIR), TIGIT, PVR, CTLA-4, NKG2A, GITR, and any other checkpoint molecule or combination thereof. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the additional immunomodulatory agent is an antibody against PD-1. According to some embodiments, the additional immunomodulatory agent is an antibody against CTLA-4.

[0077] According to some embodiments, the anti-cancer drug 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. Each possibility represents a separate embodiment of the present invention.

[0078] According to some embodiments, the methods of treating cancer include preventing or reducing the formation, growth or spread of metastases in a subject.

[0079] According to an additional aspect, the present invention provides an antibody-drug conjugate comprising a humanized antibody that specifically binds to human Nectin-2 and a toxin for use in the treatment of cancer.

[0080] Cancers and toxins are as described above.

[0081] According to some embodiments, the humanized antibody comprises a mutated Fc domain.

[0082] The present invention further provides, according to one aspect, a method for diagnosing or prognosing cancer in a subject, comprising determining the expression level of Nectin-2 in a biological sample of the subject using at least one humanized antibody, fragment or conjugate described herein.

[0083] The present invention further provides, according to another aspect, a method for determining or quantifying the expression of Nectin-2, comprising contacting a biological sample with an antibody or antibody fragment described herein and measuring the level of complex formation.

[0084] According to some embodiments, the method for detecting or quantifying the expression of Nectin-2 comprises: i. incubating the sample with an antibody specific for Nectin-2 or an antibody fragment thereof comprising at least the antigen-binding portion; ii. detecting bound Nectin-2 using a detectable probe. According to some embodiments, the method further comprises: iii. comparing the amount of (ii) with a standard curve obtained from reference samples containing known amounts of Nectin-2; and iv. calculating the amount of Nectin-2 in the sample from the standard curve.

[0085] 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.

[0086] Also provided is a kit for measuring Nectin-2 expression in a biological sample, comprising at least one antibody or antibody fragment described herein and a means for measuring Nectin-2 expression. In some embodiments, the kit further comprises instructional materials directing the use of the kit.

[0087] 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 description of the drawings]

[0088] [Figure 1] Figure 1A-1C show the correlation between Nectin-2 mRNA levels (high or low, as indicated) and survival probability in patients with low-grade glioma (Figure 1A), renal clear cell carcinoma (Figure 1B), and lung adenocarcinoma (Figure 1C). Data sets were obtained from TCGA sites and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). N indicates the number of patients included in the analysis. [Diagram 2] Figure 2 is a schematic diagram of receptors expressed on immune cells and their respective affinities for Nectin-2 (CD112) expressed by tumors or on antigen-presenting cells (APCs). TIGIT is a co-inhibitory receptor on many immune cells (e.g., T cells and NK cells); DNAM-1 (also called CD226) is an activating receptor on many immune cells (e.g., T cells), and CD112R (also called PVRIG) and TIGIT are co-inhibitory receptors on lymphoid immune cells (e.g., T cells and NK cells); Nectin-2 acts as an inhibitory ligand for immune cells mainly through its high affinity binding to CD112R (rectangle). [Diagram 3] Figure 3 is a graph showing the percentage of tumors positive for Nectin-2 expression. Data was obtained from proteinatlas.com using HPA012759 mAb (anti-Nectin-2; Sigma-Aldrich®). 17 / 20 indications show moderate to high membrane expression of Nectin-2. [Figure 4]Figures 4A-C show improved on-cell binding of humanized variants of parental (chimeric) anti-nectin-2 mAb. Nectin-2 expressing cells were analyzed by FACS analysis for binding of serially diluted mAb humanized variants. EC-50 values ​​were calculated for each variant and are reported relative to the EC-50 value of the chimeric Ab (H0K0), which was set as 1. Figure 4A - Fold change in EC-50 binding to 293T cells expressing human nectin-2. Figure 4B - Fold change in EC-50 binding to Vero cells (XP_007995342.1), derived from African green monkeys (AFG, Chlorocebus) and naturally expressing nectin-2. Figure 4C - Results of CD112R blocking by different humanized mAb variants as assessed by FACS analysis of CD112R-Fc binding to CHO(K)1 cells overexpressing human nectin-2 in the presence of anti-nectin-2 humanized clones at concentrations between 66 and 0.81 nM. Variants maintaining >70% blocking capacity at the lowest dose were considered superior. [Diagram 5] Figure 5A-C show the killing efficacy of humanized anti-nectin-2 ADC variants. The ADC is based on saporin (ZAP) and A549 target cells (lung adenocarcinoma) were confirmed to express nectin-2. Figure 5A shows the superior killing efficacy of the lead clone H3K3 compared to other humanized variants tested at two concentrations. Figure 5B shows comparable levels of ADC activity between humanized H3K3 and H4K2 variants. Figure 5C - Comparable levels of ADC activity between chimeric mAb (H0K0) and humanized H3K3 variants at three concentrations. H3K3 was also tested when grafted onto IgG2-P238H, which has an FcgR null Fc and did not affect the potency of this variant. All killing results are significant (p<0.001, two-way t-test) unless indicated by NS (not significant). [Figure 6]Figures 6A-6C demonstrate that humanized H3K3-FcgR null anti-Nectin-2 ADCs conjugated to various toxins result in potent killing of various solid tumor cell lines. The figures show significant killing activity of H3K3-FcgR null ADCs across different ADC concentrations and different cancer targets after 72 hours of incubation. Figure 6A shows killing of RKO cells (colorectal adenocarcinoma). Figure 6B shows killing of MDA-MB-231 cells (triple negative breast cancer). Figure 6C shows killing of A549 cells (lung adenocarcinoma). Target cell killing was significant (p<0.001) unless indicated by NS (not significant). All ADCs had toxic effects, but the magnitude varied considering the nature of the conjugated toxin and the target cells. Nevertheless, ADCs containing either MMAE or MMAF outperformed all others across all target cells. [Figure 7] Figure 7 demonstrates that humanized H3K3-FcgR null anti-nectin-2 ADCs are not internalized via interaction with high affinity FcgR-CD64. CHO-K1 cells, or CHO-K1 cells overexpressing human CD64 (hCD64) but not nectin-2, were incubated with two versions of H3K3-FcgR null ADCs (conjugated to either MMAE or DM4) or a control irrelevant ADC with either hIgG4(S228P) or hIgG1 Fc (both conjugated to DM4) at 12ug / ml for 72 hours. For CD64 positive cells, increased specific killing was seen when using either hIgG4 or hIgG1, but not when using the FcgR null Fc H3K3 ADC variant. Note that background non-specific killing was induced by DM4, regardless of expression of either nectin-2 or CD64 by the target cells. [Figure 8]Figure 8 demonstrates that humanized H3K3-FcgR null anti-Nectin-2 ADCs result in potent killing of hematopoietic cell lines. H3K3-FcgR null Abs with the indicated linker-payload combinations were incubated with HL-60 cells (AML model) for 72 hours at concentrations from 9-1 μg / ml. All linker-payload combinations except DM1 resulted in >90% killing at the high dose and significant killing at all doses tested (p<0.001). [Figure 9] Figure 9A-9B show the in vivo efficacy of humanized H3K3-FcgR null anti-Nectin-2 ADC against the hematological tumor cell line HL-60 sc implanted in nude female mice. Figure 9A compares the effect of treatment with different combinations of linker payloads (LPs). Two of the LP combinations had a significant effect: DM4 resulted in significant tumor growth inhibition (TGI, p<0.05), whereas complete tumor regression was seen for MMAE. The effect of the MMAE-based ADC, without other treatments, is shown in Figure 9B. [Figure 10] Figure 10 shows the in vivo efficacy of humanized H3K3-FcgR null anti-Nectin-2 ADC (1107-MC-VC-MMAE) against the solid (colorectal adenocarcinoma) tumor cell line RKO. Empty vehicle was used as a control. [Figure 11] Figure 11 shows in vitro killing of MDA-MB-231 (triple-negative breast cancer or TNBC) cells by humanized H3K3-FcgR null anti-Nectin-2 mAb (NTX1107) linked to Dxd compared to Trodelvy (sacituzumab govitecan), an approved ADC drug for TNBC. Both ADCs use the same class of cytotoxic payload (i.e., topoisomerase I (TOP1) inhibitors). MDA-MB-231 cells were incubated in the presence of these ADCs at the indicated concentrations. Killing was assessed after 120 hours. Potent killing of target cells was induced by both ADCs, with a significant advantage of Trodelvy at both concentrations (*** p<0.0005). [Figure 12]Figure 12 shows the in vivo efficacy of humanized H3K3-FcgR null anti-Nectin-2 ADC (NTX1107) against MDA-MB-231 sc tumor model in nude female mice compared to Trodelvy. NTX1107-Dxd was able to significantly inhibit tumor growth and resulted in tumor stasis, whereas Trodelvy had no effect on tumor growth with the same treatment regimen. [Figure 13] Figure 13 compares the effect of in vivo treatment with H3K3-FcgR null-Dxd (NTX1107-Dxd) to the effect of H3K3-FcgR null MMAE (NTX1107-MMAE) on MDA-MB-231 sc tumor model in nude female mice. Complete tumor regression was seen for H NTX1107-MMAE, with 7 / 7 mice having no measurable tumors at the end of the study. Tumor regression was seen for NTX1107-Dxd, with 4 / 7 mice having no measurable tumors at the end of the study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0089] The present invention provides a humanized monoclonal antibody that recognizes Nectin-2. Advantageously, the antibody of the present invention is almost completely humanized, thus avoiding the risk of adverse immune responses to the antibody, and therefore likely to be safe for use in humans. The antibody of the present invention is characterized by having unique CDR sequences and novel humanized framework sequences and designs.

[0090] The present invention further provides, in some embodiments, antibody-drug conjugates, or ADCs, comprising the humanized antibodies described herein, useful for the treatment of cancer.

[0091] In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, one 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. Moreover, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0092] As used herein, the term "about" refers to an amount close to the recited amount by no more than 10%.

[0093] The term "nectin-2" or "nectin cell adhesion molecule 2" as used herein refers to a human plasma membrane glycoprotein, also known as CD112 and PVRL2. Nectin-2 protein is a single-pass type I membrane glycoprotein with two Ig-like C2-type domains and one Ig-like V-type domain. This protein is one of the plasma membrane components of adherens junctions. It also functions as an entry point for certain mutant strains of herpes simplex virus and pseudorabies virus and is involved in the cell-to-cell spread of these viruses. Exemplary nectin-2 according to the present invention are described in SwissPort, UniPort and GenBank symbols or accession numbers: Gene ID: 5819, Q92692, I68093, NP_001036189.1, NP_002847.1, and #Q92692.

[0094] According to one aspect, the present invention provides a humanized antibody that specifically binds to Nectin-2, or a fragment thereof comprising at least an antigen-binding site, comprising 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:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; 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:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

[0095] According to another aspect, the present invention provides a humanized antibody or a fragment thereof comprising at least an antigen-binding site that specifically binds to Nectin-2, wherein the antibody or fragment thereof comprises a heavy chain variable region and a light chain variable region, (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1 to 3; and (ii) a set of four heavy chain framework sequences, wherein FR-H1 (Framework Heavy Chain Number 1) is selected from the group consisting of SEQ ID NO:21, 25 and 27; FR-H2 is SEQ ID NO:22; FR-H3 is selected from the group consisting of SEQ ID NO:23, 26, 28 and 29; and FR-H4 is SEQ ID NO:24; Including, The light chain variable region is (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4 to 6; and (ii) a set of four light chain framework sequences, wherein FR-L1 (framework light chain number 1) is selected from the group consisting of SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO: 33. The present invention provides a humanized antibody or fragment thereof comprising:

[0096] The framework is a non-CDR sequence of the chain variable region. FR-H1 is the sequence before CDR1 in the heavy variable chain, FR-H2 is the sequence between CDR1 and CDR2, FR-H3 is the sequence between CDR2 and CDR3, and FR-H4 is the sequence after CDR3. FR-L1 is the sequence before CDR1 in the light variable chain, FR-L2 is the sequence between CDR1 and CDR2, FR-L3 is the sequence between CDR2 and CDR3, and FR-L4 is the sequence after CDR3.

[0097] According to an additional aspect, the present invention provides a humanized antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: i. a set of three CDR sequences comprising the sequences SYW, SEQ ID NO:8, and SEQ ID NO:3; and ii. A set of four heavy chain framework sequences, wherein FR-H1 is selected from the group consisting of SEQ ID NO:21, 25 and 27; FR-H2 is SEQ ID NO:22; FR-H3 is selected from the group consisting of SEQ ID NO:23, 26, 28 and 29; and FR-H4 is SEQ ID NO:24; Including, The light chain variable region is i. a set of three CDR sequences comprising the sequences shown in SEQ ID NO:10, SAS, and SEQ ID NO:6; and ii. A set of four light chain framework sequences, wherein FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37, and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38, and 40; and FR-L4 is SEQ ID NO: 33. Includes.

[0098] According to some embodiments, the humanized antibody or fragment thereof comprises a heavy chain variable region comprising a set of three CDR sequences comprising the sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; and a light chain variable region comprising a set of three CDR sequences comprising the sequences of SEQ ID NO:10, SAS and SEQ ID NO:6.

[0099] Among the antibodies provided are monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. Antibodies include antibody conjugates and molecules that contain antibodies, such as chimeric molecules. Thus, antibodies include full length, as well as any specific binding portion thereof, including fragments and portions thereof that retain their binding specificity, such as those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE, and IgM); 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, any individual antibodies contained within a monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in minor amounts. A polyclonal antibody is a preparation containing different antibodies of various sequences, generally directed against two or more different determinants (epitopes). A monoclonal antibody may contain a human IgG1 constant region. A monoclonal antibody may contain a human IgG4 constant region.

[0100] 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 encompasses engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecifics, such as bispecific antibodies, diabodies, triabodies and tetrabodies, tandem di-scFv, 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 may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0101] CDR identification or determination from a given heavy or light chain variable sequence is typically performed using one of a small number of methods known in the art. For example, such 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).

[0102] When the term "CDR having a sequence" or similar terminology is used, it includes the option where the CDR comprises the sequence specified, and also the option where the CDR consists of the sequence specified.

[0103] The antigen specificity of an antibody is based on the unique CDR sequences of both the light and heavy chains which together form the hypervariable region (HVR), i.e., the antigen-binding site.

[0104] 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 that contains the variable heavy chain region and / or the variable light chain region, such as an scFv.

[0105] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all framework region (FR) amino acid residues are derived from a human FR. 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 replaced with the 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.

[0106] The amino acid residues in the Fc domain can be substituted to be null, meaning that the Fc domain does not bind to the Fc receptor or can bind with such low affinity and / or avidity that it does not cause 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 that the Fc domain can be null for binding to can be, 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 the binding of the Fc domain to the Fc receptor.

[0107] According to some embodiments, the humanized antibody has a mutated Fc domain that prevents FcγR-mediated internalization.

[0108] 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.

[0109] As used herein, "Fc null" refers to a domain that has weak or no binding to one or more of the Fcγ receptors.

[0110] Antibody-drug conjugates According to one aspect, the present invention provides a conjugate comprising a humanized antibody as described herein fused to a toxin.

[0111] According to some embodiments, the conjugate comprises an antibody or fragment thereof comprising 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:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16; 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:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20.

[0112] According to some embodiments, the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors, and RNA polymerase inhibitors, each possibility representing a separate embodiment of the present invention.

[0113] 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 monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

[0114] According to some embodiments, the toxin is a saponin.

[0115] According to some embodiments, the toxin is a maytansine derivative. According to particular embodiments, the maytansine derivative is DM4 or DM1.

[0116] According to some embodiments, the toxin is a quinoline alkaloid. According to certain embodiments, the quinoline alkaloid is SN-38.

[0117] According to additional 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, daunorubicin, 17-AAG, agrochelin A, doxorubicin, methotrexate, colchicine, cordycepin, hygrolysin, herboxidiene, ferulenol, curbulin, englerin A, taltobulin, triptolide, cryptophycin, and nemorubicin. Each possibility represents a separate embodiment of the present invention.

[0118] According to some embodiments, the toxin is a DNA topoisomerase I (TOP1) inhibitor. According to certain embodiments, the DNA topoisomerase I (TOP1) inhibitor is DXd.

[0119] Toxin names are used herein as known in the art. Non-limiting examples of suitable payloads include: -DM1-N 2 '-Deacetyl-N 2 '-(3-Mercapto-1-oxopropyl)-maytansine-tubulin inhibitor. -DM4-N 2 '-Deacetyl-N 2 '-(4-Mercapto-4-methyl-1-oxopentyl)-ravtansine-tubulin inhibitor. -SN-38 - A potent DNA topoisomerase I inhibitor, a member of the pyranoindolizinoquinoline class, which is (4S)-pyrano[3',4':6,7]indolizino[1,2-b]quinoline-3,14-dione, bearing two additional ethyl substituents at positions 4 and 11, and two additional hydroxy substituents at positions 4 and 9. -DXd-exatecan derivative, a potent DNA topoisomerase I inhibitor (e.g., MCE® catalog number: HY-13631D). -MMAF-Monomethylauristatin F - a tubulin inhibitor having the formula C39H65N5O8. -MMEA-Monomethylauristatin E - a tubulin inhibitor having the formula C39H67N5O7.

[0120] In some embodiments, the antibody is directly linked to the toxin. In other embodiments, the antibody and the toxin are linked via a linker. In some embodiments, the humanized antibody described herein is covalently linked to the toxin.

[0121] According to some embodiments, the linker is cleavable. According to additional embodiments, the linker is not cleavable.

[0122] 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.

[0123] According to another aspect, the invention provides a pharmaceutical composition comprising the humanized antibody or antigen-binding fragment described herein, or a conjugate comprising the antibody and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0124] According to another embodiment, the pharmaceutical composition according to the present invention is for use in the treatment of cancers characterized by overexpression of Nectin-2. Cancer types associated with Nectin-2 overexpression can be identified using known databases, such as the Cancer Genome Atlas (TCGA). According to a particular embodiment, the 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, READ), pancreatic ductal adenocarcinoma (PAAD), pheochromocytoma and paraganglioma (PCPG), papillary renal carcinoma (KIRP), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), prostate adenocarcinoma (PRAD), uterine endometrial carcinoma (UCEC), uterine endometrial carcinoma (U ... Selected from the group consisting of cervical cancer (CESC), cutaneous melanoma (SKCM), mesothelioma (MESO), urothelial bladder cancer (BLCA), clear cell renal 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.

[0125] As used herein, the terms "individual," "patient," or "subject" refer to an individual who has been diagnosed with, is suspected of suffering from, or is at risk of developing at least one disease that 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.

[0126] As used herein, the term "effective amount" refers to an amount of a therapeutic agent that causes a biological effect when administered to a mammal. Biological effects include, but are not limited to, inhibiting or blocking receptor-ligand interactions (e.g., PVR-TIGIT, PD-1-PD-L1 / PD-L-2), inhibiting signal transduction pathways, reducing tumor growth, reducing tumor metastasis, or prolonging the survival of tumor-bearing animals. A "therapeutic amount" is a drug combination 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 be afflicted with, suspected of, or afflicted with a tumor.

[0127] As used herein, the term "combination" or "combination treatment" can refer to either simultaneous administration of the combined products or sequential administration of the combined products. As described herein, when combination refers to sequential administration of the products, the products can be administered in any temporal order.

[0128] As used herein, "checkpoint inhibitors" refers to drugs that inhibit biological molecules produced by an organism that negatively regulate the anti-tumor / cancer activity of T cells in the organism ("checkpoint molecules"). Checkpoint molecules include, but are not limited to, PD-1, PD-L-1, PD-L-2, CTLA4, TIM-3, LAG-3, VISTA, SIGLEC7, PVR, TIGIT, IDO, KIR, A2AR, B7-H3, B7H4, CEACAM1, and CD112R.

[0129] The molecules of the present invention as active ingredients are dissolved, dispersed or mixed in excipients that are pharma- ceutically acceptable and compatible with the active ingredients, as is well known.Suitable excipients are, for example, water, saline, phosphate buffered saline (PBS), dextrose, glycerol, ethanol, and the like, 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.

[0130] 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 which the disorder is to be prevented.

[0131] The terms "cancer" and "tumor" refer to a physiological condition in a mammal that is characterized by deregulated cell proliferation. Cancer is a class of diseases in which a group of cells exhibits uncontrolled or unwanted proliferation. Cancer cells can also spread to other locations, which can result in the formation of metastases. The spread of cancer cells in the body can occur, for example, via lymph or blood. Uncontrolled proliferation, invasion, and metastasis formation are also referred to as malignant properties of cancer. These malignant properties distinguish cancer from benign tumors, which typically do not invade or metastasize.

[0132] 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.

[0133] According to some embodiments, the anti-cancer agent is selected from the group consisting of antimetabolites, antimitotics, taxanes, topoisomerase inhibitors, topoisomerase II inhibitors, asparaginases, alkylating agents, antitumor antibiotics, and combinations thereof, with each possibility representing a separate embodiment of the present invention.

[0134] According to some embodiments, the antimetabolite is selected from the group consisting of cytarabine, fludarabine, fluorouracil, mercaptopurine, methotrexate, thioguanine, gemcitabine, and hydroxyurea. According to some embodiments, the mitotic inhibitor is selected from the group consisting of vincristine, vinblastine, and vinorelbine. According to some embodiments, the topoisomerase inhibitor is selected from the group consisting of topotecan and irinotecan. According to 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. According to 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.

[0135] According to another aspect, the present invention provides a method of treating cancer in an individual suffering from cancer, comprising administering to the individual a therapeutically effective amount of a humanized antibody or antigen-binding fragment thereof or a pharmaceutical composition and an inhibitor of PD-1, PD-L1, CTLA-4 or CD112R signaling. In certain embodiments, the cancer comprises a solid tumor. In certain embodiments, the cancer is selected from the group consisting of lung cancer, colon cancer, glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, cervical cancer, or prostate cancer. In certain embodiments, the inhibitor of PD-1 signaling is an antibody or fragment thereof that binds to PD-1. In certain embodiments, the antibody or fragment thereof that binds to PD-1 is pembrolizumab, nivolumab, AMP-514, tislelizumab, spartalizumab, or a PD-1 binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling is an antibody that specifically binds to PD-L-1 or PD-L-2. In certain embodiments, the antibody that specifically binds PD-L1 or PD-L2 comprises durvalumab, atezolizumab, avelumab, BMS-936559, or FAZ053, or a PD-L1- or PD-L2-binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling comprises an Fc fusion protein that binds PD-1, PD-L1, or PD-L2. In certain embodiments, the Fc fusion protein comprises AMP-224, or a PD-1-binding fragment thereof. In certain embodiments, the inhibitor of PD-1 signaling comprises a small molecule inhibitor of PD-1, PD-L1, or PD-L2.In certain embodiments, the small molecule inhibitor of PD-1, PD-L1, or PD-L2 signaling is selected from the group consisting of N-{2-[({2-methoxy-6-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]pyridin-3-yl}methyl)amino]ethyl}acetamide (BMS 202); (2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)-5-methylbenzyl)-D-serine hydrochloride; (2R,4R)-1-(5-chloro-2-((3-cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)oxy)benzyl) -4-Hydroxypyrrolidine-2-carboxylic acid;3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenylindole;3-(4,6-dichloro-1,3,5-triazin-2-yl)-1-phenyl-1h-indole;L-α-glutamine, N2,N6-bis(L-seryl-L-asparaginyl-L-threonyl-L-seryl-L-α-glutamyl-L-seryl-L-phenylalanyl)-L-lysyl -L-phenylalanyl-L-arginyl-L-valyl-L-threonyl-L-glutaminyl-L-leucyl-L-alanyl-L-prolyl-L-lysyl-L-alanyl-L-glutaminyl-L-isoleucyl-L-lysyl;(2S)-1-[[2,6-dimethoxy-4-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinecarboxylic acid;Glycinamide, N-(2-mercaptopropyl) or one or more of the following derivatives or analogs: N-phenylalanyl, N-methyl-L-alanyl-L-asparaginyl-L-prolyl-L-histidyl-L-leucyl-N-methylglycyl-L-tryptophyl-L-seryl-L-tryptophyl-N-methyl-L-norleucyl-N-methyl-L-norleucyl-L-arginyl-L-cysteinyl-, cyclic (1→14)-thioether; or a derivative or analog thereof.

[0136] Also described herein is a method of manufacturing a composition for treating cancer in an individual suffering from cancer, comprising mixing a humanized antibody or antigen-binding fragment thereof with a pharma- ceutically 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 colon cancer, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, glioblastoma, cervical cancer, prostate cancer, and lung cancer.

[0137] Also described herein is a method of producing a humanized antibody or antigen-binding fragment thereof, comprising incubating a cell line described herein in a cell culture medium under conditions sufficient to allow expression and secretion of the humanized antibody or antigen-binding fragment thereof.

[0138] According to some specific embodiments, the additional anticancer agent is selected from the group consisting of bevacizumab, carboplatin, cyclophosphamide, doxorubicin hydrochloride, gemcitabine hydrochloride, topotecan hydrochloride, thiotepa, and combinations thereof, each possibility representing a separate embodiment of the present invention.

[0139] example Reference is now made to the following examples which, together with the above descriptions, illustrate the invention in a non-limiting manner.

[0140] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological, immunological, and recombinant DNA techniques. Many such techniques are well known in the art. Other general references referring to well-known procedures are provided throughout this document for the convenience of the reader.

[0141] Example 1. High expression of Nectin-2 mRNA correlates with poor survival probability in various cancer patients. The correlation between Nectin-2 mRNA expression level and survival probability was examined with data from the TCGA site and analyzed using the oncolnc.org site (https: / / doi.org / 10.7717 / peerj-cs.67). Nectin-2 mRNA expression level was the basis for dividing patients into two subgroups of low and high expression, as indicated by the arrows in Figure 1 for patients with low-grade glioma (Figure 1A; p=5.22E-5), renal clear cell carcinoma (Figure 1B; p=0.00037) and lung adenocarcinoma (Figure 1C; p=0.0319).

[0142] Example 2. Nectin-2 binds to and influences immune cells via specific receptors. The schematic diagram in Figure 2 demonstrates the receptors expressed on immune cells and their respective affinities for Nectin-2 expressed by tumors or on antigen-presenting cells (APCs). TIGIT is associated with a co-inhibitory receptor on immune cells such as T cells and NK cells; DNAM-1 (also called CD226) is associated with an activating receptor on immune cells (e.g., T cells), and CD112R (also called PVRIG) is associated with a co-inhibitory receptor on lymphoid immune cells (e.g., T cells and NK cells); Nectin-2 (CD112) serves as an inhibitory ligand for immune cells, primarily through its binding to CD112R. According to the present invention, humanized anti-Nectin-2 mAbs can block Nectin-2 interaction with its high affinity receptor CD112R and target cancer cells for specific toxin delivery and killing.

[0143] Example 3. Nectin-2 is expressed in the majority of solid tumors The database Proteinatlas.com was searched for all aliases of Nectin-2 (NECTIN2, CD112, HVEB, PRR2, PVRL2, PVRR2). Data were found using three different mAbs under pathology experiments. HPA0127569 mAb has the highest validation score (enhanced) since it was validated by orthogonal methods. Therefore, expression data across different tumors were selected only for this clone and are shown in Figure 3. The graph shows the percentage of tumors positive for Nectin-2 expression. In 17 / 20 indications, a moderate to high level of membrane expression of Nectin-2 is seen.

[0144] Example 4. Improved properties of humanized anti-nectin-2 mAb Mouse anti-human nectin-2 clone 2.11, disclosed in patent application publication number WO2020144697, was selected as the lead mAb for humanization. Based on structural analysis, a large preliminary set of sequence segments was identified that were used to generate the humanized variants. These segments were selected and analyzed using iTope™ technology for in silico analysis of peptide binding to human MHC class II alleles (Perry et al., 2008) and using TCED™ of known antibody sequence-associated T cell epitopes (Bryson et al., 2010). Sequence segments that were identified as significant non-human germline binders to human MHC class II or scored significant hits to TCED™ were discarded. This reduced the set of segments, and these combinations were further analyzed as described above to ensure that the junctions between the segments did not contain potential T cell epitopes. The selected sequence segments were assembled into complete V-region sequences lacking significant T cell epitopes. Five heavy chain (VH1-VH5) and five light chain (VK1-VK5) sequences were then selected. The data in Table 1 show the readout of the iTope™ algorithm. To reduce the possibility of anti-drug antibody (ADA) generation, the sequences of the mAbs were analyzed for potential binding of MHCII. The main risk is associated with high affinity binding peptides, which are separated from the medium affinity binding peptides. [Table 1]

[0145] The parent heavy chain (VH0) has 8 predicted high affinity motifs and the parent light chain (Vk0) has 4 such motifs. Following the humanization process, the number of predicted high affinity MHCII epitopes for the heavy chain was reduced to 3 (VH2, VH3) or even 1 (VH4, VH5) and for the light chain the predicted high affinity MHCII motifs were reduced to 2 (VK3-5).

[0146] Thus, the humanization process eliminated most of the predicted high affinity MHCII motifs that may induce immunogenicity to the mAb.

[0147] The stability of the humanized variants was evaluated by thermal gradient stability experiments, which is a well-established method 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 evaluating modifications of the structure and formulation buffer. A widely used measure of the thermal structural stability of a protein is the temperature at which the protein unfolds from the native state to the denatured state.

[0148] 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. An increase in Tm is a desirable feature in Ab lead selection, as it predicts more stable Abs.

[0149] Purified lead humanized antibody variants were diluted in duplicate to a final test concentration of 0.5 mg / ml in PBS to which Sypro Orange (160x stock solution) was added to a final concentration of 20x solution. 9 μL of each sample mixture was loaded in duplicate into UNi microcuvettes. Samples were subjected to a thermal ramp from 15 to 95 °C with a ramp rate of 0.3 °C / min and excitation at 473 nm. Full emission spectra were collected from 250-720 nm and the area under the curve from 510-680 nm was used to calculate the inflection points (Tonset and Tm) of the transition curves. As seen in Table 2, for all antibodies tested, Tm1 and Tonset are higher for the humanized variants compared to the chimeric antibodies (VH0 / Vκ0). [Table 2]

[0150] Example 5. Improved binding to nectin-2 and blocking of CD112R binding by humanized anti-nectin-2 mAb Humanized anti-nectin-2 mAbs were evaluated for binding to human nectin-2 expressed by 293T cells (protein id: Q92692) and Chlorocebus (African green monkey, AFG) Vero cells and EC50 values ​​were established. AFG expresses the nectin-2 protein (XP_007995342.1) with 97% similarity to human nectin-2. Figure 4A and Figure 4B show the fold change in EC50 values ​​for human and AFG cells expressing nectin-2, respectively. Both cell lines were incubated at 0.5x10 per well. 5 Cells were seeded with 100 μg / ml of humanized mAb. Chimeric mAb (H0K0) and humanized mAb were added at concentrations ranging from 20 to 0.001 nM. For detection, anti-human APC Ab was used at 1:200 dilution (Jackson Immunoresearch AB_2340526). Cells were analyzed by FACS and EC50 was calculated and compared to chimeric Ab (fold improvement). This analysis revealed improved binding of the humanized variant compared to the chimeric mAb, and a similar degree of cross-reactivity to monkey nectin-2 (Figure 4A vs. 4B). To assess the blocking capacity of the humanized Ab, human nectin-2 was overexpressed in Chinese Hamster Ovary (CHO) cells. Figure 4C shows the results of 10 μg / ml of humanized Ab after 10 min of incubation. 5 Shown is data generated when 1000 CHO-hNectin-2 / well were incubated with 30 nM human CD112R-mIgG2a in the presence of the indicated humanized Abs, at concentrations ranging from 66 to 0.81 nM. Bound CD112R was detected by using αmIgG2a-647 (Jackson Immunoresearch catalog 115-607-186) at a 1:200 dilution, followed by FACS analysis. As can be seen, all humanized variants maintained full blocking capacity.

[0151] Example 6. Anti-hNectin-2 humanized variant H3K3 can act as an ADC driver in an Fc-independent manner To evaluate the ability of the mAbs to act as ADCs, streptavidin-saporin (ZAP), IT-27-250 (ATS) was used. The mAbs shown in Figure 5 were biotinylated using the biotinylation kit Ab207195 (Abcam) at a 1:1 ratio. Figure 5A shows the results of an initial screen involving the top humanized clones and A549 (lung adenocarcinoma) cells as targets. 2x10 per well 3 Cells were seeded and allowed to adhere for 4-6 hours. ADC was added and cells were incubated with ADC. After 72 hours, the assay was harvested and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay- (Promega G9242) according to standard protocols. Only clone H3K3 showed strong killing. Clone H3K3 was then directly compared to clone H4K2 (Figure 5B). Both clones showed strong killing and H3K3 was selected for future analysis due to its superior developmental properties. FcgR of chimeric parent clones and clone H3K3 (IgG1) ヌル Variant (IgG2-P238H)(FcgR ヌル In this case, g represents gamma or γ) were tested under the above conditions and the results are shown in Figure 5C. Significant killing (p<0.001) by both chimeric and humanized H3K3 variants was seen at all concentrations of these ADCs, with FcgR ヌル The variants produced identical levels of target cell killing compared to the hIgG1 clone.

[0152] Example 7. Anti-hNectin-2 H3K3-FcgR ヌル The ADCs provide potent killing of solid tumor cell lines in vitro. As shown in Table 3, additional H3K3-FcgRs were identified by mutating key residues in the hinge region of human IgG1. ヌル mAbs were generated. [Table 3]

[0153] The presented linker-payload combinations were selected according to the desired release mechanism and produced following standard protocols by Abzena LTD. Briefly, mAbs were reduced and incubated with excess linker-payload to obtain the desired drug-antibody ratio (DAR) of 4 for all linker-payloads except SN-38, which has a target DAR of 8. The final products were then purified and the DAR was established by LC / MS methodology. The potency of the different linker-payload combinations was evaluated in vitro using selected tumor cell lines representing various solid tumors. The indicated target cells were incubated at 2x10 per well. 3 Cells were seeded with IgG and allowed to adhere for 4-6 hours. ADC was added at concentrations of 4-0.16 μg / ml using 5-fold dilutions and cells were incubated with ADC. After 72 hours, the assay was harvested and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay- (Promega G9242) according to standard protocols. Potent killing of RKO cells (colorectal adenocarcinoma), MDA-MB-231 cells (triple-negative breast cancer) and A549 cells (lung adenocarcinoma) is shown in Figure 6A-6C, respectively. Target cell killing was significant (p<0.001) by two-tailed Student's t-test unless indicated by NS (not significant, p>0.05).

[0154] Example 8. Selected FcgR ヌル The mutation prevents FcgR-mediated ADC killing To assess the extent of non-target specific killing by the humanized anti-Nectin-2 ADC, which may be due to Ab Fc binding to its FcγR and contribute to the non-specific effects of the ADC, CHO cells overexpressing the high affinity Fc receptor hCD64 (CHO-hCD64) were generated. These cells do not express human Nectin-2 and therefore target specific killing is not expected. Parental CHO cells and CHO-hCD64 were cultured at 2x10 per well. 3Cells were seeded and allowed to adhere for 4-6 hours. Different Fc variant ADCs were added at 12 μg / ml. After 72 hours of incubation, the assays were harvested and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay- (Promega G9242).

[0155] As can be seen in Figure 7, both IgG4-S228P and the hIgG1 positive control resulted in a significantly higher degree of killing of CHO-hCD64 cells compared to parental CHO cells, as expected. On the other hand, the IgG1 G237I substitution resulted in a similar low level of killing of both parental and hCD64 overexpressing cells (non-specific killing). These results support the conclusion that FcgR ヌル Ensure that the variant is not bound and internalized by the high affinity FcgR CD64.

[0156] Example 9. Anti-hNectin-2 humanized H3K3-FcgR ヌル The ADC results in potent killing of the hematological tumor cell line HL-60 in vitro HL-60 cells (AML model) at 2x10 per well 3 Cells were seeded and the ADC was added at concentrations of 9-1 μg / ml using 3-fold dilutions. Cells were incubated with the ADC for 72 hours. The assay was then harvested and tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay- (Promega G9242) as described above. As seen in Figure 8, with the exception of the DM1 payload (shown on the graph), all other compounds resulted in significant killing of targets at all concentrations tested, with all exceeding 90% at the highest concentration (>60 nM). Based on these results, the SMCC-DM1 linker payload was excluded from future experiments.

[0157] Example 10. Anti-hNectin-2 H3K3-FcgR ヌル -MC-VC-PAB-MMAE ADC leads to tumor regression in aggressive AML models Nude female mice (n=25) were treated with 10x10 6 HL-60 cells were injected sc. The tumors were 205 mm 3 Once the mean volume of 100 μg / kg was reached, mice were randomized into 5 groups (n=5 / group) and received PBS (vehicle), H3K3-FcgR ヌル -MMAE, H3K3-FcgR ヌル -MMAF, H3K3-FcgR ヌル -DM4 or H3K3-FcgR ヌル -SN-38 in a blinded fashion. All treatments were at 5 mg / kg and given every 4 days for 4 consecutive doses. As seen in Figure 9A, on day 23 after randomization, H3K3-FcgR ヌル -MMAF and SN-38 variants did not attenuate tumor growth. ヌル -DM4 induced a significant tumor growth inhibition (TGI) of 49%, whereas H3K3-FcgR ヌル -MMAE resulted in significant tumor regression. ヌル - Mean effect of MMAE is shown.

[0158] Example 11. Anti-hNectin-2 H3K3-FcgR ヌル -MC-VC-PAB-MMAE ADC induces tumor regression in an invasive colon adenocarcinoma model Nude female mice (n=5 / group) were treated with 5x10 6 RKO cells were injected SC. Tumors were 160 mm 3 Once the mean volume of 100 μg / kg was reached, mice were randomized into two groups and received either PBS (vehicle) or H3K3-FcgR. ヌル Patients were treated in a blinded fashion with either 100 mg / kg IV injection of either 100 mg / kg MMAE (1107-MC-VC-MMAE) or 100 mg / kg MMAE (1107-MC-VC-MMAE) at 5 mg / kg for four consecutive doses every four days. ヌル -MMAE resulted in significant tumor regression as seen in Figure 10, demonstrating >80% TGI compared to the vehicle-treated group.

[0159] Example 12. Anti-hNectin-2 H3K3-FcgR ヌル The mAb has similar in vitro killing activity compared to Trodelvy For the in vitro killing assay (Figure 11), MDA-MB-231 target cells were cultured at 2x10 per well. 3 Cells were seeded and allowed to adhere for 4–6 h. Next, H3K3-FcgR linked to Dxd was ヌル Anti-Nectin-2 mAb (NTX1107) and Trodelvy (sacituzumab govitecan), an approved ADC drug for TNBC, were added at concentrations of 12 μg / ml (dark grey bars) and 3 μg / ml (light grey bars). Of note, both ADCs use the same class of cytotoxic payload, i.e., topoisomerase I (TOP1) inhibitors. Cells were incubated with the ADCs for 120 h, after which tumor cell killing was assessed using the CellTiter-Glo® 2.0 Cell Viability Assay- (Promega G9242) according to standard protocols. Potent killing of MDA-MB-231 cells was induced by both ADCs, with a significant advantage of Trodelvy at both concentrations (*** p<0.0005).

[0160] Example 13. Anti-hNectin-2 H3K3-FcgR conjugated to a topoisomerase 1 inhibitor but not Trodelvy ヌル inhibits the growth of invasive MDA-MB-231 tumors in vivo Nude female mice (n=7 / group) were treated with 5x10 6 MDA-MB-231 cells were injected sc. Tumors grew to an average volume of 230 mm 3 Once tumor growth was reached, mice were randomized into treatment groups and treated with either PBS (vehicle), NTX1107-Dxd, or Trodelvy by iv injection at 5 mg / kg in a blinded fashion for three consecutive doses every four days. There was no effect of Trodelvy treatment on tumor growth.

[0161] As seen in Figure 12, NTX1107-Dxd was able to significantly inhibit tumor growth (*p<0.02, **<0.005.*** p<0.0005). These findings were unexpected, as the in vitro killing potency of Trodelvy was higher than that of NTX1107-Dxd (Figure 11), and Trodelvy is an approved ADC for TNBC. These findings suggest that anti-Nectin-2 (NTX1107) is a uniquely potent ADC for the treatment of solid tumors.

[0162] Example 14. Anti-hNectin-2 H3K3-FcgR ヌル can regress aggressive MDA-MB-231 tumors in vivo when conjugated to tubulin or TOP1 targeting agents Nude female mice (n=7 / group) were treated with 5x10 6 MDA-MB-231 cells were injected sc. Tumors grew to an average volume of 210 mm 3 Once the mice reached 100% CI, they were randomized into treatment groups: PBS (vehicle), H3K3-FcgR ヌル Animals were treated with either (NTX1107)-MMAE or NTX1107-Dxd by iv injection every 4 days for 5 consecutive doses in a blinded fashion. As seen in Figure 13, all animals treated with NTX1107-MMAE were free of detectable tumors at the end of the study. For animals treated with NTX1107-Dxd, 4 were tumor-free, while the remaining 3 had a mean tumor volume of 210 mm 3 and demonstrated tumor stasis. These findings suggest that the humanized antibody (NTX1107) targeting Nectin-2 described herein may form a uniquely potent ADC for the treatment of solid tumors when using different classes of payloads.

[0163] array [Table 4] SEQ ID NO:11- Heavy chain variable region NT20-11_VH3

number

number

number

number

number

number

number

number

number

number

number

number

number

number

Claims

1. A humanized antibody that specifically binds to human Nectin-2 or a fragment thereof that contains at least an antigen-binding site, the antibody or fragment thereof comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 1-3; and (ii) a set of four heavy chain (HC) framework (FR) sequences, wherein FR-H1 is selected from the group consisting of SEQ ID NOs: 21, 25, and 27; FR-H2 is SEQ ID NO: 22; FR-H3 is selected from the group consisting of SEQ ID NOs: 23, 26, 28, and 29; and FR-H4 is SEQ ID NO: 24; Including, The light chain variable region comprises: (i) a set of three CDR sequences comprising the sequences set forth in SEQ ID NOs: 4-6; and (ii) a set of four light chain (LC) framework (FR) sequences, wherein FR-L1 is selected from the group consisting of SEQ ID NOs: 30, 34, 37 and 39; FR-L2 is selected from the group consisting of SEQ ID NOs: 31 and 35; FR-L3 is selected from the group consisting of SEQ ID NOs: 32, 36, 38 and 40; and FR-L4 is SEQ ID NO:

33. A humanized antibody or fragment thereof comprising:

2. 2. The humanized antibody of claim 1, wherein the heavy chain comprises a variable region sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16, and the light chain comprises a variable region sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:

20.

3. The humanized antibody or fragment thereof comprises: (i) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO:11 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO:12; (ii) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 15 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 18; (iii) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 13 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 17; (iv) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 14 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 18; (v) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 15 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 19; and (vi) the heavy chain comprises a variable region having the amino acid sequence of SEQ ID NO: 16 and the light chain comprises a variable region having the amino acid sequence of SEQ ID NO: 20; 3. The humanized antibody of claim 1, comprising a heavy chain and a light chain selected from the group consisting of:

4. The humanized antibody of any one of claims 1 to 3, wherein the humanized 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 SEQ ID NO:11, and the light chain comprising a variable region having an amino acid sequence at least about 95% identical to SEQ ID NO:

12.

5. The humanized antibody of claim 4, wherein the humanized antibody or fragment thereof comprises a heavy chain and a light chain, the heavy chain comprising a variable region having the amino acid sequence of SEQ ID NO: 11, and the light chain comprising a variable region having the amino acid sequence of SEQ ID NO:

12.

6. The humanized antibody of any one of claims 1 to 5, wherein the humanized monoclonal antibody has a heavy chain constant region selected from IgG1, IgG4 and IgG2.

7. The humanized antibody of any one of claims 1 to 6, having a mutated Fc domain that prevents FcγR-mediated internalization.

8. The humanized antibody of claim 7, wherein the Fc domain is null for binding to Fcγ receptors.

9. A conjugate comprising the antibody or fragment thereof according to any one of claims 1 to 8.

10. The conjugate of claim 9 , wherein the humanized antibody or fragment thereof is conjugated to a cytotoxic moiety, a radioactive moiety, an affinity moiety or a labeling moiety.

11. The conjugate of claim 9 , wherein the humanized antibody or fragment thereof is conjugated to a toxin (payload).

12. The conjugate of claim 11 , wherein the toxin is selected from the group consisting of microtubule inhibitors, DNA synthesis inhibitors, topoisomerase inhibitors and RNA polymerase inhibitors.

13. The conjugate of claim 12, wherein the microtubule inhibitor is an auristatin or a derivative thereof.

14. 14. The conjugate of claim 13, wherein the auristatin derivative is monomethylauristatin E (MMAE) or monomethylauristatin F (MMAF).

15. The conjugate of claim 12 , wherein the toxin is a maytansine derivative.

16. The conjugate of claim 15, wherein the maytansine derivative is DM4 or DM1.

17. The conjugate of claim 12, wherein the toxin is a DNA topoisomerase I (TOP1) inhibitor.

18. The conjugate of claim 17, wherein the DNA topoisomerase I (TOP1) inhibitor is DXd.

19. 18. The conjugate of claim 17, wherein the DNA topoisomerase I (TOP1) inhibitor is SN-38.

20. The conjugate of claim 11 , wherein the antibody and the toxin are connected via a linker.

21. The conjugate of claim 20 , wherein the linker is cleavable.

22. A polynucleotide sequence encoding the amino acid sequence of the heavy chain variable region and the light chain variable region of the humanized antibody or fragment thereof according to any one of claims 1 to 8.

23. A pharmaceutical composition comprising the humanized antibody or antigen-binding fragment of any one of claims 1 to 8 and / or the conjugate of any one of claims 9 to 20, and a pharma- ceutically acceptable excipient, carrier or diluent.

24. 24. The pharmaceutical composition of claim 23 for use in the treatment of cancer.

25. 22. A method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of at least one humanized antibody or fragment thereof according to any one of claims 1 to 8 or a conjugate according to any one of claims 9 to 21.

26. 26. The method of claim 25, wherein the cancer is a solid cancer.

27. 26. The method of claim 25, wherein the cancer is selected from the group consisting of prostate cancer, colorectal cancer, liver cancer, ovarian cancer, endometrial cancer, gastric cancer, thyroid cancer, carcinoid tumors, head and neck cancer, breast cancer, pancreatic cancer, testicular cancer, urothelial cancer, cervical cancer, melanoma, lymphoma, and lung cancer.

28. 26. The method of claim 25, wherein the cancer is colorectal adenocarcinoma or lung adenocarcinoma.

29. 26. The method of claim 25, wherein the cancer is a hematological cancer.

30. 30. The method of any one of claims 25 to 29, further comprising administering or administering at least one additional anti-cancer treatment.

31. A method for diagnosing or prognosing cancer in a subject, comprising determining the expression level of Nectin-2 in a biological sample of said subject using at least one humanized antibody or fragment according to any one of claims 1 to 8.

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